CN103641810A - Polyketone compound, preparation method and application thereof - Google Patents

Abstract

The invention discloses a polyketide compound whose chemical structural formula is:

The present invention also discloses a preparation method of the above-mentioned polyketide compound: fermenting and culturing Fusarium proliferatum ZS07 with the preservation number of CCTCC NO: M2013257; filtering the obtained fermentation liquid, extracting the filtrate with ethyl acetate, concentrating and drying in vacuo , to obtain a crude extract; the crude extract was subjected to silica gel column chromatography for segmentation, and gradient elution was performed with dichloromethane/methanol; the eluted part F2 was concentrated and then recrystallized to obtain polyketides. The polyketide compound can be used as a herbicide, specifically, it can inhibit the growth of young roots of Amaranthus reflexae.

Description

Polyketides and their preparation and use

technical field

The invention relates to the field of biotechnology, in particular to the preparation and herbicide application of katydid intestinal symbiotic bacterial strains and new active metabolites thereof.

Background technique

Amaranth (Amaranthus retroflexus L) is a typical vicious invasive weed in my country, which seriously damages dryland crops and affects crop yield. Chemical herbicides used to be the main measures to control the harm of Amaranth retroflexus, but the long-term use of chemical herbicides has caused many negative effects such as environmental pollution and ecological imbalance. In the context of human beings' thirst for safe, effective, and pollution-free new herbicides, the research on efficient and environmentally friendly microbial herbicides has attracted more and more attention from experts and scholars around the world.

Insect symbionts are a class of special environmental microorganisms that are less studied. Compared with terrestrial soil-derived microorganisms, it has a wide range of sources and rich diversity. At the same time, symbiotic bacteria have formed unique physiological characteristics and metabolic pathways during the long-term co-evolution with the host, and are ideal sources of new active metabolites.

An existing compound whose structural formula is shown in formula 1; in the literature Ma S M, Zhan J, Xie X, et al. Redirecting the cycling steps of fungal polyketide synthase [J]. Journal of the American Chemical Society, 2008, 130(1):38-39 has a corresponding notification, but there is no herbicidal activity and any other use reports.

Strain F. Proliferatum (FJ648201) is a crop pathogen in agricultural production, which can cause corn ear rot (Lu Weihong, Huang Siliang, Si Shengjuan, etc.; Proceedings of the 2010 Academic Annual Conference of the Chinese Society of Phytopathology), F .Proliferatum can produce fumonisins, which can cause various species-specific toxicities to livestock and experimental animals (Molecular biodiversity of mycotoxigenic fungi that threaten food safety. Int. J. Food microbiol. 2013, 167, 57-66 ).

The patent with the publication number 101928672A informs that Fusarium laminarum can induce Dracaena to produce dried blood.

The patent with the publication number 103271022A informs that the swollen-legged wasp carrying Fusarium laminarum can be used to prevent and control the larvae of the poplar ten-spotted beetle;

The article published by Ji Zhiqin of Northwest A&F University informed that the metabolites of the endophytic fungus Fusarium sp. have obvious bactericidal activity against a variety of agricultural pathogenic fungi.

Contents of the invention

The technical problem to be solved by the present invention is to provide a polyketide compound and its application.

In order to solve the above-mentioned technical problems, the invention provides a kind of polyketide compound, its chemical structural formula is:

The present invention also simultaneously provides a Fusarium proliferatum ZS07 (Fusarium proliferatum ZS07), the preservation number of which is: CCTCCNO: M2013257.

