Preparation method of pyripyroene derivative and application of pyripyroene derivative in preparation of medicine for preventing and treating plant diseases

Abstract

The invention discloses a preparation method of a pyripyroene derivative and application of the pyripyroene derivative in preparation of a medicine for preventing and treating plant diseases. According to the present invention, a solid fermentation extract of Aspergillus penicillioides is subjected to separation and purification so as to obtain the pyripyropene derivative, and the specific structure of the pyripyropene derivative is represented by any one of formulas (I-III); tests prove that the derivative has a remarkable antagonistic effect on plant pathogenic bacteria xanthomonas campestris, and can be used for preparing a medicine for preventing and treating plant diseases caused by xanthomonas campestris. # imgabs0 #

Description

Technical Field:

[0001] The present invention relates to the field of biological or agricultural technologies, and particularly relates to a preparation method of pyripyropene derivatives and their application in the preparation of drugs for preventing and treating plant diseases. Background Art:

[0002] Pyripyropene is a class of characteristic sesquiterpenoids containing an α-pyrone ring, and its diverse biological activities have attracted extensive attention. It is reported that pyripyropene derivatives have significant selective inhibitory effects on acyltransferase cholesterolacyltransferase 2 (ACAT2), and their inhibitory activities reach the nM level. In addition, pyripyropene derivatives have also been applied to pest control in agriculture or horticulture. Xanthomonas campestris pv. campestris is a class of plant pathogenic bacteria and the main pathogen of black rot in cruciferous crops, which is extremely harmful in agricultural production, especially vegetable production. Therefore, it is very important to develop antibacterial drugs with low toxicity, high efficiency and antagonistic effects against Xanthomonas campestris pv. campestris. Summary of the Invention:

[0003] In order to solve the problems existing in the prior art, the present invention provides a preparation method of pyripyropene derivatives and their application in the preparation of drugs for preventing and treating plant diseases. The present invention plays an important role in the preparation of pyripyropene secondary metabolites in Aspergillus penicillioides and the research of drugs for preventing and treating plant diseases caused by the plant pathogen Xanthomonas campestris pv. campestris.

[0004] The first object of the present invention is to provide the application of pyripyropene derivatives, or their pharmaceutically acceptable salts, in the preparation of drugs for preventing and treating plant diseases, and the structures of the pyripyropene derivatives are as shown in any one of formulas (I-III):

[0005]

[0006] Preferably, the drug for preventing and treating plant diseases is specifically a drug for preventing and treating plant diseases caused by Xanthomonas campestris pv. campestris.

[0007] The second object of the present invention is to provide a drug for preventing and treating plant diseases, which contains pyripyropene derivatives or their pharmaceutically acceptable salts as active ingredients, and the structures of the pyripyropene derivatives are as shown in any one of formulas (I-III):

[0008]

[0009] The third object of the present invention is to provide a method for preparing pyripyropene derivatives, which are isolated and prepared from the fermentation culture of Aspergillus penicillioides, and the structures of the pyripyropene derivatives are shown as any one of formulas (I-III):

[0010]

[0011] Preferably, the method for preparing the pyripyropene derivatives specifically comprises the following steps:

[0012] (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;

[0013] (2) Subject the obtained extract to silica gel column chromatography, and 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, and collect the fractions Fr.2 obtained by elution with petroleum ether: ethyl acetate volume ratios of 2:1 and 1:1 and developed by TLC thin layer chromatography with dichloromethane: methanol = 20:1 v / v to obtain R f = 0.5-0.7; collect the fractions Fr.3 obtained by elution with petroleum ether: ethyl acetate volume ratios of 1:3 to 0:1 and developed by TLC thin layer chromatography with dichloromethane: methanol = 20:1 v / v to obtain R f = 0.4-0.6;

[0014] Subject the fraction Fr.2 to Sephadex LH-20 column chromatography separation, elute with dichloromethane: methanol with a volume ratio of 1:1 as the eluent, and collect the fraction Fr.2-3 obtained by TLC thin layer chromatography with dichloromethane: methanol = 20:1 v / v to obtain R f = 0.4-0.6;

