CN108299277B - Preparation and application of indole derivative - Google Patents

Abstract

The invention discloses the preparation and application of a chlamydosporin indole derivative. The structure of the compound is shown in formula (1), and it has good herbicidal activity. At a concentration of 5 μg/mL, the growth inhibition rate of the radicle of Echinochloa crusgalli (L.) Beauv. /mL, the inhibition rate of coleoptile growth of Amaranthus retroflexus was 81.8%. Therefore, it can be used as a leading compound or a new pesticide ingredient with herbicidal effect. The compound represented by formula (1) was prepared by strain Fusarium chlamydosporum QJP1 (deposited in China General Microorganism Culture Collection and Management Center, preservation date: December 9, 2016, preservation number: CGMCC No. 13198) through fermentation, culture, extraction and isolation , and its chemical structure was identified by nuclear magnetic resonance and mass spectrometry.

Description

Preparation and application of a kind of indole derivative

technical field

The invention relates to a microbial herbicide, in particular to the preparation and application of a chlamydosporin indole derivative.

Background technique

Weeds have always been a major problem plaguing agricultural production, affecting crop yields or disrupting ecological balance. Barnyardgrass [ Echinochloa crusgalli (L.) Beauv . ] is the most important weed in paddy fields and one of the 18 malignant weeds in the world. It is widely distributed and highly competitive, and its production mainly relies on herbicides for control. Amaranthus retroflexus ( Amaranthus retroflexus ) is a common weed in farmland, orchard, roadside and wasteland. It often pollutes the seeds of crops. If not effectively controlled, the yield of corn, soybean, spring wheat, rape and vegetables will be significantly damaged.

In recent years, chemical synthetic pesticides have been widely used to control weeds in crops. However, for a long time, the application of chemical pesticides has caused a series of problems such as environmental pollution and increased resistance of weeds. At the same time, excessive pesticide residues will also endanger the safety of agricultural products, and even endanger human health. Therefore, it is urgent to find low toxicity, effective and environmentally friendly herbicides from natural resources.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a chlamydosporin indole derivative and its preparation method and application.

For achieving the above object, the technical scheme adopted in the present invention is:

A chlamydosporin indole derivative, the chlamydosporin indole derivative is shown in formula (1),

。

.

A preparation method of chlamydosporin class indole derivatives,

1) The strain Fusarium chlamydosporum QJP1 (deposited at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, General Microbiology Center of China Microbial Culture Collection Management Committee, preservation date: December 9, 2016, preservation number: CGMCC No. 13198) inoculated in a fungal solid medium for static fermentation, and extracted with an organic solvent to obtain an extract, which is ready for use;

2) The above extract is subjected to silica gel column chromatography, and gradient elution is carried out with petroleum ether-ethyl acetate in a volume ratio of 50:1 to 1:1 and chloroform-methanol in a volume ratio of 50:1 to 1:1. Deliquoring, the eluate was detected by thin layer chromatography;

3) The elution fraction of the petroleum ether-ethyl acetate volume ratio 1:1 gradient in the above step 2) is separated and purified by reversed-phase silica gel column chromatography and preparative high performance liquid chromatography in sequence, and the collection retention time t _R value is The component of 15.4 min, the chlamydosporin indole derivative represented by formula (1) is obtained;

In the step 1), the strain Fusarium chlamydosporum QJP1 is inoculated into a fungal solid medium for fermentation for 30-40 days, and extracted with one or more of ethyl acetate, acetone, chloroform, methanol, ethanol and water as an organic solvent to obtain fermentation product.

The fungal solid medium in step 1) is a rice medium.

In step 2), the elution gradient of petroleum ether-ethyl acetate is 50:1 to 1:1, and the elution gradient of chloroform-methanol is 50:1 to 1:1.

In the step 3), the reverse silica gel column chromatography eluent is methanol-water, and the elution gradient is 1:9 to 1:0; the conditions for preparing high performance liquid chromatography are methanol-water with a volume ratio of 3:10, and the flow rate is was 3 mL/min, and the detection wavelength was 230 nm.

An application of a chlamydosporin indole derivative, the chlamydosporin indole derivative represented by formula (1) can be used as a microbial herbicide for weed control.

The advantages of the present invention:

1) The chlamydosporin indole derivatives involved in the present invention have good herbicidal activity, and the herbicidal activity of the compound represented by formula (1): at a concentration of 5 μg/mL, the inhibition rate of the radicle growth of Echinochloa crusgalli is as follows: 95.8%, at 10 μg/mL concentration, the inhibition rates of coleoptile and radicle growth of Amaranthus retroflexus were 81.8% and 77.0%, respectively.

