CN107722032B - Anhydride compound with prodenia litura poisoning activity and preparation method and application thereof - Google Patents

Abstract

The invention discloses an anhydride compound with a prodenia litura poisoning activity, which has a structure shown in the following formula (I):

the compound has good prodenia litura poisoning activity. The invention also discloses a preparation method of the anhydride compound and application of the anhydride compound in preparing a poisoning agent with a prodenia litura poisoning effect.

Description

An acid anhydride compound with Spodoptera litura poisoning activity and preparation method thereof application

technical field

The invention belongs to the technical field of acid anhydride compounds, in particular to an acid anhydride compound with Spodoptera litura poisoning activity and a preparation method and application thereof.

Background technique

Spodoptera litura Fabricius, belonging to the Noctuidae family of Lepidoptera, also known as Spodoptera litura, commonly known as night robbers, aconites, etc., is an omnivorous and gluttonous species that occurs worldwide. Sexual crop pests are distributed in tropical and subtropical Asia, Mediterranean Europe and Africa. It occurs in all parts of my country, especially in the warm areas south of the Huaihe River, the middle and lower reaches of the Yangtze River and southern China have a large number of insect populations, and occasionally occurs in the north. Spodoptera litura is not only widely distributed geographically, but also damages a wide range of hosts. According to reports, Spodoptera litura can harm 389 species (including varieties) of host plants in 109 families, except for cabbage, cabbage, radish, mustard and other cruciferous plants In addition to vegetables, it also includes spinach and leeks, onions, etc., and involves ferns, gymnosperms, dicots and monocots. In North China, there are 4 to 5 generations per year, and 6 to 9 generations in South China, with serious overlapping of generations and no diapause characteristics. Female adults can lay 8 to 17 eggs in a lifetime, about 1 000 to 2 000 eggs, and up to 3 000 eggs. As long as the environmental conditions are suitable, their population density can increase rapidly, and outbreaks often occur. In recent years, the frequency of Spodoptera litura outbreaks has increased significantly, the area has expanded year by year, and the damage has become increasingly serious. Tomatoes, peppers, eggplants, watermelons, bitter gourds, cowpeas, cabbage, cabbage, etc., which are planted on a large scale in Hainan Island, are all damaged by this insect, which seriously affects the planting and production of crops and their economic benefits, causing great harm.

The occurrence law of Spodoptera litura is closely related to temperature and things. Compared with some other pests, Spodoptera litura prefers high temperature. Usually at a temperature of 28-30 °C, it can grow rapidly and develop rapidly, so in summer it is often It is the season of high incidence of Spodoptera litura, and it is also a critical period for the prevention and control of Spodoptera litura. On the other hand, the food of Spodoptera litura, that is, its host, also has a great influence on its occurrence. The larvae of Spodoptera litura have a wide range of food habits, and many crops and plants cannot escape its harm, but they also have certain food preferences. , different foods have a greater impact on its growth and development. Spodoptera litura larvae have a large amount of food, and when it occurs in a certain place, it often leads to serious crop yield reduction, or even no harvest.

In actual production, chemical control has become an indispensable measure to control Spodoptera litura. It is precisely because of the frequent and irregular use of chemical pesticides that kill and injure a large number of natural enemies in farmland, weakening the control effect of natural control factors in farmland ecosystem on Spodoptera litura, resulting in successive outbreaks of Spodoptera litura. For the control of Spodoptera litura, people generally give priority to chemical pesticides. Previous studies have shown that carbaryl not only has high penetrating ability and insecticidal activity to Spodoptera litura larvae, but also reduces the fecundity of Spodoptera litura adults. At the same time, the study found that some pyrethroid pesticides (such as fenvalerate, deltamethrin) have better control effect on Spodoptera litura larvae. After that, the field efficacy test of pyrethroid pesticides and their compounded chemical pesticides on Spodoptera litura larvae has been continuously reported in the literature. However, it was soon found that Spodoptera litura had different degrees of resistance to various chemical pesticides, and the control effect was reduced. Drug resistance is one of the important reasons for the rampant growth of this insect, and it is also a key factor affecting the effect of pesticide use. In India, 1995 compared with 1961, 1970 and 1987, respectively, the Spodoptera litura against pyrethrum, endosulfan, lindane, monocrotophos, moth, dimethoate, malathion, fenthion, methyl The resistance to parathion, cypermethrin, fenvalerate and deltamethrin was significantly enhanced. It has also been reported in China that Spodoptera litura has developed resistance to pyrethroids and organophosphorus pesticides. Abamectin, chlorpyrifos, beta-cypermethrin and indoxacarb have been used in fields with high frequency in Hainan Island for a long time, so serious drug resistance has occurred. At the same time, it was found that there were some differences in the resistance of Spodoptera litura larvae to pesticides feeding on different host plants. The problems caused by the extensive use of pesticides are becoming more and more serious, thus forcing people to explore new ways to control Spodoptera litura that are environmentally friendly and harmonious.

