Indole compound as well as preparation method and application thereof

Abstract

The invention discloses an indole compound, the chemical structural formula of which is as follows: # imgabs0 #. The invention obtains a novel indole compound which has a remarkable growth inhibition effect on radicles of gramineous weeds barnyard grass and has a remarkable damage effect on root tip cells of the gramineous weeds barnyard grass.

Description

Technical Field

[0001] The present invention relates to the field of natural products, in particular to new compounds of natural origin which can be used for weed control. Background Art

[0002] Echinochloa crus-galli (L.) P.Beauv. is an annual grass weed with strong adaptability and tolerance to adverse environments. It forms a fierce competition for rice water, light and nutrients. It is one of the malignant weeds in rice fields across the country. Chemical control is currently the main measure for the prevention and control of barnyardgrass, but the long-term use and abuse of herbicides will cause environmental pollution and pose potential risks to human health. At the same time, the problem of weed resistance is becoming increasingly prominent. Therefore, finding new compounds to inhibit the growth of barnyardgrass is an urgent problem to solve the harm and resistance of barnyardgrass. Summary of the invention

[0003] The purpose of the present invention is to solve the defects in the prior art and provide a novel compound which can effectively inhibit the growth of barnyard grass.

[0004] In order to achieve the above object, the technical solution provided by the present invention is as follows:

[0005] An indole compound, whose chemical structure is as follows:

[0006]

[0007] The invention obtains a new indole compound (hereinafter named as Colletotriauxin H), which has a significant growth inhibition effect on the young roots of the gramineous weed barnyard grass and a significant damage effect on the root tip cells thereof.

[0008] The indole compounds are prepared by the following method: co-culturing and fermenting the endophytic fungus A. destruens CIY4 of the genus Alternaria isolated from chicory leaves and the wolfberry anthracnose pathogen C. acutatum, extracting the metabolites with methanol, and then separating and obtaining the indole compounds.

[0009] The present invention can stimulate the silent genes of microorganisms through fungal co-culture fermentation, thereby obtaining hidden natural products, and adopts natural product chemical methods to extract and separate to obtain new indole compounds, which can be used for the preparation of natural source herbicides.

[0010] More specifically, the preparation method is as follows: the Alternaria endophytic fungus A. destruens CIY4 isolated from chicory leaves and the wolfberry anthracnose pathogen C. acutatum are co-cultured and fermented, the metabolites are extracted with methanol, and then extracted with ethyl acetate, silica gel column chromatography, and preparative liquid column chromatography to obtain the indole compound.

[0011] The co-culture fermentation method is as follows: A. destruens CIY4 and C. acutatum are inoculated into potato dextrose agar medium (PDA) respectively, and cultured at 27±1°C for 5 days; then, the bacterial blocks are inoculated into potato dextrose broth medium (PDB) respectively, and cultured at 27-28°C and 160r / min for 7 days to obtain seed liquid; A. destruens CIY4 and C. acutatum seed liquid are inoculated into high-temperature sterilized and cooled rice culture medium at a ratio of 1:1, and fermented and cultured at room temperature of about 25°C for 30 days.

[0012] Furthermore, after the above-mentioned co-cultivation and fermentation, the following method is used to extract and separate the indole compounds of the present invention: the co-cultivated rice fermentation product is extracted 3 times with 3 volumes of analytical pure methanol, each time for 1 day, the 3 extracts are combined and concentrated under reduced pressure by a rotary evaporator until there is no methanol taste to obtain a total extract; ultrapure water is added for suspension, and the same volume of ethyl acetate is used for extraction, and a total of 3 extractions are obtained to obtain an ethyl acetate extract; the ethyl acetate extract is preliminarily separated by silica gel column chromatography, the sample is mixed with 100-200 mesh silica gel, and the column is loaded with 100-200 mesh silica gel, and a petroleum ether / ethyl acetate solvent system of 100:0-100:100 and a dichloromethane / methanol solvent system of 100:4-0:100 are used for gradient elution, and the same components are combined by HPLC detection to obtain components Fr.1-Fr.5 in total; component Fr.5 is gradient eluted by a silica gel column, and the eluent is CH 2 Cl 2 :CH 3 OH=100:4-100:16, to obtain subcomponents Fr 5.1-Fr 5.5, and the Fr 5.5 portion is purified by semi-preparative high performance liquid chromatography using 40% acetonitrile water to obtain the indole compound.

[0013] The present invention also provides application of the indole compound in weed control.

[0014] In some embodiments, more preferably, the above-mentioned indole compounds are used to prepare natural herbicides, wherein the concentration of the indole compounds is 12.5 μg / mL to 100 μg / mL, more preferably 50 μg / mL to 100 μg / mL.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The present invention uses fungus co-culture to activate microbial silent gene clusters to produce secondary metabolites, thereby obtaining active hidden natural products, and extracting and separating them through natural product chemical methods, thereby screening out new compounds with excellent weed control effects.