[0015] The fraction Fr.2-3 was separated by ODS reversed-phase column chromatography. Methanol-water with volume ratios of 30:70, 40:60, 50:50, 60:40, 80:20, 100:0 were used as eluents in sequence for gradient elution. The fractions Fr.2-3-22 and Fr.2-3-24 which were obtained by elution with methanol:water volume ratio of 50:50 and 60:40 and had Rf values of 0.5 - 0.6 when developed by TLC (thin-layer chromatography) with dichloromethane:methanol = 20:1 v / v were collected; f The fractions Fr.2-3-22 and Fr.2-3-24 were separately purified by HPLC to obtain Compound 1 shown in Formula (Ⅰ) and Compound 2 shown in Formula (Ⅱ);

[0016] The fraction Fr.3 was separated by Sephadex LH-20 column chromatography and eluted with pure methanol. The fraction Fr.3-3 which had an Rf value of 0.3 - 0.6 when developed by TLC with dichloromethane:methanol = 20:1 v / v was collected; f =0.3 - 0.6 of fraction Fr.3-3;

[0017] The fraction Fr.3-3 was separated by ODS reversed-phase column chromatography. Methanol-water with volume ratios of 25:75, 35:65, 45:55, 55:45, 65:35, 75:25, 100:0 were used as eluents in sequence for gradient elution. The fraction Fr.3-3-14 which was obtained by elution with methanol:water volume ratio of 55:45 and had an Rf value of 0.4 - 0.5 when developed by TLC with dichloromethane:methanol = 20:1 v / v was collected; f The fraction Fr.3-3-14 was purified by HPLC to obtain Compound 3 shown in Formula (Ⅲ).

[0018] Further 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 potato dextrose agar medium, statically culture at 28 °C for 5 days, then take the agar block with mycelium and inoculate it into rice solid medium, statically culture 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 to dissolve, and sterilize; 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.

[0019] More preferably, the separation and purification of fraction Fr.2-3-22 in step (2) by HPLC is specifically as follows: fraction Fr.2-3-22 is separated by semi-preparative HPLC using a YMC-Pack ODS-A column, with the mobile phase being acetonitrile-water with a volume ratio of 55:45, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 12.2 min is collected to obtain compound 1 shown in formula (I).

[0020] More preferably, the separation and purification of fraction Fr.2-3-24 in step (2) by HPLC is specifically as follows: fraction Fr.2-3-24 is separated by semi-preparative HPLC using a YMC-Pack ODS-A column, with the mobile phase being acetonitrile-water with a volume ratio of 55:45, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 11.9 min is collected to obtain compound 2 shown in formula (II).

[0021] More preferably, the separation and purification of fraction Fr.3-3-14 in step (2) by HPLC is specifically as follows: fraction Fr.3-3-24 is separated by semi-preparative HPLC using a YMC-Pack ODS-A column, with the mobile phase being acetonitrile-water with a volume ratio of 45:55, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 29.6 min is collected to obtain compound 3 shown in formula (III).

[0022] The fourth object of the present invention is to provide the use of Aspergillus penicillioides in the preparation of pyripyropene derivatives; the structures of the pyripyropene derivatives are as shown in any one of formulas (I-III):

[0023]

[0024] The present invention has the following advantages compared with the prior art:

[0025] 1. In vitro activity studies have shown that compounds 1-3 proposed by the present invention have significant antagonistic effects, and they have significant antagonistic effects against the plant pathogen Xanthomonas campestris pv. campestris.

[0026] 2. The present invention separates and prepares pyripyropene derivatives from Aspergillus penicillioides. These derivatives have significant antagonistic effects against Xanthomonas campestris pv. campestris and can be used to prepare drugs for preventing and treating plant diseases caused by Xanthomonas campestris pv. campestris, providing candidate compounds for the research and development of new antibacterial drugs.

[0027] The Aspergillus penicillioides of the present invention is deposited in Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, zip code: 510070, and the deposit number is: GDMCC No: 3.587. Description of the drawings:

[0028] Figure 1 is compound 1 1 H NMR spectrum;

[0029] Figure 2 is the HR-ESI-MS spectrum of compound 1;

[0030] Figure 3 It is compound 2 1 H NMR spectrum;

[0031] Figure 4 is the HR-ESI-MS spectrum of compound 2;

[0032] Figure 5 is compound 3 1 H NMR spectrum;

[0033] Figure 6 is the HR-ESI-MS spectrum of compound 3. Specific implementation method:

[0034] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0035] Example 1

[0036] 1. The Aspergillus penicillioides of the present invention is deposited in Guangdong Microbiological Culture Collection Center (GDMCC).