For a long time, high-efficiency and broad-spectrum chemical pesticides have been widely used in the control of agricultural diseases and insect pests. While killing the diseases and insect pests, many beneficial natural enemies have also been eliminated, causing serious damage to the ecological balance of nature. Moreover, due to the continuous use in one area, the pathogenic microorganisms gradually become resistant to drugs, resulting in the use of chemical pesticides not only can not achieve the expected effect, but will pollute agricultural products and the environment, thereby endangering human health. Today, when the awareness of environmental protection and the idea of sustainable agricultural development are generally accepted by people, it is necessary to find other alternative, safe and effective biogenic natural product pesticides that are compatible with the environment, with less toxic and side effects, to prevent and control crop diseases, and to ensure pollution-free products and Production of green food.

In recent years, screening for insecticides from natural products has received widespread attention and is one of the important ways to discover new biological pesticides. Metabolites produced by microorganisms as pesticides, namely agricultural antibiotics, have developed rapidly in recent years, and have become one of the mainstays of microbial pesticides, which can be used for biological control or provide a new source of environmentally friendly pesticides. Studying the secondary metabolites of microorganisms with anti-agricultural pest and disease activity has opened up a new way for the research and development of biological natural product pesticides, and has great application value and development potential.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an acid anhydride compound with Spodoptera litura poisoning activity, and the compound has better Spodoptera litura poisoning activity.

The object of the present invention is also to provide a preparation method of the above-mentioned acid anhydride compounds with Spodoptera litura poisoning activity, and the preparation method is simple in process and low in cost.

The third object of the present invention is to provide the application of the above acid anhydride compounds with Spodoptera litura poisoning activity in the preparation of a poisonous agent with Spodoptera litura poisoning effect.

The first object of the present invention is achieved through the following technical solutions: a kind of acid anhydride compound with Spodoptera litura poisoning activity, it has the structure in the following formula (I):

The structural formula of the compound is C ₁₈ H ₂₀ O ₆ , and the measured molecular weight is 331.1184 ([MH]-molecular ion peak, its [MH] ^-theoretical molecular weight is ^331.1182 ).

The acid anhydride compounds are produced by fermentation of Aspergillus fumigatus JRJ111048 with a deposit number of CGMCC No. 14155.

Aspergillus fumigatus JRJ111048 with the preservation number of CGMCC No. 14155 was obtained through the culture and screening of Aspergillus spp It was isolated and obtained from the leaves of the tree plant Halogena cuspidatum, and identified as Aspergillus fumigatus by molecular biology research.

The classification name of the strain of Aspergillus fumigatus JRJ111048: Aspergillus fumigatus, preservation unit: General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences), deposit address: Beijing, China, deposit date: September 4, 2017, deposit number: CGMCC No. 14155.