The present invention also provides a preparation method for the above-mentioned polyketide compound, comprising the following steps:

1) Inoculate Fusarium proliferatum ZS07 (Fusarium proliferatum ZS07) with the preservation number of CCTCC NO: M2013257 on ME medium on a shaker at 160-200rpm (preferably 180rpm) and 27.5-28.5°C (preferably 28°C) for 2 to 3 days to obtain seed solution;

2) Inoculate the seed solution into ME medium (generally, inoculate 10ml of seed solution into 350-450ml of ME medium), at 160-200rpm (preferably 180rpm), 27.5-28.5°C (compared to preferably at 28°C) for 6.5 to 7.5 days (preferably 7 days); to obtain a fermentation liquid;

3), filter the fermented liquid obtained in step 2), extract the filtrate with ethyl acetate (equal volume of ethyl acetate), concentrate and dry in vacuo (at a vacuum degree of 0.1 negative pressure, dry at 45°C for 30-50 minutes), get crude extract;

4), the crude extract obtained in step 3) is subjected to silica gel column chromatography, and gradient elution is performed using dichloromethane/methanol, and the volume ratio of dichloromethane to methanol is 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32; thereby obtaining 7 elution fractions F1-F7 respectively;

5) Concentrating (distillation and concentration) the eluted fraction F2 and then recrystallizing to obtain polyketides.

As an improvement of the preparation method of the polyketide compound of the present invention: the ME medium is: 20 g of malt extract, 20 g of sucrose, 1 g of peptone, and the volume is adjusted to 1 L with distilled water.

The present invention also simultaneously provides the use of the above-mentioned polyketide compound: as a herbicide.

As an improvement of the use of the polyketide compound of the present invention: inhibiting the growth of young roots of Amaranthus reflexae.

The invention adopts the petri dish-filter paper method to measure the inhibitory effect of the new compound on the young roots of Amaranthus reflexae seeds.

The invention relates to the preparation and herbicide application of a new active metabolite of katydid intestinal symbiotic bacteria. The commensal fungal strain Fusarium proliferatum ZS07 was isolated from the intestinal tract of healthy katydids. The strain was preserved in the China Center for Type Culture Collection (CCTCC), and its preservation number is CCTCC NO: M2013257. The compound is a new structure compound, which has not been reported before. The use is embodied in that the new structural compound can inhibit the growth of young roots of the invasive weed Amaranthus retroflexi. The fermentation and cultivation conditions of the bacterial strain of the invention are simple, the extraction process of the new compound is clear, and the effect of inhibiting the growth of young roots of Amaranthus retroflexus is obvious. Can be used as agricultural herbicide.

The usage and dosage of the polyketide compound of the present invention as a herbicide: dilute to 0.05-0.1g/L for spraying; the dosage (effective amount) per hectare is about 50-100g.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is a diagram of the culture characteristics and spore morphology of the katydid intestinal commensal bacteria (F. Proliferatum ZS07) strain;

The picture on the left shows the culture characteristics of F. Proliferatum ZS07 strain;

The figure on the right shows the morphological characteristics of spores of F. Proliferatum ZS07 strain.

Figure 2 is a comparison chart of the herbicidal activity of polyketides and 2,4-D.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The reagents are all commercially available.

Embodiment 1, bacterial strain screening:

In November 2012, healthy katydid samples were captured from the suburbs of Jinhua, and the samples were brought back to the laboratory for processing as soon as possible.

The medium is MEA medium: 20g of malt extract, 20g of sucrose, 20g of agar, 1g of peptone, distilled water to 1L, pH7.0. 1.1 atmospheric pressure, sterilized at 121°C for 20min (for conventional sterilization).

Methods: Healthy katydid insects were starved for 24 hours and then sterilized with 75% (volume %) alcohol for 2 minutes under aseptic conditions, rinsed with sterile water for 3 times, and then dissected. The suspension was serially diluted 10 ^-1 , 10 ^-2 , and 10 ^-3 times with sterile water, respectively, and 0.2 mL of each serial dilution was spread on an MEA medium plate, and cultured in a 28°C incubator for 2-3 days. After the colony grows, pick a small amount of mycelium and transfer it to a new MEA medium plate to purify it to obtain a single colony, and inoculate the colony --- symbiotic bacteria ZS07 into the MEA inclined test tube and save it for later use.