[0037] 2. Solid fermentation of Aspergillus penicillioides

[0038] The mycelium of Aspergillus penicillioides was inoculated into potato dextrose agar medium and cultured at 28°C for 5 days. 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 dextrose 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.

[0039] 3. Preparation of the compound

[0040] (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 was extracted three times with petroleum ether and ethyl acetate solution respectively. The extract was concentrated to obtain about 45 g of the extract.

[0041] (2) The extract was subjected to silica gel column chromatography, using petroleum ether - ethyl acetate 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 volume ratios of 100:1, 50:1, 20:1, 10:1, 0:1 as eluents in turn for gradient elution. The fractions eluted with petroleum ether:ethyl acetate volume ratio of 2:1 and 1:1 and developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v to obtain R f = 0.5 - 0.7 were Fr.2; The fractions eluted with petroleum ether:ethyl acetate volume ratio of 1:3 to 0:1 and developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v to obtain R f = 0.4 - 0.6 were Fr.3.

[0042] (3) Component Fr.2 was separated by Sephadex LH - 20 gel column chromatography, eluted with dichloromethane:methanol (1:1) as the eluent, and the fractions eluted with dichloromethane:methanol = 20:1 v / v by TLC thin layer chromatography to obtain R f = 0.4 - 0.6 were Fr.2 - 3;

[0043] Component Fr.2 - 3 was separated by ODS reverse phase column chromatography, using methanol - water volume ratios of 30:70, 40:60, 50:50, 60:40, 80:20, 100:0 as eluents in turn for gradient elution. The fractions eluted with methanol:water volume ratio of 50:50 and 60:40 and developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v to obtain R fComponents Fr.2-3-22 and Fr.2-3-24 with a ratio of 0.5 - 0.6; Component Fr.2-3-22 was separated and purified by HPLC using a YMC-Pack ODS-A column, with a mobile phase of acetonitrile - water (0.1% TFA) at a volume ratio of 55:45, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 12.2 min was collected to obtain Compound 1; Component Fr.2-3-24 was separated and purified by HPLC using a YMC-Pack ODS-A column, with a mobile phase of acetonitrile - water (0.1% TFA) at a volume ratio of 55:45, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 11.9 min was collected to obtain Compound 2.

[0044] (4) Component Fr.3 was separated by Sephadex LH-20 gel column chromatography and eluted with pure methanol as the eluent, and the fraction Fr.3-3 with an R f value of 0.3 - 0.6 was collected;

[0045] Component Fr.3-3 was separated by ODS reverse-phase column chromatography and gradient eluted with methanol - water at volume ratios of 25:75, 35:65, 45:55, 55:45, 65:35, 75:25, and 100:0 in sequence as the eluents. The fraction Fr.3-3-14 with an R f value of 0.4 - 0.5 was collected. Component Fr.3-3-14 was separated and purified by HPLC using a YMC-Pack ODS-A column, with a mobile phase of acetonitrile - water (0.1% Formic acid) at a volume ratio of 45:55, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 29.6 min was collected to obtain Compound 3.

[0046] 4. Structure Identification of Compounds

[0047] The NMR nuclear magnetic resonance spectra were measured using a Bruker AscendTM 400M nuclear magnetic resonance spectrometer with tetramethylsilane (TMS) as the internal standard; the HR-ESI-MS data were measured using a Thermo Fisher Orbitrap Exploris 120 high-resolution liquid chromatography - mass spectrometry instrument. The structure identification data are as follows:

[0048] As Figure 1-6 shown, Figure 1 this is the 1 1H NMR spectrum of Compound 1; Figure 2 this is the HR-ESI-MS spectrum of Compound 1; Figure 3 this is the1 1H NMR spectrum; Figure 4 is the HR-ESI-MS spectrum of Compound 2; Figure 5 is that of Compound 3 1 1H NMR spectrum; Figure 6 is the HR-ESI-MS spectrum of Compound 3.