The second object of the present invention is achieved by the following technical solutions: the above-mentioned preparation method of the acid anhydride compound with Spodoptera litura poisoning activity, comprises the following steps:

(1) Aspergillus fumigatus JRJ111048 was inoculated into ME medium after slant activation, and cultured with shaking at 180 to 250 r·min ^-1 for 3 to 5 d at 26 to 30 ° C, and then according to 3 to 10% of the inoculum volume (volume). percentage) inoculated into ME medium, 26～30℃, 180～250r·min ^-1 shaking culture for 12～15d to obtain fermentation broth;

(2) Extract the fermentation broth with an equal volume of ethyl acetate, and concentrate the ethyl acetate extract to dryness under reduced pressure to obtain a crude extract;

(3) suspend the crude extract with distilled water, then extract with equal volumes of chloroform and ethyl acetate successively to obtain the chloroform extract layer A, the ethyl acetate extract layer B and the water layer part C, and then the ethyl acetate extract layer is B was subjected to silica gel open column chromatography, eluted with a chloroform-methanol gradient with a volume ratio of 100:0, 100:1, 100:2, 100:4, 100:8, 0:100 to obtain subfractions B1, B2, B3, B4, B5 and B6, the subfraction B2 is separated by ODS column chromatography and Sephadex-LH20 column chromatography to obtain acid anhydride compounds with Spodoptera litura poisoning activity.

In the preparation method of the acid anhydride compound with Spodoptera litura poisoning activity:

1 L of ME medium described in step (1) contains the following components: 8-10 g of malt extract, 16-20 g of sucrose, 2-4 g of peptone, and the pH of the ME medium is 7.0-7.2 .

In step (2), the fermentation broth is extracted 2 to 3 times with an equal volume of ethyl acetate, and the temperature for concentration under reduced pressure is 40 to 60°C.

The third object of the present invention is achieved through the following technical solutions: the application of the above acid anhydride compounds in the preparation of a poisonous agent with the poisoning effect of Spodoptera litura.

Preferably, the poisonous agent is applied to crops such as cotton, tobacco, soybean, taro, peanut, sesame, lotus root, cabbage, cabbage, radish, mustard, spinach, leek or green onion that are harmed by Spodoptera litura.

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

(1) The acid anhydride compound of the present invention with the structure shown in formula (I) has not been isolated from microorganisms, and the structure has not been reported, and it is a new structure compound;

(2) The present invention shows that the acid anhydride compounds with the structure shown in formula (I) have strong Spodoptera litura poisoning activity through biological activity test experiments, and these compounds are used as Spodoptera litura poisonous agents, which can be used for controlling Spodoptera litura Spodoptera litura is a biological pesticide that harms crops such as cotton, tobacco, soybean, taro, peanut, sesame, lotus root, cabbage, cabbage, radish, mustard, spinach, leek, and onion.

Description of drawings

Fig. 1 is the high-resolution mass spectrum in embodiment 4;

Fig. 2 is the ^1H one-dimensional nuclear magnetic resonance spectrum in Example 4;

Fig. 3 is ¹³ C one-dimensional nuclear magnetic resonance spectrum in embodiment 4;

Fig. 4 is HSQC two-dimensional nuclear magnetic resonance spectrum in embodiment 4;

Fig. 5 is HMBC two-dimensional nuclear magnetic resonance spectrum in embodiment 4;

Fig. 6 is ¹ H- ¹ H COSY two-dimensional nuclear magnetic resonance spectrum in embodiment 4;

Fig. 7 is ROESY two-dimensional nuclear magnetic resonance spectrum among the embodiment 4;

Detailed ways

In the following examples, the mass spectrometers were Bruker amaZon SL (low resolution) and Agilent 6210 TOF LC-MS (high resolution) mass spectrometers. The superconducting NMR instrument was Bruker AVIII-500. Silica gel GF254 for thin layer chromatography and silica gel for column chromatography (200-300 mesh) are products of Qingdao Ocean Chemical Factory. Reversed-phase ODS fillers and Sephadex LH-20 are products of Merck Company in the United States. Water was double distilled water, and other reagents were of analytical grade. In the biological activity test experiment, the insecticidal activity test model for Spodoptera litura was based on Medicinal Chemistry Research, 2014, 23(7): 3347-3352 (CA-4, a natural cis-stibene compound with potential insecticidal activity. Xiu-QingLv, Gang Feng, Ying-Qian Liu, Xiang Nan, Liu Yang). The positive insecticide azadirachtin was purchased from Haikou Shengjing Technology Co., Ltd.