[0017] The indole compounds obtained by the invention have a significant growth inhibition effect on young roots of the gramineous weed barnyard grass and a significant damage effect on its root tip cells, can be used for the preparation of natural source herbicides, and provide new lead compounds for the research and development of herbicide biopesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the chemical structural formula of the indole compound Colletotriauxin H of the present invention;

[0019] Figure 2 The key to the indole compound Colletotriauxin H of the present invention 1 H- 1 H COSY, HMBC correlation diagram;

[0020] Figure 3 The figure is a flow chart for preparing the indole compound Colletotriauxin H of the present invention;

[0021] Figure 4 is the mass spectrum of the indole compound Colletotriauxin H of the present invention;

[0022] Figure 5 is the IR spectrum of the indole compound Colletotriauxin H of the present invention;

[0023] Figure 6 is the UV spectrum of the indole compound Colletotriauxin H of the present invention;

[0024] Figure 7 is the hydrogen spectrum of the indole compound Colletotriauxin H of the present invention;

[0025] Figure 8 is the carbon spectrum of the indole compound Colletotriauxin H of the present invention;

[0026] Fig. 9 The indole compound Colletotriauxin H of the present invention 1 H- 1 H COSY diagram;

[0027] Fig.10is the HMBC diagram of the indole compound Colletotriauxin H of the present invention;

[0028] Fig.11 is the HSQC chart of the indole compound Colletotriauxin H of the present invention;

[0029] Fig.12 It is the NOESY diagram of the indole compound Colletotriauxin H of the present invention;

[0030] Fig.13 This is the activity diagram of the indole compound Colletotriauxin H of the present invention in inhibiting the growth of young roots of barnyard grass;

[0031] Fig.14 The effect of the indole compound Colletotriauxin H of the present invention on the growth of young roots of barnyard grass;

[0032] Fig.15 The present invention shows the damage to young root cells of Echinochloa crusgalli treated with the indole compound Colletotriauxin H of the present invention. DETAILED DESCRIPTION

[0033] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0034] Example 1

[0035] Preparation Example of the Indole Compound Colletotriauxin H of the Present Invention

[0036] like Figure 3As shown, the present invention activates and cultures the endophytic fungus A. destruens CIY4 of the genus Alternaria isolated from chicory leaves and the anthracnose pathogen C. acutatum (see Liu Yang, Meng Xiuhua, Lv Han, et al. Isolation of endophytic fungi from chicory leaves and analysis of their anti-plant pathogen activity [J / OL]. Journal of Plant Resources and Environment, 1-4 [2024-12-10]. http: / / kns.cnki.net / kcms / detail / 32.1339.S.20240912.1009.002.html.). The activation culture conditions are: A. destruens CIY4 and C. acutatum are inoculated in potato dextrose agar medium (PDA) at 27±1°C for 5 days. Then, the bacterial blocks are inoculated in potato dextrose broth medium (PDB) respectively, and cultured at 27-28°C and 160r / min for 7 days to obtain seed liquid. The seed liquid of A.destruens CIY4 and C.acutatum was inoculated into the rice culture medium after high temperature sterilization and cooling at a ratio of 1:1, and fermented and cultured at room temperature of about 25℃ for 30 days. Then, it was extracted 3 times with 3 volumes of analytical pure methanol (MeOH) to make the rice and fermentation products completely immersed in the solvent, and each time was soaked for 1 day. The 3 extracts were combined and concentrated under reduced pressure by a rotary evaporator until there was no methanol smell to obtain a total extract of 1.6 kg. An appropriate amount of ultrapure water was added for suspension, and the same volume of ethyl acetate (EtOAc) was used for extraction. The total extraction was 3 times to obtain 41.6 g of ethyl acetate extract. The ethyl acetate extract was initially separated by silica gel column chromatography, mixed with 100-200 mesh silica gel, loaded with 100-200 mesh silica gel, and a petroleum ether (PE) / ethyl acetate (EtOAc) solvent system (100:0~100:100) and dichloromethane (CH 2 Cl 2 ) / Methanol(CH 3 OH) solvent system (100:4~0:100) was used for gradient elution, and the same components were combined by HPLC detection to obtain components Fr.1-Fr.5. Component Fr.5 was gradient eluted by silica gel column (CH 2 Cl 2 :CH 3 OH=100:4~100:16), and obtained subcomponents Fr 5.1-Fr 5.5. The Fr 5.5 part was subjected to semi-preparative high performance liquid chromatography (40% CH 3 CN) was purified, and the obtained monomer compounds were screened for activity, and the compound Col letotriauxin H with barnyard grass growth inhibitory activity was obtained.

