CN104804071A - Depsipeptide compound, and preparation method and application thereof - Google Patents

Abstract

The invention relates to a microbe-derived pesticide, and concretely relates to a depsipeptide compound, and a preparation method and an application thereof. The depsipeptide compound is represented by formula (1). The depsipeptide compound is obtained by through fermenting culture of Beauveria felina AS-70 (preserved in China General Microbiological Culture Collection Center on Sep. 29, 2012 with the preservation number of CGMCC NO.6643), and through extraction and separation. A result of identification of the chemical structure of the compound through nuclear magnetic resonance and mass spectrum shows that the compound has good insecticidal activity.

Description

A kind of depsipeptide compound and its preparation method and application

technical field

The invention relates to a microbial source insecticide, in particular to a depsipeptide compound and its preparation method and application. the

Background technique

With the extensive use of chemical pesticides, the resistance of plant diseases and insect pests to them has become more and more obvious, which has also brought problems such as environmental pollution and human and animal poisoning. In recent years, the "green barrier" caused by chemical pesticide residues has seriously restricted the export of agricultural products in my country, and the traditional agricultural industry is also facing greater risks and challenges. Compared with traditional chemically synthesized pesticides, microbial-derived pesticides have the advantages of being safe for humans, animals and non-target organisms, good environmental compatibility, less likely to develop resistance, and easy for large-scale production. Therefore, the development and application of microbial pesticides is of great significance to human health, environmental protection and sustainable development of agriculture. the

Contents of the invention

The object of the present invention is to provide a kind of depsipeptide compound and its preparation method and application. the

To achieve the above object, the technical solution adopted in the present invention is:

A kind of depsipeptide compound, the depsipeptide compound is shown in formula (1),

A preparation method of depsipeptide compound,

1) Beauveria felina AS-70 (preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee, the address of the preservation unit is Datun Road, Chaoyang District, Beijing, the preservation date: September 29, 2012, preservation No.: CGMCC6643, taxonomically named Beauveria felina) was inoculated in the fungal solid medium for static fermentation, extracted with an organic solvent, and the extract was obtained for use;

2) The above extracts were subjected to silica gel column chromatography, followed by gradient elution with petroleum ether-ethyl acetate and chloroform-methanol as eluents, and the eluents were collected and detected by thin-layer chromatography;

3) Separation and purification of the eluted components with a gradient of chloroform-methanol volume ratio of 20-10:1 in the above step 2) followed by reversed-phase silica gel column chromatography, gel column chromatography and preparative high-performance liquid chromatography, collected and retained Time t _R value is the component of 10.8-14.8min, obtains the depsipeptide compound shown in formula (1);

The step 3) collects the components with a retention time t _R value of 12.8 min to obtain the depsipeptide compound shown in formula (1).

Said step 1) inoculate Beauveria felina AS-70 in the fungal solid medium for 35-40 days, and ferment and culture it for 35-40 days. Extracted as an organic solvent to obtain an extract,

The step 1) the fungus solid medium is rice medium. the

In step 2), the petroleum ether-ethyl acetate elution gradient is 20:1 to 1:1, and the chloroform-methanol elution gradient is 40:1 to 1:1. the

In the step 3), the reverse silica gel column chromatography eluent is methanol-water with a volume ratio of 1:1; the gel column chromatography eluent is acetone; the preparative high performance liquid chromatography condition is a volume ratio of 9:11 Acetonitrile-water, the flow rate is 16mL/min, and the detection wavelength is 210nm. the

An application of a depsipeptide compound, the depsipeptide compound shown in formula (1) can be used to prepare agricultural insecticides. the

The advantages that the present invention has:

1) The depsipeptide compounds involved in the present invention have good insecticidal activity, and the LD ₅₀ value against Artemia is 26.6 μM.