Embodiment 2, bacterial strain identification

ITS rDNA analysis:

According to the instructions of BioTeke's new rapid plant genomic DNA extraction kit, the genomic DNA of the symbiotic bacteria ZS07 obtained in the above-mentioned Example 1 was extracted, using ITS universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'forward) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3 ', reverse) to amplify the gene sequence in the rDNA ITS region and purify it.

The similarity of the sequencing results was compared with the BLAST program; the analysis results showed that the symbiotic fungus ZS07 had a high similarity with the strain F.Proliferatum (FJ648201), with a similarity of 99.5%. Combined with the morphological characteristics of the bacterium, the bacterium was identified as F. Proliferatum.

The bacterium was preserved, the preservation unit: China Center for Type Culture Collection; preservation name: Fusarium proliferatum ZS07 (Fusarium proliferatum ZS07), preservation address: Wuhan University, Wuhan, China; preservation date: 2013.06.13; preservation number: CCTCC NO: M2013257.

Embodiment 3, the preparation of polyketide compound

Inoculate fresh Fusarium proliferatum ZS07 (Fusarium proliferatum ZS07) mycelium block (0.5×0.5cm ² , about 0.5g) into 250mL Erlenmeyer flasks containing 150mL ME medium in each bottle, inoculate 5-6 bottles, 180rpm , Cultivate at 28°C for 2-3 days as seed solution.

Remarks: Fresh Fusarium ZS07 can be obtained after activation of Fusarium ZS07 by conventional malt solid medium (MEA medium).

10 mL of seed solution was inoculated into a 1000 mL Erlenmeyer flask containing 400 mL of ME medium, and fermented for 7 days at 180 rpm and 28°C.

The fermentation broth was filtered with two layers of gauze, the filtrate was extracted with an equal volume of ethyl acetate, the number of extractions was 3 times, concentrated and dried in vacuo (at a vacuum degree of 0.1 negative pressure, 45°C for 30-50 minutes), and a crude extract 40.5 g.

The above ME medium is: 20g of malt extract, 20g of sucrose, 1g of peptone, distilled water to 1L, natural pH, and then sterilized at 121°C for 20min at 1.1 atmospheric pressure (for conventional sterilization).

The 40.5g crude extract was subjected to silica gel column chromatography segmented (the silica gel column was selected from 200-300 mesh silica gel, with a weight of 150g), and the dichloromethane/methanol gradient was used for elution, and the volume ratio of dichloromethane/methanol was sequentially 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, the amount of each eluent is about 1500ml, about 1200ml, about 1000ml, about 1000ml, about 800ml, about 800ml, about 800ml; Seven elution fractions F1-F7. The obtained second elution fraction F2 was concentrated by distillation (dried at 45°C for 40-55 minutes), and then recrystallized in methanol. The obtained pale yellow crystals were polyketides.

Embodiment 4, the structural identification of polyketide compound

The polyketide compound obtained in Example 3 is a light yellow crystal. The molecular weight of the compound obtained by ESI-MS is m/z 357 [M+H] ⁺ , and the molecular formula is C ₁₉ H ₁₆ O ₇ . ¹ H-NMR, ¹³ C-NMR combined with 2D-NMR spectrum analysis to determine the structure of the compound.

Its chemical structural formula is:

The spectroscopic data are: ¹ H NMR(Acetone-d ₆ )δ: 2.43(3H,s,H-18),4.25(2H,s,H-10),6.30(1H,d,J=2.3Hz,H -8),6.32(1H,d,J=2.3Hz,H-16),6.51(1H,d,J=2.3Hz,H-14),6.59(1H,d,J=2.3Hz,H-4 ),6.62(1H,s,H-6),11.14(1H,s,3-OH),3.93(3H,s,OCH ₃ ). ¹³ C NMR(Acetone-d ₆ )δ:21.5(C-18 ), 47.8(C-10), 55.4(5-OCH ₃ ), 100.1(C-2), 100.4(C-14), 101.3(C-4), 102.9(C-6), 107.0(C-8 ),111.0(C-16),118.9(C-12),139.5(C-7),141.2(C-17),152.4(C-9),156.4(C-13),160.8(C-15) , 163.4 (C-3), 166.9 (C-5), 167.1 (C-1), 198.9 (C-11).