Example 1

The fungal strain JRJ111048 was isolated from the leaves of the mangrove plant Halogena cuspidatum collected from the Dongzhai mangrove forest in Haikou, Hainan, China, and identified as Aspergillus fumigatus by molecular biology research. The taxonomic name of the strain: Aspergillus fumigatus fumigatus), preservation unit: General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), preservation address: Beijing, China, date of preservation : September 4, 2017, accession number: CGMCC No. 14155.

The GenBank accession number of the 18S rRNA gene sequence of this strain is MF817615, and its sequence is:

Example 2 Mass fermentation of strain JRJ111048 and pretreatment method of fermented product samples

Aspergillus fumigatus JRJ111048 was activated by slant, inoculated into ME medium, shaken for 3 days at 28°C, 210r·min ^-1 , and inoculated into 20L of ME medium according to 5% of the inoculum, 28°C, 220r·min ^-1 Shake culture for 13 days to obtain fermentation broth. The fermentation broth was extracted with an equal volume of ethyl acetate (20 L), the extraction was repeated three times, and the ethyl acetate extracts were combined and concentrated to dryness under reduced pressure at 60°C to obtain a crude extract. The ME medium was composed of the following components in a weight-to-volume ratio: malt extract 10 g/L, sucrose 20 g/L, peptone 2 g/L, and the pH value was adjusted to 7.0.

Isolation of Example 3 Compounds

The crude extract obtained in Example 2 was suspended with distilled water, and then extracted with equal volumes of chloroform and ethyl acetate successively to obtain a chloroform extract layer (A), an ethyl acetate extract layer (B) and an aqueous layer part (C). The ethyl acetate layer (B) was subjected to silica gel column chromatography, eluted with a chloroform-methanol gradient with a volume ratio of 100:0, 100:1, 100:2, 100:4, 100:8, and 0:100 to obtain There are 6 subfractions B1, B2, B3, B4, B5 and B6. Fraction B2 (833.5 mg) was separated by ODS column chromatography and Sephadex-LH20 column chromatography to obtain acid anhydride compound 1 (9.6 mg).

Structural identification of the compound of Example 4

The structures of the above compounds have been determined by high-resolution mass spectrometry (as shown in Figure 1), nuclear magnetic resonance (including one-dimensional and two-dimensional NMR experiments) (as shown in Figures 2-7) and other spectroscopic methods, which are not reported. The acid anhydride compound with a new structure was named glaucinic acid.

Its ¹ H, ¹³ C NMR data are shown in Table 1 below:

Table 1 NMR data and attribution of compound 1 (CDCl ₃ is the test solvent, δ unit is ppm)

The structure is as follows:

Example 5 Test of Spodoptera litura poisoning activity of extract samples, fraction samples and compound 1

5.1 Test organisms: Spodoptera litura eggs were collected from the unsprayed lotus root leaves in Yangfuyun Village, Guilin, Meilan District, Longhua District, Haikou, Hainan. They were reared and bred indoors for many generations, and the newly hatched larvae that had just hatched for 2 hours were picked from the same egg mass during the test. For testing.

5.2 Test method

Insecticidal activity adopts the feed mixing method. First, the test samples (crude extract in Example 2, middle fraction B2 in Example 3, compound 1) were prepared with acetone to 1 mg/mL, and a small amount of DMSO (content below 1%) was added to individual insoluble samples to prepare twill night. For moth semi-artificial diet, add 1 mL of semi-artificial diet to each well of a 12-well culture plate while it is still hot. After the feed was solidified, 20 μL of the sample to be tested was added to each well, and each sample was added to 10 wells, and repeated 3 times. After the solvent volatilized, each hole was connected to a newly hatched larva of Spodoptera litura 2 hours after hatching, and put it into an insect breeding room with a temperature of 25 ± 1 °C and a humidity of 80%, and acetone (acetone + cosolvent) was used as a solvent. For the control, azadirachtin was used as the drug control, and the death of the test worms was checked at 7d, 10d, 14d and 20d after the infestation, and the mortality was calculated.