2) The depsipeptide compounds involved in the present invention can be used as new pesticide components or lead compounds with insecticidal effects. the

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

Detailed ways

In order to clarify the understanding of the characteristics of the present invention, the present invention will be further elaborated below in conjunction with some non-limiting implementation examples. the

Example 1: The depsipeptide compound is shown in formula (1) (the Arabic numerals and Greek letters in the structural formula are the carbon atoms in the chemical structure). the

Example 2: Fermentative production and separation and purification of depsipeptide compounds:

1) Fermentation culture

Strain culture: According to the conventional culture method of microorganisms, a small amount of Beauveria felina AS-70 strains preserved in agar-malt extract medium was picked, inoculated on the surface of PDA plates, and cultured at 28°C for 3 days as a scale. The strains cultured by fermentation are ready for use. the

An appropriate amount of bacteria on the surface of the above-mentioned PDA plate was cut, inoculated into a sterilized Erlenmeyer flask filled with rice culture medium, and cultured at room temperature for 35 days. Add ethyl acetate to sterilize and set aside. the

Described rice culture medium is rice 100g/bottle, peptone 0.6g/bottle, natural sea water 100mL/bottle, 1000mL Erlenmeyer Erlenmeyer flask. the

2) Separation and purification of compounds

The above-mentioned rice culture medium was ultrasonically extracted 3 times with ethyl acetate, the ethyl acetate extract was combined, and the extract was obtained by distillation under reduced pressure. It was subjected to silica gel VLC (vacuum liquid chromatography) flash column chromatography, according to the order of increasing polarity of the eluent, with a volume ratio of 20:1 to 1:1 petroleum ether-ethyl acetate (flow rate 150mL/min), volume Gradient elution was carried out sequentially at a ratio of 40:1 to 1:1 chloroform-methanol (flow rate 150mL/min). The eluate was collected and detected by thin layer chromatography (TLC). During the detection, anisaldehyde-concentrated sulfuric acid was used as a chromogen, and the same or similar fractions were combined according to the Rf value and color development. Collect chloroform-methanol volume ratio 20-10:1 gradient elution fraction, carry out reverse-phase silica gel column chromatography to the collected fraction, with volume ratio 1:9 to 1:0 methanol-water (flow rate is 5mL/ min) gradient elution was carried out sequentially, and the methanol-water eluted fraction with a volume ratio of 1:1 was collected. The component was subjected to gel column chromatography, using acetone as the eluent (flow rate: 2mL/min), and finally separated and purified by preparative high-performance liquid chromatography (the chromatographic separation condition was acetonitrile-water with a volume ratio of 9:11, and the flow rate was 16mL/min, the detection wavelength is 210nm), and the components with a retention time t _R value of 12.8min are collected to obtain the depsipeptide compound shown in formula (1).

Depsipeptide compounds shown in formula (1), colorless crystals; UV(MeOH)λ _max (logε)202(4.45)nm; ESIMS m/z540[M+H] ⁺ ; HRESIMS m/z540.3392[ M+H] ⁺ (calcd for C ₂₆ H ₄₆ N ₅ O ₇ ⁺ ,540.3392); ¹ H and ¹³ C NMR, see Table 1.

Table 1. ¹ H (500MHz) and ¹³ C NMR (125MHz) spectrum data of the depsipeptide compound shown in formula (1)

Solvent used in NMR test: DMSO-d ₆ .

Embodiment 3: Insecticidal activity test

Artemia (brine shrimp), also known as saltwater Artemia, belongs to the phylum Arthropoda, Crustacea, Order Anocaria, Saltwater Artemia family, and the genus Artemia. As a model for insecticide screening, Artemia has been reported at home and abroad. Wang Zaiqiang et al. [Pesticides, 2011, 50(4): 261–263] used Artemia as a model organism to evaluate the killing effect of 14 common insecticides. Insecticidal activity, the results show that the method of screening compounds with insecticidal activity with Artemia is simple and sensitive to a variety of insecticides with different mechanisms of action; Hu Zhiyu et al. Artemia as an indicator organism, rapid screening of compounds with insecticidal activity in marine actinomycetes; Blizzard T.A. et al [J.Antibiot,1989,42(8):1304–1307] rapid screening of insecticide Avermella with Artemia Analogues of the element. In conclusion, as a model organism for the determination of insecticidal activity, Artemia has the advantages of a wide range of sources, simple operation, and a small amount of required compounds, which can improve the screening efficiency and is of great significance to the development of new agricultural insecticides. the