[0037] The structure of the obtained compound was identified:

[0038] The compound Colletotriauxin H is a white powder. The structure of the compound Colletotriauxin H is identified by combining various spectral data (such as MS, NMR, UV, IR, etc.). Figure 2 , Figures 4 to 12 ): [α] 25 D =+49.333°(c0.075, MeOH); UV(CH 3 OH)λ max 216.5, 279.2nm; IR (KBr) ν max 3402, 1715, 1456, 1185, 1079, 1036cm -1 ; m / z 376.1402[M+Na] from HR-ESI-MS data + (Calcd.for C 17 H 23 NO 7 Na 376.1367), the molecular formula of the compound is inferred to be C 17 H 23 NO 7 , the unsaturation is 7. 1 The H-NMR data (as shown in Table 1 below) show that δ H 7.17 (1H, s, H-2), 7.62 (1H, d, J = 8.0, H-4), 7.01 (1H, t, J = 7.0, H-5), 7.09 (1H, t, J = 6.9, H-6), 7.33 (1H, d, J = 8.2, H-7) There is a typical 3-substituted indole part, a methyl δ H 1.60 (3H, d, J = 7.2, H-3'), a methine group connected to a methyl group H 4.09 (1H, m, H-2'), in δ H 4.12 (1H, m, H-1") and δ H 4.46(1H,dd,11.6,2.7,H-1”), δ H 3.61 (1H, m, H-6") and δ H 3.78 (1H, m, H-6") two oxymethylene signals, δ H 3.87(1H,ddd,J=8.9,6.4,2.8,H-2”), δ H 3.76 (1H, m, H-3”), δ H 3.76 (1H, m, H-4"), δ H Four oxymethine signals at 3.65 (1H, m, H-5”).

[0039] Table 1 NMR spectrum data of compound Colletotriauxin H

[0040]

[0041] As shown in Table 1 above, 13 C-NMR showed the presence of 17 carbon signals, including an ester carbonyl carbon δ C 177.5 (C-1'), one methyl carbon delta C 18.4 (C-3'), two oxymethylene carbons δ C 68.1(C-1”),δ C 65.1 (C-6"), one methylene carbon δ C 38.4 (C-2'), 4 oxygen methylene carbon δ C 70.5(C-2”),δ C 71.0(C-3”),δ C 71.0(C-4”),δ C 73.0 (C-5"), 8 indole carbons C 123.1(C-2),δ C 115.6(C-3),δ C 127.7(C-3a), δ C 119.7(C-4),δ C 119.9(C-5),δ C 122.5(C-6), δ C 112.3(C-7), δ C 138.1 (C-7a). HMBC correlations of H-5 / C-3a, H-6 / C-4, C-7a, H-7 / C-3a, H-2 / C-3a, and C-7a showed the presence of indole, and the HMBC spectrum showed δ C The aromatic carbon C-3a at 127.7 and δ C The methylene carbon C-2' at 38.4 showed a correlation, which confirmed the C-3 substitution of the indole moiety. The HMBC correlation of H-3' / C-3, C-1' indicated the presence of a methylated 3-acetoxy moiety. The COSY correlation indicated the presence of a hexitol, which was obtained from δ H The oxymethylene at 4.12 and 4.46 starts at H-2", H-3", H-4", H-5", and ends at δ H 3.61, 3.78 at the oxymethylene, from δ H 4.14(H1”) / C-1’ and δ HThe key HMBC correlation of the carbonyl carbon at 4.48 (H1”) / C-1’ proves that the terminal connection of hexitol and indole is also connected through an ester bond. The above NMR data analysis shows that the compound is a 3-(2’-methyl)-ethyl ester-hexitol substituted indole, which is a new compound named Colletotriauxin H. Its chemical structure is as follows Figure 1 shown.

[0042] Example 2

[0043] Test of the growth inhibition activity of the compound Colletotriauxin H on the young roots of the grass weed Echinochloa crusgalli

