CN114907434B

CN114907434B - Sorbicillinoid derivative and preparation method and application thereof

Info

Publication number: CN114907434B
Application number: CN202210494476.XA
Authority: CN
Inventors: 张鹏; 袁晓龙; 高丽伟; 赵栋霖; 申国明; 郝贤伟
Original assignee: Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
Current assignee: Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2023-03-31
Anticipated expiration: 2042-05-07
Also published as: CN114907434A

Abstract

The invention discloses a sorbicillinoid derivative and a preparation method and application thereof. Belongs to the technical field of phytochemistry. The sorbicillinoid derivative is prepared from tobacco endophytic fungus Aspergillus neensensis 1022 LEF. The novel structural sorbitinid derivative is named tennessenoid A through high-resolution mass spectrum and nuclear magnetic resonance spectrum. The sorbicillinoid derivative tennessenoid A has obvious inhibitory activity on various plant pathogenic fungi. Has the potential of being developed into novel microbial natural product pesticides.

Description

Sorbicillinoid derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of phytochemistry, in particular to a sorbicillinoid derivative and a preparation method and application thereof.

Background

The traditional chemical synthetic pesticide for preventing and treating agricultural diseases brings great economic benefits to agricultural production. However, due to the large application amount and high toxicity of chemical pesticides, the non-target toxic effect on beneficial organisms and the like, the land fertility is often reduced, the biological population is degraded, pesticide residues harm the health of people and livestock, and the like, and finally, the structure and the function of an ecological system are seriously damaged. Natural products play an important role in the design of pesticide molecules. According to statistics, about 24.5 percent of 102 pesticides which are registered for the first time in 2011-2017 worldwide are derived from natural products or derivatives thereof.

The microbial natural product pesticide has the characteristics of high efficiency, safety and good environmental compatibility, has an indispensable strategic position in the fields of agricultural pest control and protection and food safety protection in China, has developed various products such as spinosad, abamectin, validamycin, kasugamycin and the like at present, and is widely applied to various links of agricultural production. The metabolites derived from fungi not only account for about half of the whole microbial metabolites in number, but also have the advantages of high yield, novel structure, remarkable activity, high innovation index, high drug-like property and the like, and become a new bright point in the current microbial research. Therefore, the deep discovery of agricultural lead compounds with new structures in fungi becomes a breakthrough for the creation and development of novel microbial pesticides.

The sorbicillinoid compound is a novel polyketide compound, is mainly produced by fungi, and has wide biological activity, such as anti-tumor, insecticidal and the like.

Therefore, how to provide a soricillinoid derivative for controlling plant diseases is a problem which needs to be solved urgently by the technical personnel in the field.

Disclosure of Invention

In view of the above, the invention provides a sorbicillinoid derivative, and a preparation method and application thereof.

The invention provides a novel sorbicillinoid derivative; the second purpose is to provide a preparation method of the sorbicinoliid derivative; the third purpose is to provide the application of the soricillinoid derivative in the control of plant fungal diseases, and provide a new compound entity for the effective control of plant fungal diseases.

The strain 1022LEF is preserved by the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and the preservation number is as follows: CGMCC No.21469, and is classified and named Aspergillus tennessensis. The preservation date is as follows: 16/3/2021, storage location: beijing, chaoyang district, beichen Xilu No. 1 institute, institute of microbiology, china academy of sciences.

In order to achieve the purpose, the invention adopts the following technical scheme:

a sorbicillinoid derivative has a molecular structural formula as follows:

named tenness oil A.

The invention also provides a preparation method of the sorbicillinoid derivative, which comprises the steps of selecting 1022LEF mycelia for fermentation culture, and separating and purifying fermentation liquor;

1022LEF was deposited by the general microbiological culture collection center of the china committee for culture collection of microorganisms with the following collection numbers: CGMCC No.21469, is classified and named Aspergillus tennessensis, and has a preservation date: 16/3/2021, storage site: xilu No. 1, beijing, chaoyang, beijing, and institute for microbiology, china academy of sciences.

Further, the strain is disclosed in the patent publication (CN 202110633349.9).