2) The chlamydosporin indole derivatives involved in the present invention can be used as the preparation of leading compounds with herbicidal effect.

3) The chlamydosporin indole derivatives involved in the present invention can be fermented on a large scale by microorganisms, and have the characteristics of simple production process, short cycle and low product cost.

Detailed ways

In order to clarify the understanding of the features of the present invention, the present invention will be further described below with reference to some non-limiting embodiments.

Example 1: chlamydosporin indole derivatives are shown in formula (1) (the Arabic numerals in the structure are the carbon atoms),

。

.

Example 2: Fermentative production and separation and purification of chlamydosporin indole derivatives:

1) Fermentation culture

Strain culture: According to the conventional culture method of microorganisms, a small amount of strain Fusarium chlamydosporum QJP1 preserved in agar-malt extract medium was selected, inoculated on the surface of PDA plate, and cultivated at 28°C for 3 days as a strain for large-scale fermentation and culture, ready to use .

An appropriate amount of the bacterial species on the surface of the above-mentioned PDA plate was cut, inoculated into a sterilized conical flask containing a rice culture medium, and cultured at room temperature for 30 days. Add ethyl acetate to sterilize and set aside.

The rice culture medium is 100 g/bottle of rice, 0.6 g/bottle of peptone, and 100 mL/bottle of artificial seawater.

2) Separation and purification of compounds

The above-mentioned rice culture medium was ultrasonically extracted three times with ethyl acetate, the ethyl acetate extracts were combined, and the extract was obtained by distillation under reduced pressure. It was subjected to silica gel VLC (vacuum liquid chromatography) flash column chromatography, in accordance with the increasing order of eluent polarity, with petroleum ether-ethyl acetate in a volume ratio of 50:1 to 1:1 (the flow rate was 150 mL/min), Gradient elution was performed with a volume ratio of 50:1 to 1:1 chloroform-methanol (flow rate 150 mL/min). The eluate was collected and detected by thin layer chromatography. Anisaldehyde-concentrated sulfuric acid was used as the color developing agent during the detection, and the same or similar parts were combined according to the R _f value and color development. The fractions of petroleum ether-ethyl acetate volume ratio 1:1 gradient elution were collected, and the collected fractions were subjected to reverse-phase silica gel column chromatography, with methanol-water (flow rate of 5 mL) in a volume ratio of 1:9 to 1:0. 6-2。 /min) gradient elution was performed sequentially, and the methanol-water elution fraction Fr. 6-2 with a volume ratio of 1:1 was collected. The components were separated and purified by preparative high performance liquid chromatography (chromatographic conditions were methanol-water with a volume ratio of 3:10 , the flow rate was 3 mL/min, and the detection wavelength was 230 nm) _. Then, the chlamydosporin indole derivatives represented by formula (1) are obtained.

Chlamydosporin indole derivatives represented by formula (1), pale yellow powder, HR-ESI-MS m/z 248.1277 [M + H] ⁺ , suggesting that the molecular formula is C ₁₄ H ₁₇ NO ₃ , its ¹ H- and ¹³ C -NMR data are shown in Table 1.

Example 3: Herbicidal activity test

The agar mixed powder method established by Luo Xiaoyong et al. [Journal of Qingdao Agricultural University, 2007; 24(4): 267-270] is used to rapidly determine the herbicidal activity of compounds, with high sensitivity, simple operation, short measurement time, and good repeatability. , The characteristics of large-throughput and extensive screening, and it is easy to observe the growth status of the indicated plants. Luo Xiaoyong et al. used this method to rapidly determine the herbicidal activity of leaves of 40 garden plants. Zhang Lijuan [Plant Protection, 2016; 42:63-66.] and Su Fansheng [Applied Chemistry, 2014;31:290-295.] Using barnyardgrass as the test weed, the rapid determination of corn stalk by agar mixed powder method and herbicidal activity of imidazoles. Zhang Yun [Journal of Microbiology, 2015; 55(3): 292-298.] and Li Shuai [Journal of Agricultural and Food Chemistry, 2014; 62(14): 8997-9001.] took Amaranthus as the test weed, The inhibitory effect of fungal metabolites on the root growth of Amaranthus amaranthus was studied.

1) Germination of weed seeds

Weed seeds were first disinfected with 0.2% sodium hypochlorite for 15 minutes, then repeatedly washed with sterile distilled water, soaked for 4-6 hours, and germinated in a medical tray with sterile wet filter paper for 12 hours in the dark.

2) Preparation of sample solution

The compounds to be tested were dissolved in dimethyl sulfoxide (DMSO) to prepare a 4 mg/mL solution, which was diluted with distilled water containing 50% DMSO to a certain concentration for use.

