Diboside cyclic ether compound, preparation method thereof and application of compound in preparation of medicine for preventing and treating plant diseases

Abstract

The invention discloses a depside cyclic ether compound, a preparation method thereof and application of the depside cyclic ether compound in preparation of a medicine for preventing and treating plant diseases. According to the present invention, Aspergillus penicillioides solid fermentation extract is subjected to separation and purification to obtain the depsilate cyclic ether compound, and the specific structure of the depsilate cyclic ether compound is represented by a formula (I); tests prove that the compound has a remarkable antagonistic effect on plant pathogenic bacteria xanthomonas campestris and can be used for preparing the medicine for preventing and treating plant diseases caused by xanthomonas campestris. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biological or agricultural technologies, and particularly relates to a phloroglucinol cyclic ether compound, a preparation method thereof, and an application thereof in the preparation of a drug for preventing and treating plant diseases. Background Art

[0002] Xanthomonas campestris pv. campestris is a plant pathogenic bacterium and is the main pathogen of black rot disease in cruciferous crops, causing great harm in vegetable production and horticultural plants. Therefore, it is very important to develop a drug with low toxicity, high efficiency and antagonistic effect against Xanthomonas campestris pv. campestris. Fungi of the genus Aspergillus are one of the main microbial species that produce a large number of bioactive natural products. Aspergillus penicillioides GDMCC 3.587 is an Aspergillus fungus isolated from soil. So far, there has been little research on the secondary metabolites of this strain, and the biological activities of the metabolites of this strain have not attracted attention. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the present invention provides a phloroglucinol cyclic ether compound, a preparation method thereof, and an application thereof in the preparation of a drug for preventing and treating plant diseases. The Aspergillus penicillioides strain and its phloroglucinol cyclic ether products provided by the present invention will play a key role in the development of drugs for preventing and treating plant diseases caused by Xanthomonas campestris pv. campestris.

[0004] The first object of the present invention is to provide a phloroglucinol cyclic ether compound having an antagonistic effect on plant pathogenic bacteria.

[0005] The phloroglucinol cyclic ether compound of the present invention has a structure as shown in formula (Ι):

[0006]

[0007]

[0008] The second object of the present invention is to provide a preparation method of the phloroglucinol cyclic ether compound as shown in formula (Ⅰ), and the compound is isolated and prepared from the fermentation culture of Aspergillus penicillioides.

[0009] Preferably, the preparation method of the phloroglucinol cyclic ether compound specifically comprises the following steps:

[0010] (1) Prepare a solid fermentation culture of Aspergillus penicillioides, extract the solid fermentation culture with ethanol and filter it. After concentration, a crude extract is obtained. The crude extract is dissolved in water and extracted separately with petroleum ether and ethyl acetate solutions. The extractant is concentrated to obtain an extract;

[0011] (2) Subject the extract obtained in step (1) to silica gel column chromatography. Use petroleum ether - ethyl acetate with volume ratios of 60:1, 30:1, 15:1, 10:1, 5:1, 2:1, 1:1, 1:3, 1:5, 0:1, and ethyl acetate - methanol with volume ratios of 100:1, 50:1, 20:1, 10:1, 0:1 as eluents in turn for gradient elution. Collect the fractions eluted with ethyl acetate:methanol volume ratios of 50:1 and 20:1 and having R f = 0.3 - 0.5, which is fraction Fr.5;

[0012] Subject fraction Fr.5 to ODS reversed-phase column chromatography separation. Use methanol - water with volume ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 75:25, 100:0 as eluents in turn for gradient elution. Collect the fractions eluted with methanol:water volume ratios of 40:60 and 50:50 and having R f = 0.3 - 0.8, which is fraction Fr.5 - 7;

[0013] Subject fraction Fr.5 - 7 to Sephadex LH-20 column chromatography separation. Use pure methanol as the eluent for elution. Collect the fractions having R f = 0.4 - 0.5, which is fraction Fr.5 - 7 - 6; Subject fraction Fr.5 - 7 - 6 to HPLC separation and purification to obtain a compound.