The polyketide compound of embodiment 5, embodiment 3 gained is to the inhibitory effect of retroflexus radix young root

Take an appropriate amount of acetone to dissolve the compound to prepare 10 μg/mL and 1 μg/mL dilutions, add 5ml of the dilutions to a petri dish covered with filter paper to fully saturate the filter paper and the drug solution, and add 5ml of sterile water after the acetone volatilizes. The blank control was treated with acetone in the same way. The same concentration (ie, 10 μg/mL, 1 μg/mL) of 2,4-D was used as a positive control. Select 150 grains (10 grains per dish) of freshly white Amaranthus reflexae seeds and place them on the above-mentioned treated filter paper, and cultivate them in an artificial climate incubator under the conditions of 27°C, 80% relative humidity, and regular light (12h light, 12h dark) conditions for 4 days . The length of young roots was measured, and the results were expressed as the average root growth inhibition rate, and each treatment was repeated for 3 dishes.

Average growth inhibition rate (%)=[average root length of control-average root length of treatment]/average root length of control×100%.

The experimental results are shown in Figure 2, and the results show that the polyketide compound has an inhibitory effect on the growth of Amaranthus retroflexus. At a concentration of 1 μg/mL, the inhibition rate of the new compound on the growth of young roots of A. retroflexus was 42.1%, which was slightly higher than that of the positive control 2,4-D (40.1%) at the same concentration. When the concentration increased to 10 μg/mL, both the new compound and the positive drug showed strong growth inhibitory activity on young roots of A. ,4-D inhibition rate was 72.7%.

The above results suggest that the polyketide compound has inhibitory activity on the young root growth of Amaranthus retroflexus and can be used as a herbicide.

Comparative example 1, the compound described in formula 1 informed in the background technology is detected according to the method described in example 5, no matter when the concentration is 1 μg/mL or when the concentration is 10 μg/mL, the compound described in formula 1 The inhibitory rates of the compounds on the young roots of Amaranthus reflexae were all 0%. That is, the compound described in formula 1 has no inhibitory activity on the young root growth of Amaranthus retroflexus, and cannot be used as a herbicide.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

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Claims (6)

1. polyketide compounds, characterized in that the chemical structural formula is:

2. Fusarium proliferatum ZS07 (Fusarium proliferatum ZS07), its preservation number is: CCTCC NO: M2013257. 3. the preparation method of polyketide compound as claimed in claim 1 is characterized in that comprising the steps: 1) Inoculate Fusarium proliferatum ZS07 (Fusarium proliferatum ZS07) with the preservation number of CCTCC NO:M2013257 on ME medium, and culture it on a shaker at 160-200rpm and 27.5-28.5°C for 2-3 days to obtain a seed liquid; 2) Inoculate the seed solution in ME medium, ferment at 160-200rpm, 27.5-28.5°C for 6.5-7.5 days; obtain the fermentation liquid; 3) Filtrate the fermented liquid obtained in step 2), extract the filtrate with ethyl acetate, concentrate and dry in vacuo to obtain a crude extract; 4), the crude extract obtained in step 3) is subjected to silica gel column chromatography, and gradient elution is performed using dichloromethane/methanol, and the volume ratio of dichloromethane to methanol is 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32; thereby obtaining 7 elution fractions F1-F7 respectively; 5) Concentrating the eluted fraction F2 and then recrystallizing to obtain polyketides. 4 . The method for preparing polyketides according to claim 3 , wherein the ME medium comprises: 20 g of malt extract, 20 g of sucrose, 1 g of peptone, and distilled water to a volume of 1 L. 5. The use of the polyketide compound as claimed in claim 1, characterized in that: as a herbicide. 6. The purposes of polyketide compound according to claim 5, it is characterized in that: the growth of young root of Amaranthus reflexae is inhibited.

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