3) Test method

Mix 1 mL of sample solution and 99 mL of sterile agar aqueous solution, and pour them into three 25 mL small beakers to condense. Use sharp-nose tweezers to insert 5 small openings on the surface of the condensed agar, respectively pick up the germinated weed seeds with radicles up to 4-5 mm long, and gently insert the radicles into the small openings vertically. 5 capsules per beaker, repeat 3 times. Then, all the beakers were sealed with tin foil and placed in a sterilized small carton box, and placed in an artificial climate box for 2 d in the shade. The artificial climate box was set up with an automatic cycle of 14 h light (25 °C) and 10 h dark (20 °C), and the relative humidity inside the box was 60%. After 2 days, the lengths of radicle and hypocotyl were investigated, and the growth inhibition rate of weeds was calculated.

The calculation formula is [Chinese Agricultural Science Bulletin, 2013;29(15):177-182.]:

Growth amount = radicle (cotyl) length after treatment − radicle (cotyl) length before treatment;

Inhibition rate (%) = [(average growth of control − average growth of treatment)/average growth of control]×100%.

The test result is the herbicidal activity of the compound represented by formula (1): at the concentration of 5 μg/mL, the inhibition rate of the radicle growth of barnyardgrass was 95.8%, and at the concentration of 10 μg/mL, it was effective on the coleoptile and The inhibition rates of radicle growth were 81.8% and 77.0%, respectively. Has good herbicidal activity.

The above experimental results prove that the compounds involved in the present invention have good herbicidal activity against barnyard grass and amaranthus, and can be used for the preparation of herbicidal active lead compounds and microbial herbicides with weedicidal activity.

sequence listing

<110> Qingdao Agricultural University

<120> A kind of preparation and application of indole derivative

<130> 2010

<141> 2018-02-08

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 528

<212> DNA

<213> Fusarium chlamydosporum QJP1

<400> 1

tccgtaggtg aaccttgcgg ttaaactccc aaacccctgt gacataccta tacgttgcct 60

cggcggatca gcccgcgccc cgtaaaacgg gacggcccgc ccgaggaccc ctaaactctg 120

tttttagtgg aacttctgag taaaacaaac aaataaatca aaactttcaa caacggatct 180

cttggttctg gcatcgatga agaacgcagc aaaatgcgat aagtaatgtg aattgcagaa 240

ttcagtgaat catcgaatct ttgaacgcac attgcgcccg ccagtattct ggcgggcatg 300

cctgttcgag cgtcatttca accctcaagc tcagcttggt gttgggactc gcggtaaccc 360

gcgttcccca aatcgattgg cggtcacgtc gagcttccat agcgtagtaa tcatacacct 420

cgttactggt aatcgtcgcg gccacgccgt aaaaccccaa cttctgaatg ttgacctcgg 480

atcaggtagg aatacccgct gaacttaagc atatcaataa gcggagga 528

Claims (3)

1. an indole derivative, is characterized in that: indole derivative is shown in formula (1),

. 2. a preparation method of indole derivative according to claim 1, is characterized in that: 1) The strain Fusarium chlamydosporum QJP1 was inoculated into a rice medium for 30 days of static fermentation, and extracted with ethyl acetate to obtain an extract, which was ready for use; the strain Fusarium chlamydosporum QJP1 was deposited in the General Microbiology Center of the China Committee for the Storage and Administration of Microorganisms , preservation date: December 9, 2016, preservation number: CGMCC No.13198; 2) The above extract is subjected to silica gel column chromatography, and gradient elution is carried out with petroleum ether-ethyl acetate and chloroform-methanol, and the eluent is collected, and the eluent is detected by thin layer chromatography; the petroleum ether-ethyl acetate The ester elution gradient was 50:1 to 1:1 and the chloroform-methanol elution gradient was 50:1 to 1:1; 3) The elution fraction of the petroleum ether-ethyl acetate volume ratio 1:1 gradient in the above step 2) is separated and purified by reversed-phase silica gel column chromatography and preparative high performance liquid chromatography in sequence, and the collection retention time t _R value is 15.4 min components, the indole derivative represented by formula (1) is obtained; the eluent of the reversed-phase silica gel column chromatography is methanol-water, and the elution gradient is 1:9 to 1:0; high-efficiency liquid is prepared The chromatographic conditions were methanol-water with a volume ratio of 3:10, a flow rate of 3 mL/min, and a detection wavelength of 230 nm. 3 . The application of the indole derivative according to claim 1 , wherein the indole derivative represented by the formula (1) is used to prepare a microbial herbicide for barnyardgrass or amaranth. 4 .