[0014] More preferably, the specific steps for preparing the solid fermentation culture of Aspergillus penicillioides in step (1) are as follows: Pick the mycelium of Aspergillus penicillioides and inoculate it into a potato dextrose agar medium, statically culture it at 28°C for 5 days, then take the agar block with mycelium and inoculate it into a rice solid medium, and statically culture it at 28°C for 30 days to obtain the solid fermentation culture; the potato dextrose agar medium is prepared per liter by the following method: Add 200 g of potato powder, 20 g of glucose and 20 g of agar to 1000 mL of distilled water, heat and dissolve it, and sterilize it; the rice solid medium is prepared by the following method: Mix 200 g of rice, 3 g of peptone and 200 mL of water and sterilize it.

[0015] More preferably, the specific purification of component Fr.5-7-6 by HPLC in step (2) is as follows: Component Fr.5-7-6 is separated by semi-preparative HPLC, using a Kromasil C18 column, with the mobile phase being acetonitrile-water with a volume ratio of 42:58, a flow rate of 3 mL / min, and collecting the eluted component with a retention time of 22.02 min to obtain the compound.

[0016] The third object of the present invention is to provide the application of the described depside ring ethers compound or its medicinal salt in the preparation of drugs for preventing and treating plant diseases.

[0017] Preferably, the drug for preventing and treating plant pathogenic bacteria is specifically a drug for preventing and treating plant diseases caused by Xanthomonas campestris.

[0018] The fourth object of the present invention is to provide a drug for preventing and treating plant diseases, comprising the described depside ring ethers compound or its medicinal salt as an active ingredient.

[0019] The fifth object of the present invention is to provide the application of Aspergillus penicillioides in the preparation of the described depside ring ethers compound.

[0020] Compared with the prior art, the advantages of the present invention are as follows:

[0021] 1. In vitro activity studies have shown that the depside ring ethers compound proposed by the present invention has a significant antagonistic effect, and its inhibitory effect on the plant pathogen Xanthomonas campestris pv. campestris is very obvious.

[0022] 2. The depside ether compounds are isolated and prepared from Aspergillus penicillioides in the present invention. These compounds have significant antagonistic effects against Xanthomonas campestris, and can be used to prepare drugs for preventing and treating plant diseases caused by Xanthomonas campestris, providing candidate compounds for the research and development of new drugs.

[0023] Aspergillus penicillioides of the present invention is deposited in Guangdong Microbial Culture Collection Center (GDMCC), Address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Zip Code: 510070, Deposit Number: GDMCC No: 3.587. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the 1 1H NMR spectrum of the depside ether compounds;

[0025] Figure 2 is the 13 13C NMR spectrum of the depside ether compounds;

[0026] Figure 3 is the HR-ESI-MS spectrum of the depside ether compounds. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be further explained below in conjunction with the embodiments, but the embodiments do not limit the present invention in any form.

[0028] Example 1: Preparation and Structure Identification of Compounds

[0029] 1. Aspergillus penicillioides of the present invention is deposited in Guangdong Microbial Culture Collection Center (GDMCC), Deposit Number: GDMCC No: 3.587.

[0030] 2. Solid Fermentation of Aspergillus penicillioides

[0031] The mycelium of Aspergillus penicillioides is inoculated into potato dextrose agar medium and statically cultured at 28 °C for 5 days. Then, take about 1×1 cm 2The agar blocks with hyphae were inoculated into the solid rice medium and statically cultured at 28 °C for 30 days to obtain the solid fermentation culture. The potato dextrose agar medium was prepared per liter by the following method: 200 g of potato powder, 20 g of glucose and 20 g of agar were added to 1000 mL of distilled water, heated and melted, and sterilized; the rice medium was prepared by the following method: 200 g of rice, 3 g of peptone and 200 mL of water were mixed and sterilized.

[0032] 3. Preparation of the compound

[0033] (1) The solid fermentation culture of Aspergillus penicillioides was extracted with ethanol, and the concentrated crude extract was obtained. The crude extract was dissolved in water to obtain a suspension, and each was extracted 3 times with petroleum ether and ethyl acetate solutions respectively. The extract was concentrated to obtain about 45 g of the extract.

[0034] (2) The obtained extract was subjected to silica gel column chromatography, and petroleum ether - ethyl acetate with volume ratios of 60:1, 30:1, 15:1, 10:1, 5:1, 2:1, 1:1, 1:3, 1:5, 0:1, and ethyl acetate - methanol with volume ratios of 100:1, 50:1, 20:1, 10:1, 0:1 were used as eluents in turn for gradient elution. The fractions eluted with ethyl acetate:methanol volume ratio of 50:1 and 20:1 and developed by TLC thin layer chromatography with dichloromethane:methanol = 30:1 v / v to obtain R f = 0.3 - 0.5 fraction Fr.5.