1) Hatching of Artemia eggs

Take 100 mg of Artemia eggs and place them in a 500 mL beaker, add 400 mL of artificial seawater, inflate slowly with an air pump, incubate at room temperature for 24 hours, remove egg shells and unhatched eggs, continue to culture Artemia for 24 hours, and set aside. the

2) Preparation of sample solution

The compound to be tested was dissolved in DMSO to prepare a 4mg/mL solution, and then diluted to 2, 1 and 0.5mg/mL solutions for later use. the

3) Test method

According to the improved method of Solis, take a 96-well cell culture plate and add 195 μL of artificial seawater solution containing 10-15 Artemia to each well to make a test culture plate. Three parallel wells were set up for the blank control group and each concentration sample group, 5 μL of artificial seawater was added to the blank control group, and 5 μL of the sample solution of the required concentration was added to the sample group. After incubation at room temperature for 24 hours, the number of dead individuals of Artemia was detected and counted under a binocular dissecting microscope. the

The lethal activity of Artemia is expressed by the corrected mortality rate, and the calculation formula is as follows:

Corrected mortality rate = (survival rate of control group - survival rate of treatment group)/survival rate of control group × 100%, and calculate the median lethal rate LD ₅₀ value.

The test result shows that the LD ₅₀ value of the depsipeptide compound shown in formula (1) is 26.6 μM, which has good insecticidal activity.

The above experimental results prove that the compounds involved in the present invention have good insecticidal activity, and they can be used to prepare agricultural insecticides. the

Claims (8)

1. a depsipeptide compounds, is characterized in that: depsipeptide compounds such as formula shown in (one),

2. a preparation method for depsipeptide compounds according to claim 1, is characterized in that:

1) muscardine (Beauveria felina AS-70) (is preserved in China General Microbiological culture presevation administrative center, preservation date: on September 29th, 2012, preserving number: CGMCC6643) be inoculated in standing for fermentation in fungus solids substratum, through organic solvent extraction, obtain extract, stand-by;

2) said extracted thing is carried out silica gel column chromatography, successively with Shi You Mi – ethyl acetate and Lv Fang – methyl alcohol for elutriant carries out gradient elution, collect elutriant, elutriant detects through thin-layer chromatography;

3) by above-mentioned steps 2) in the elution fraction of Lv Fang – methyl alcohol volume ratio 20 – 10:1 gradient, carry out reversed-phase silica gel column chromatography, gel filtration chromatography and preparative high-performance liquid chromatographic separation and purification successively, collection retention time t _rvalue is the component of 10.8 – 14.8min, obtains depsipeptide compounds shown in formula ().

3. by the preparation method of depsipeptide compounds according to claim 2, it is characterized in that: described step 3) collects retention time t _rvalue is the component of 12.8min, obtains depsipeptide compounds shown in formula ().

4. by the preparation method of depsipeptide compounds according to claim 2, it is characterized in that: muscardine (Beauveria felina AS-70) is inoculated in fermentation culture 35-40 days in fungus solids substratum by described step 1), one or more in ethyl acetate, acetone, chloroform, methyl alcohol, ethanol, water are extracted as organic solvent, obtain extract.

5., by the preparation method of depsipeptide compounds according to claim 2, it is characterized in that: described step 1) fungus solids substratum is rice medium.

6., by the preparation method of depsipeptide compounds according to claim 2, it is characterized in that: described step 2) PetroChina Company Limited. Mi – eluent ethyl acetate gradient be 20:1 to 1:1, Lv Fang – methanol elution gradient is 40:1 to 1:1.

7., by the preparation method of depsipeptide compounds according to claim 2, it is characterized in that: in described step 3), reverse silica gel column chromatography elutriant is the Jia Chun – water of volume ratio 1:1; Gel filtration chromatography elutriant is acetone; Preparative high-performance liquid chromatographic condition is the Yi Jing – water of volume ratio 9:11, and flow velocity is 16mL/min, and determined wavelength is 210nm.

8. an application for depsipeptide compounds according to claim 1, is characterized in that: shown in described formula (), depsipeptide compounds can be used for preparing agricultural insecticide.