Preferably: the method comprises the following steps:

(1) Selecting 1022LEF mycelia, and performing fermentation culture to obtain fermentation liquor;

(2) Extracting the fermentation liquor by ethyl acetate to obtain an ethyl acetate extract;

(3) Mixing the ethyl acetate extract with silica gel, and performing gradient elution by reduced pressure column chromatography;

(4) Collecting and eluting conditions that the volume ratio of dichloromethane to methanol is 20: eluting the component Fr.6 eluted at 1 by an ODS reverse phase silica gel column;

(5) Purifying the subfraction Fr.6.3 eluted under the elution condition of 30% methanol-water by preparative thin layer chromatography and gel column chromatography to obtain tennessoid A, wherein the molecular structural formula is as follows:

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preferably: specifically, the step (1) is to select 1022LEF mycelia and transfer the mycelia to a liquid PDB culture medium, and standing, fermenting and culturing the mycelia for 30d at 28 ℃ to obtain fermentation liquor.

Further, the specification of the mycelium is 0.5 multiplied by 0.5cm; the amount of liquid PDB medium used was 300mL.

Preferably: the specific operation of the step (2) is that the fermentation liquor is extracted by equal volume of ethyl acetate, repeated for 3 times, 3 times of extraction liquid is merged, and vacuum decompression concentration is carried out: vacuum degree of-0.1 Mpa, and concentrating to obtain non-fluid extract to obtain ethyl acetate extract.

Preferably: the specific operation of the step (3) is to add the ethyl acetate extract into 100-200 mesh silica gel for sample mixing, then carry out gradient elution through reduced pressure column chromatography, and the elution system is to use ethyl acetate in turn: petroleum ether is 30: 1. 20: 1. 10: 1. 5: 1. 2: 1. 1:1, dichloromethane: the methanol content is 20: 1. 10: 1. 5: 1. 1:1, eluting to obtain 10 components Fr.1-Fr.10 with the polarity from small to large.

Preferably, the following components: the specific operation of the step (4) is that the volume ratio of the collected ethyl acetate to the collected petroleum ether is 20: the component Fr.6 eluted at 1 is sequentially eluted by 30 percent methanol-water to 100 percent methanol solution through an ODS reverse phase silica gel column to obtain 10 subcomponents Fr.6.1 to Fr.6.10 with the polarity from large to small.

Preferably: in the step (5), the elution system is that the volume ratio of dichloromethane to methanol is 25:1; the developing system for preparing the thin layer chromatography is that the volume ratio of dichloromethane to methanol is 20:1; the gel column was sephadex LH-20 and eluted with methanol.

The invention also provides the application of the phthalide compounds in the prevention and treatment of plant fungal diseases.

Preferably: the sorbicillinoid derivative tennessoidA is dissolved in DMSO to prepare a mother solution with the concentration of 10.0 mg/L.

According to the technical scheme, compared with the prior art, the invention discloses and provides the soricillinoid derivative and the preparation method and application thereof, and the technical effect is that the soricillinoid derivative is prepared from the tobacco endophytic fungus Aspergillus tennessensis 1022 LEF. The novel structural sorbitinid derivative is named tennessenoid A through high-resolution mass spectrum and nuclear magnetic resonance spectrum. The sorbieritoid derivative tennessenoid A has obvious inhibitory activity on various plant pathogenic fungi. Has the potential of developing into novel microbial natural product pesticides.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a molecular structure diagram of the novel compound tennessenoid A provided by the present invention.

FIG. 2 is a high resolution mass spectrum of tennessenoid A compound provided by the present invention.

FIG. 3 is a drawing showing the hydrogen spectrum (500MHz, CDCl) of tennessenoid dA compound provided by the invention ₃ ) Figure (a).

FIG. 4 is a graph showing the carbon spectrum (125MHz, CDCl) of tennessenoid dA compound provided by the invention ₃ ) Figure (a).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a sorbicillinoid derivative and a preparation method and application thereof.

Example 1

A preparation method of a sorbicillinoid derivative has a molecular structural formula as follows:

named teness oil A, see FIG. 1.