[0049] The characterization data of Compounds 1-3 are as follows:

[0050] HR-ESI-MS (m / z) of Compound 1: 452.2418 [M+H] + C 27 H 34 NO 5 , and the calculated value is 452.2431.

[0051] HR-ESI-MS (m / z) of Compound 2: 468.2376 [M+H] + C 27 H 34 NO 6 , and the calculated value is 468.2381.

[0052] HR-ESI-MS (m / z) of Compound 3: 568.2511 [M+H] + C 31 H 38 NO 9 , and the calculated value is 568.2541.

[0053] The structural formulas of Compounds 1-3 and the assignment of their NMR data are as follows:

[0054] Table 1. 1 1H-NMR data (δ in ppm, J in Hz, CDCl 3 )

[0055]

[0056]

[0057] The structural formulas of the target Compounds 1-3 separated by the above method are shown in Formulas (I-III):

[0058]

[0059] Example 2

[0060] Test the antagonistic effects of Compounds 1-3 against the phytopathogenic bacterium Xanthomonas campestris pv. campestris.

[0061] The plant pathogenic bacterium detected in this experiment was Xanthomonas campestris pv. campestris.

[0062] 1. Detection principle of the microbroth dilution method: This method uses a 96-well plate for detection and measures the absorbance value (OD 600 ) at 600 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and estimates the number of viable bacteria based on the OD value.

[0063] 2. Experimental method

[0064] (1) Preparation of the bacterial suspension: Dilute the liquid fermentation broth of the test bacteria in the logarithmic phase 1000-fold with nutrient broth (NB) culture medium to prepare a bacterial suspension with an OD 600 value less than 0.01. Transfer the bacterial suspension to a 96-well plate, with a volume of 200 μL in the first well, 100 μL in each of the 2nd - 11th wells, and the last well as a blank.

[0065] (2) Addition of the test compound solution: Compound 1 was dissolved in DMSO, and 1 - 2 μL of compound 1 was pipetted into the first well to make the final concentration in the first well 200 μg / mL, and mixed well. Pipette 100 μL of the bacterial solution from the first well into the second well, and dilute successively to the 12th well in this way, making the final volume in each well 100 μL. Each treatment was set with 3 replicates, and incubated at 37 °C for 24 h, and then the absorbance value (OD 600 ) at 600 nm was measured after the reaction was complete. The experimental procedures for compounds 2 and 3 were the same as those for compound 1.

[0066] (3) Positive control compound: Streptomycin was used as the positive compound in each experiment. A growth curve was plotted with the concentration 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).

[0067] The experimental results are shown in Table 2:

[0068] Table 2. Test results of the antibacterial effects of compounds 1 - 3 (IC 50 , in μg / mL)

[0069]

[0070] As can be seen from the results shown in Table 2, compounds 1 - 3 of the present invention have an antagonistic effect against the plant pathogen Xanthomonas campestris pv. campestris. Among them, compounds 1 and 2 have a significant antagonistic effect, and the antagonistic effect is better than that of the positive control drug streptomycin.

[0071] The results indicate that compounds 1-3 of the present invention have significant antagonistic effects against the plant pathogenic bacterium Xanthomonas campestris pv. campestris. Therefore, the present invention provides candidate compounds for the research and development of new drugs for preventing and treating plant diseases caused by Xanthomonas campestris pv. campestris.

[0072] The description of the above embodiments is only used to help understand the technical solution and its core idea of the present invention. It should be noted that for those skilled in the art of this technology, without departing from the principle of the present invention, several improvements and modifications can still be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

Use of a pyripyropene derivative or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and treating plant diseases, characterized in that, the pyripyropene derivative has a structure shown in any one of formulas (I-III):

2. The use according to claim 1, characterized in that, the medicament for preventing and treating plant diseases is specifically a medicament for preventing and treating plant diseases caused by Xanthomonas campestris.