5.3 Results of insecticidal activity

Fraction B2 and compound 1 had strong poisoning effects on Spodoptera litura larvae, slightly lower than the control agent azadirachtin. With the prolongation of treatment time, the mortality rate showed an upward trend, and the specific activity results are shown in Table 2 below.

Observation of toxic symptoms showed that fraction B2 and compound 1 had a certain inhibitory effect on the growth and development of Spodoptera litura. The specific manifestations were that the body weight of the treated insects increased slowly and the individuals were smaller. Entering the pre-pupa stage, the control test worms can pupate normally, and the pupa is full. After the test worms in the treatment group entered the pre-pupal stage, they could not shed their epidermis to form mature larvae; some larvae could only shed part of their epidermis to form a half-larva and half-pupa state; The body is too small, and individual pupae appear deformed. When entering the adult stage, the control surviving test worms can emerge normally. The eclosion rate of the test worms in the treatment group is lower, and the moth body is smaller.

Table 2 The poisoning effect of the tested samples on Spodoptera litura larvae

Some specific embodiments are listed above to illustrate the present invention. It is necessary to point out that the above and below specific embodiments are only used to further illustrate the present invention, and do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the present invention still belong to the protection scope of the present invention.

sequence listing

<110> Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences

<120> An acid anhydride compound with Spodoptera litura poisoning activity and its preparation method and application

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Claims (6)

1. an acid anhydride compound with Spodoptera litura poisoning activity is characterized in that: it has the structure in the following formula (I):

(

). 2. the preparation method of the acid anhydride compound with Spodoptera litura poisoning activity according to claim 1, is characterized in that comprising the following steps: (1) Aspergillus fumigatus JRJ111048 with preservation number of CGMCC No. 14155 was inoculated into ME medium after slant activation, and cultured with shaking at 180-250 r·min-1 at 26-30 °C for 3-5 d, and then according to the volume The inoculum with a percentage content of 3-10% is inoculated into ME medium, 26-30 ℃, 180-250r·min-1 shaking culture for 12-15d, to obtain a fermentation broth; (2) Extract the fermentation broth with an equal volume of ethyl acetate, and concentrate the ethyl acetate extract to dryness under reduced pressure to obtain a crude extract; (3) Suspend the crude extract with distilled water, then extract with equal volumes of chloroform and ethyl acetate successively to obtain the chloroform extraction layer A, the ethyl acetate extraction layer B and the water layer part C, and then the ethyl acetate extraction layer is B was subjected to silica gel open column chromatography, eluted with a chloroform-methanol gradient with a volume ratio of 100:0, 100:1, 100:2, 100:4, 100:8, 0:100 to obtain subfractions B1, B2, B3, B4, B5 and B6, the subfraction B2 is separated by ODS column chromatography and Sephadex-LH20 column chromatography to obtain acid anhydride compounds with Spodoptera litura poisoning activity. 3. the preparation method of the acid anhydride compound with Spodoptera litura poisoning activity according to claim 2, is characterized in that: ME medium 1L described in step (1) contains the component of following quality: malt extract 8-10 g, 16-20 g sucrose, 2-4 g peptone, and the pH of the ME medium is 7.0-7.2. 4. the preparation method of the acid anhydride compound with Spodoptera litura poisoning activity according to claim 2, is characterized in that: in step (2), extract fermentation broth 2～3 times with equal volume of ethyl acetate, decompression The temperature for concentration is 40-60°C. 5. the application of the acid anhydride compound according to claim 1 in the preparation of the poisoning agent with Spodoptera litura poisoning effect. 6. application according to claim 5, it is characterized in that: described poisonous agent is applied to the cotton, tobacco, soybean, taro, peanut, sesame, lotus root, cabbage, cabbage, radish, In crops of mustard, spinach, leeks or green onions.

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