[0035] (3) The fraction Fr.5 was separated by ODS reverse phase column chromatography, and methanol - water with volume ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 75:25, 100:0 were used as eluents in turn for gradient elution. The fractions eluted with methanol:water volume ratio of 40:60 and 50:50 and developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v to obtain R f = 0.3 - 0.8 fraction Fr.5 - 7;

[0036] (4) The fraction Fr.5 - 7 was separated by Sephadex LH - 20 column chromatography and eluted with pure methanol as the eluent. The fraction eluted with dichloromethane:methanol = 20:1 v / v and developed by TLC thin layer chromatography to obtain R f = 0.4 - 0.5 fraction Fr.5 - 7 - 6; the fraction Fr.5 - 7 - 6 was separated by semi - preparative HPLC using a Kromasil C18 column, the mobile phase was acetonitrile - water with a volume ratio of 42:58, the flow rate was 3 mL / min, and the eluted fraction with a retention time of 22.02 min was collected to obtain the compound.

[0037] 4. Structural Identification of Compounds

[0038] The NMR nuclear magnetic resonance spectrum was measured using a Bruker AscendTM 600M nuclear magnetic resonance spectrometer, and the deuterated reagent was methanol-d 4 ; The HR-ESI-MS data was measured using a Thermo Fisher Orbitrap Exploris 120 high-resolution liquid chromatography-mass spectrometry instrument. The structural identification data is as follows:

[0039] As Figures 1-3 shown, Figure 1 is the 1 1H NMR spectrum of the compound; Figure 2 is the 13 13C NMR spectrum of the compound; Figure 3 is the HR-ESI-MS spectrum of the compound.

[0040] The characterization data of the compound is as follows:

[0041] HR-ESI-MS (m / z) of the compound: 301.1052 [M+H] + C 17 H 17 O 5 , and the calculated value is 301.1071.

[0042] The structural formula of the target compound separated by the above method and the NMR data attribution are as follows:

[0043] Table 1. NMR data of the compound (δ in ppm, J in Hz, methanol-d 4 )

[0044]

[0045] The structural formula of the target compound separated by the above method is as shown in formula (I):

[0046]

[0047] Example 2

[0048] Test the antagonistic effect of the compound against the plant pathogenic bacterium Xanthomonas campestris pv. campestris.

[0049] The plant pathogenic bacterium detected in this experiment is Xanthomonas campestris pv. campestris.

[0050] 1. Detection principle of the microbroth dilution method: This method uses a 96-well plate for detection and measures the absorbance value (OD 600) The number of viable bacteria was estimated based on the optical density (OD) value.

[0051] 2. Experimental methods

[0052] (1) Preparation of bacterial suspension: The bacterial liquid fermentation broth in the logarithmic phase was diluted 1000-fold with nutrient broth (NB) culture medium to prepare a bacterial suspension with an OD 600 value less than 0.01. The bacterial suspension was transferred to a 96-well plate, with the volume of the first well being 200 μL, the volume of each of the 2nd - 11th wells being 100 μL, and the last well being blank.

[0053] (2) Addition of the test compound solution: The compound was dissolved in DMSO, and 1 - 2 μL of the compound was pipetted into the first well to make the final concentration of the test compound in the first well 200 μg / mL. After thorough mixing, 100 μL of the bacterial solution was pipetted from the first well into the second well, and this method was used to serially dilute to the 12th well, making the final volume of each well 100 μL. Each treatment was set with 3 replicates, and incubation was continued at 37 °C for 24 h. After the reaction was complete, the absorbance value (OD 600 ) at 600 nm was measured.

[0054] (3) Positive control compound: Streptomycin was used as the positive compound in each experiment. A growth curve was plotted with the concentration of the test compound as the abscissa and the bacterial survival rate as the ordinate, and the IC 50 value of the compound was calculated using the two-point method (Reed and Muench method).

[0055] The experimental results showed that the compound had an antagonistic effect against the plant pathogen Xanthomonas campestris pv. campestris, with an IC 50 = 6.1 ± 0.4 μg / mL, and the IC 50 of the positive control drug streptomycin = 3.8 ± 0.4 μg / mL.