The preparation method comprises the following steps:

selecting 1022LEF mycelia for fermentation culture, and separating and purifying the fermentation liquor;

in order to further optimize the technical scheme: the method comprises the following steps:

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in order to further optimize the technical scheme: specifically, the step (1) is to select 1022LEF mycelia and transfer the mycelia to a liquid PDB culture medium, and standing, fermenting and culturing the mycelia for 30d at 28 ℃ to obtain fermentation liquor.

In order to further optimize the technical scheme: the concrete operation of the step (2) is that the fermentation liquor is extracted by equal volume of ethyl acetate, the extraction is repeated for 3 times, 3 times of extraction liquid is merged, and vacuum decompression concentration is carried out: vacuum degree of-0.1 Mpa, and concentrating to obtain non-fluid extract to obtain ethyl acetate extract.

In order to further optimize the technical scheme: the specific operation of the step (3) is to add the ethyl acetate extract into 100-200 mesh silica gel for sample mixing, then carry out gradient elution through reduced pressure column chromatography, and the elution system is to use ethyl acetate in turn: petroleum ether is 30: 1. 20: 1. 10: 1. 5: 1. 2: 1. 1:1, dichloromethane: the methanol content is 20: 1. 10: 1. 5: 1. 1:1, eluting to obtain 10 components Fr.1-Fr.10 with the polarity from small to large.

In order to further optimize the technical scheme: the specific operation of the step (4) is that the volume ratio of the collected ethyl acetate to the collected petroleum ether is 20: the component Fr.6 eluted at 1 is sequentially eluted by 30 percent methanol-water to 100 percent methanol solution through an ODS reverse phase silica gel column to obtain 10 subcomponents Fr.6.1 to Fr.6.10 with the polarity from large to small.

In order to further optimize the technical scheme: in the step (5), the elution system is that the volume ratio of dichloromethane to methanol is 25:1; the developing system for preparing the thin layer chromatography is that the volume ratio of dichloromethane to methanol is 20:1; the gel column was sephadex LH-20 and eluted with methanol.

Example 2

The structure of tennessoid a was determined by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. See fig. 2, 3, 4.

The physicochemical properties of the compound tennessoid a are as follows:

the characteristics are as follows: white amorphous powder; solubility: easily soluble in dichloromethane and acetone, slightly soluble in methanol, and insoluble in petroleum ether; the molecular formula is as follows: c ₄₂ H ₅₈ O ₅ (ii) a Specific rotation degree: [ alpha ] to] ²⁰ D +15.6 (concentration c =0.10, methanol); ultraviolet absorption spectrum (UV absorbance spectrum) λ max (methanol, log ∈) 201 (3.29), 268 (3.05), 290 (4.19), 305 (3.10) nm; high resolution mass spectrometry (HR-ESI-MS): m/z 665.4183[ m ] +Na]+ (theoretical value C) ₄₂ H ₅₈ O ₅ Na, 665.4182); the hydrogen and carbon spectra data are shown in table 1;

table 1: process for preparing tennessenoid A compound ¹ H NMR (500 MHz,. Delta. Inppm, J in Hz) and ¹³ c NMR (125 MHz, delta inppm) data

Example 3

The growth rate method is adopted to evaluate the inhibition effect of the novel compound tennessoid A on 8 plant pathogenic fungi, and the specific process is as follows:

the 8 test plant pathogenic fungi are respectively peanut Bai Juan germ Sclerotium rolfsii Sacc, cucumber Fusarium oxysporum (Schl.) F.sp.cumerinum Owen, grape white rot germ Coniella diplodiella Petrak et Sydow, apple ring rot germ Physalospora piricola Nose, wheat scab germ schw, apple Alternaria alternata mali rob, cucumber Colletotrichum orbicularis arx, garlic purple rot germ Alternaria pori (E11 iott) Cife. The compound tennessoid A was dissolved in DMSO to prepare a mother liquor having a concentration of 10.0 mg/L.

100 mul of the mother liquor was evenly spread on a PDA plate, 3 fungus cakes with a diameter of 5mm were evenly placed on the plate from the edges of the colonies of the activated plant pathogenic fungi, DMSO was used as a blank control instead of the sample, and carbendazim (1.0 mg/mL) was used as a positive control. Culturing at 28 deg.C for 72h, recording the growth diameter of the fungus cake by cross method, and calculating the inhibition rate.