[0044] The present invention adopts the plate method to determine the inhibitory activity of the compound Colletotriauxin H on the young root growth of barnyard grass, and glyphosate is used as a positive control drug. First, 3 mg of the compound is weighed and dissolved in a small amount of DMSO, so that the final content of DMSO in the test solution is below 0.1%, 0.1% Tween 20 is added to 3 mL, and a 1 mg / mL mother solution is prepared, and then the test solution of the corresponding concentration is prepared according to Table 2. The barnyard grass seeds are pre-treated by soaking in sterile deionized water for 1 day in advance, disinfected with 75% alcohol for 5 minutes, washed with sterile deionized water 3 times, each time for 30 seconds, two filter papers are laid flat on each culture dish, and 5 mL of the above test solution is added, and an aqueous solution containing 0.1% (v / v) dimethyl sulfoxide and 0.1% (v / v) Tween 20 is used as a solvent control ( Fig.13 , 14 Solvent group) and sterile deionized water as blank control ( Fig.13 CK group as shown), 10 seeds were placed in each dish, sealed with stretch film, and cultured at 23℃, 70% humidity, 14000 light intensity, light / dark (16h / 8h) for 4 days, and each concentration was repeated 3 times. After 4 days, photos were taken, and the length of the young roots of barnyard grass was calculated using Image J, and the data were analyzed using GraphPad Prism 8.0.2. Results (as shown in Fig.13 , 14) showed that the compound Colletotriauxin H could significantly inhibit the growth of young roots of barnyard grass at a concentration of 25 μg / mL. When the concentration reached 50 μg / mL, its effect in inhibiting the growth of barnyard grass roots was equivalent to that of glyphosate, showing a strong inhibitory activity.

[0045] Table 2 Preparation method of gradient concentration test solution

[0046]

[0047] Example 3

[0048] Effects of Colletotriauxin H on Root Tip Cells of Echinochloa crusgalli Seedlings

[0049] The present invention uses propidium iodide (PI) staining to detect the effect of the compound on the root tip cells of barnyard grass seedlings. The barnyard grass seedlings treated with the compound in Example 2 for 4 days were stained with 10 μg / mL PI stain for 30 minutes, washed with 0.01M PBS buffer, and placed under a laser scanning confocal microscope to observe the treated root tips at a wavelength of 561nm. The experiment was repeated 3 times and images were recorded. Results (such as Fig.15 As shown in the data, compared with the blank control group (CK group), the barnyard grass seedlings treated with the compound Colletotriauxin H showed obvious damage to the root tip cells of the barnyard grass at 12.5 μg / mL, and its damaging effect was stronger than that of the positive control glyphosate, indicating that it has significant root tip cell damaging activity and shows excellent potential for weed control applications.

Claims

1. An indole compound, whose chemical structure is as follows:

2. The method for preparing the indole compound according to claim 1, characterized in that: The preparation method is as follows: the endophytic fungus A. destruens CIY4 of the genus Alternaria isolated from chicory leaves and the lycium anthracnose fungus C. acutatum by co-culturing and fermenting, extracting the metabolites with methanol, and then separating to obtain the indole compounds.

3. The preparation method according to claim 2, characterized in that: The preparation method is as follows: the endophytic fungus A. destruens CIY4 of the genus Alternaria isolated from chicory leaves and the wolfberry anthracnose pathogen C. acutatum are co-cultured and fermented, the metabolites are extracted with methanol, and then extracted with ethyl acetate, chromatographed with silica gel column, and prepared by liquid phase preparation to obtain the indole compound.

4. The preparation method according to claim 3, characterized in that: The co-culture fermentation method comprises: A. destruens CIY4 and C.acutatum were inoculated into potato dextrose agar medium, cultured at 27±1℃ for 5 days; then, the bacterial blocks were inoculated into potato dextrose broth medium, cultured at 27-28℃, 160r / min for 7 days to obtain seed liquid; A.destruens CIY4 and C.acutatum seed liquid were mixed in a 1:1 ratio and inoculated into high-temperature sterilized and cooled rice culture medium, and fermented and cultured at room temperature of about 25℃ for 30 days.

5. The preparation method according to claim 4, characterized in that: After the co-culture and fermentation, the indole compound is obtained by the following method: the co-cultured rice fermentation product is extracted three times with 3 times the volume of analytical pure methanol, each time for 1 day, the three extracts are combined and concentrated by a rotary evaporator under reduced pressure until there is no methanol to obtain a total extract; ultrapure water is added for suspension, and the same volume of ethyl acetate is used for extraction, and the total extraction is extracted three times to obtain an ethyl acetate extract; the ethyl acetate extract is preliminarily separated by silica gel column chromatography, the sample is mixed with 100-200 mesh silica gel, and the column is loaded with 100-200 mesh silica gel, and a petroleum ether / ethyl acetate solvent system of 100:0-100:100 and a dichloromethane / methanol solvent system of 100:4-0:100 are 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 a silica gel column, and the eluent is CH2Cl2:CH3OH=100:4-100:16 to obtain subcomponents Fr 5.1-Fr 5.5, Fr Part 5.5 is purified by preparative high performance liquid chromatography using 40% acetonitrile water for isocratic elution to obtain the indole compound.

6. Use of the indole compound according to claim 1 in weed control.

7. The use according to claim 6, characterized in that: The indole compound is used for preparing a natural source herbicide, wherein the concentration of the indole compound is 12.5 μg / mL to 100 μg / mL.

8. The use according to claim 7, characterized in that: The concentration of the indole compound in the natural herbicide is 50 μg / mL to 100 μg / mL.

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