[0056] This result indicates that the compound proposed in the present invention has a significant antagonistic effect against the plant pathogenic bacterium Xanthomonas campestris pv. campestris. Therefore, the present invention provides a candidate compound for the research and development of new drugs for controlling plant pathogens caused by Xanthomonas campestris pv. campestris.

[0057] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be regarded as a limitation of the present invention. The protection scope of the present invention should be determined by the scope defined by the claims. For those of ordinary skill in the art of this technology, without departing from the spirit and scope of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. The depsidone cyclic ether compound shown in formula (Ι):

2. The preparation method of the depsidone cyclic ether compound according to claim 1, characterized in that, the compound is isolated and prepared from the fermentation culture of Aspergillus penicillioides.

3. According to the preparation method described in claim 2, characterized in that, it specifically includes the following steps: (1) Prepare the solid fermentation culture of Aspergillus penicillioides, extract the solid fermentation culture with ethanol and filter it, concentrate to obtain a crude extract, dissolve the crude extract in water, and extract it with petroleum ether and ethyl acetate solutions respectively, and concentrate the extract to obtain an extract; (2) The extract obtained in step (1) is subjected to silica gel column chromatography, and petroleum ether-ethyl acetate with volume ratios of 60:1, 30:1, 15:1, 10:1, 5:1, 2:1, 1:1, 1:3, 1:5, 0:1, and ethyl acetate-methanol with volume ratios of 100:1, 50:1, 20:1, 10:1, 0:1 are used as eluents in turn for gradient elution. Collect the fraction Fr.5 obtained by elution with ethyl acetate:methanol volume ratios of 50:1 and 20:1 and developed by TLC thin layer chromatography with dichloromethane:methanol = 30:1 v / v to obtain R f = 0.3 - 0.5; The fraction Fr.5 was separated by ODS reversed-phase column chromatography. Methanol-water with volume ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 75:25, and 100:0 were used as eluents in turn for gradient elution. The fractions obtained by elution with methanol:water volume ratios of 40:60 and 50:50 and developed by TLC thin-layer chromatography with dichloromethane:methanol = 20:1 v / v to give R f = 0.3 - 0.8 of the fraction Fr.5-7; The fraction Fr.5-7 was separated by Sephadex LH-20 column chromatography, eluted with pure methanol as the eluent, and the fraction Fr.5-7-6 with R f = 0.4 - 0.5 was collected by TLC thin layer chromatography developed with dichloromethane:methanol = 20:1 v / v; the fraction Fr.5-7-6 was separated and purified by HPLC to obtain the compound.

4. According to the preparation method described in claim 3, characterized in that, the specific steps for preparing the solid fermentation culture of Aspergillus penicillioides in step (1) are: pick the mycelium of Aspergillus penicillioides and inoculate it into a potato dextrose agar medium, statically culture it at 28°C for 5 days, and then take the agar block with mycelium and inoculate it into a rice solid medium, statically culture it at 28°C for 30 days to obtain a solid fermentation culture; the potato dextrose agar medium is prepared per liter by the following method: add 200 g of potato powder, 20 g of glucose and 20 g of agar, add 1000 mL of distilled water, heat and dissolve, and sterilize to obtain; the rice solid medium is prepared by the following method: mix 200 g of rice, 3 g of peptone and 200 mL of water and sterilize.

5. According to the preparation method described in claim 3, characterized in that, the specific purification of component Fr.5-7-6 in step (2) by HPLC is as follows: separate component Fr.5-7-6 by semi-preparative HPLC, use a Kromasil C18 column, the mobile phase is acetonitrile-water with a volume ratio of 42:58, the flow rate is 3 mL / min, collect the eluted component with a retention time of 22.02 min to obtain the compound.

6. The application of the depsidone cyclic ether compound according to claim 1, or its medicinal salt in the preparation of drugs for preventing and treating plant diseases.

7. According to the application described in claim 6, characterized in that, the drug for preventing and treating plant diseases is specifically a drug for preventing and treating plant diseases caused by Xanthomonas campestris.

8. A drug for preventing and treating plant diseases, characterized in that, it contains the depsidone cyclic ether compound according to claim 1 or its medicinal salt as an active ingredient.

9. The application of Aspergillus penicillioides in the preparation of the depsidone cyclic ether compound according to claim 1.

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