Inhibition (%) = (1-treatment group pure growth amount/blank group pure growth amount) × 100

Pure growth amount (mm) = growth diameter of fungus cake (mm) -5

The results show that the inhibition rate of the compound tennessoid a against 8 phytopathogenic fungi is between 46.2% and 95.3% as shown in table 2. In particular, the inhibition rate of the Bai Juan germ Sclerotium rolfsii Sacc reaches 92.1 percent, which is slightly superior to that of the positive medicament carbendazim (90.3 percent). In addition, the compound also showed strong activity (95.3%) against Fusarium oxysporum (schl.) f.sp.cumerinum Owen, which is comparable to the activity of the positive drug carbendazim (95.6%).

Table 2: inhibitory Effect of Compound TennessenoidA on various plant pathogenic fungi (% inhibition)

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A sorbicillinoid derivative is characterized in that the molecular structural formula is as follows:

named tennessoid a.

2. The method for preparing a sorbicillinoid derivative as defined in claim 1, wherein 1022LEF mycelia are selected for fermentation culture, and the fermentation broth is separated and purified;

the 1022LEF is preserved by the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number: CGMCC No.21469, and is named as Aspergillus tianasicusAspergillus tennesseensisThe preservation date: 16/3/2021, storage location: beijing, haoyang district, xilu No. 1, ministry of microbiology, china academy of sciences, 3;

the method specifically comprises the following steps:

(5) And (2) purifying the subfraction Fr.6.3 eluted under the elution condition of 30% methanol-water by preparative thin layer chromatography and gel column chromatography to obtain tennessoid A, wherein the molecular structural formula of the tennessoid A is as follows:

。

3. the method for preparing a sorbicillinoid derivative as claimed in claim 2, wherein the specific operation of step (1) is to pick 1022LEF mycelium and transfer it to liquid PDB medium, and to perform static fermentation culture at 28 ℃ for 30d to obtain fermentation broth.

4. The method for preparing the sorbicillinoid derivative as claimed in claim 3, wherein the step (2) is specifically performed by extracting the fermentation broth with an equal volume of ethyl acetate, repeating for 3 times, combining the 3 extracts, and concentrating under reduced pressure: vacuum degree of-0.1 Mpa, and concentrating to obtain non-fluid extract to obtain ethyl acetate extract.

5. The preparation method of the sorbicinoliid derivative as claimed in claim 4, wherein the specific operation in the step (3) is to add the ethyl acetate extract into silica gel of 100 to 200 meshes for sample stirring, and then perform gradient elution through reduced pressure column chromatography, wherein the elution system is that ethyl acetate is sequentially used: petroleum ether is 30: 1. 20: 1. 10: 1. 5: 1. 2: 1. 1:1, dichloromethane: the methanol content is 20: 1. 10: 1. 5: 1. 1:1, eluting to obtain 10 components Fr.1-Fr.10 with the polarity from small to large.

6. The method for preparing the sorbicillinoid derivative as claimed in claim 5, wherein the step (4) is carried out by collecting the ethyl acetate and the petroleum ether at a volume ratio of 20: and eluting the component Fr.6 eluted at the time of 1 by using 30 percent methanol-water-100 percent methanol solution through an ODS reverse phase silica gel column in sequence to obtain 10 subcomponents Fr.6.1-Fr.6.10 with the polarity from large to small.

7. The method for preparing the sorbicillinoid derivative as set forth in claim 6, wherein the elution system in the step (5) is that the volume ratio of dichloromethane to methanol is 25:1; the developing system for preparing the thin layer chromatography is that the volume ratio of dichloromethane to methanol is 20:1; the gel column was sephadex LH-20 and eluted with methanol.

8. Use of the soricillinoid derivative as defined in claim 1 for the control of fungal diseases in plants.

9. The use according to claim 8, wherein said soricillinoid derivative tennessoid A is dissolved in DMSO to finally prepare a mother liquor with a concentration of 10.0 mg/L.