3. A medicament for preventing and treating plant diseases, characterized in that, it contains a pyripyropene derivative or a pharmaceutically acceptable salt thereof as an active ingredient, and the pyripyropene derivative has a structure shown in any one of formulas (I-III):

4. Preparation method of a pyripyropene derivative, characterized in that, the pyripyropene derivative is isolated and prepared from the fermentation culture of Aspergillus penicillioides, and the pyripyropene derivative has a structure shown in any one of formulas (I-III):

5. The preparation method according to claim 4, 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, concentrate to obtain a crude extract, dissolve the crude extract in water, and extract with petroleum ether and ethyl acetate solutions respectively, and concentrate the extract to obtain an extract; (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 petroleum ether:ethyl acetate volume ratios of 2:1 and 1:1 and developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v to obtain R f = 0.5 - 0.7 of fraction Fr.2; The fractions eluted with petroleum ether:ethyl acetate volume ratios from 1:3 to 0:1 and developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v to obtain R f = 0.4 - 0.6 of fraction Fr.3; The fraction Fr.2 was separated by Sephadex LH-20 column chromatography, eluted with dichloromethane:methanol at a volume ratio of 1:1 as the eluent, and the fraction Fr.2-3 with an R f value of 0.4 - 0.6 was collected when developed by TLC thin layer chromatography with dichloromethane:methanol = 20:1 v / v; The fraction Fr.2-3 was separated by ODS reverse-phase column chromatography, and methanol-water with volume ratios of 30:70, 40:60, 50:50, 60:40, 80:20, and 100:0 were used as eluents in sequence for gradient elution. The fractions Fr.2-3-22 and Fr.2-3-24 obtained by elution with methanol:water volume ratios of 50:50 and 60:40 and developed by TLC thin-layer chromatography with dichloromethane:methanol = 20:1 v / v to give R f values of 0.5 - 0.6 were collected; the fractions Fr.2-3-22 and Fr.2-3-24 were separately purified by HPLC to obtain compound 1 shown in formula (I) and compound 2 shown in formula (II); The fraction Fr.3 was separated by Sephadex LH-20 column chromatography and eluted with pure methanol as the eluent. The fraction Fr.3-3 with an R f value of 0.3 - 0.6 was collected by TLC thin layer chromatography developed with dichloromethane:methanol = 20:1 v / v; The fraction Fr.3-3 was separated by ODS reversed-phase column chromatography. Methanol-water with volume ratios of 25:75, 35:65, 45:55, 55:45, 65:35, 75:25, and 100:0 were used as eluents in sequence for gradient elution. The fraction Fr.3-3-14 obtained by elution with a methanol:water volume ratio of 55:45 and having an R f value of 0.4 - 0.5 as developed by TLC thin-layer chromatography with dichloromethane:methanol = 20:1 v / v was collected; the fraction Fr.3-3-14 was separated and purified by HPLC to obtain the compound 3 shown in formula (Ⅲ).

6. The preparation method according to claim 5, 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 at 28 °C for 5 days, then take the agar block with mycelium and inoculate it into a rice solid medium, and statically culture 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 to 1000 mL of distilled water, heat and dissolve, and sterilize; 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.

7. The preparation method according to claim 5, characterized in that, the specific method for separating and purifying the component Fr.2-3-22 by HPLC in step (2) is: separate the component Fr.2-3-22 by semi-preparative HPLC, use a YMC-Pack ODS-A column, the mobile phase is acetonitrile-water with a volume ratio of 55:45, the flow rate is 3 mL / min, collect the eluted component with a retention time of 12.2 min, and obtain compound 1 shown in formula (I).

8. The preparation method according to claim 5, characterized in that, The specific operation of separating and purifying fraction Fr.2-3-24 in step (2) by HPLC is as follows: fraction Fr.2-3-24 is separated by semi-preparative HPLC using a YMC-Pack ODS-A column, with the mobile phase being acetonitrile-water with a volume ratio of 55:45, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 11.9 min is collected to obtain compound 2 shown in formula (ⅠⅠ).

9. According to the preparation method described in claim 5, it is characterized in that the specific operation of separating and purifying fraction Fr.3-3-14 in step (2) by HPLC is as follows: fraction Fr.3-3-24 is separated by semi-preparative HPLC using a YMC-Pack ODS-A column, with the mobile phase being acetonitrile-water with a volume ratio of 45:55, a flow rate of 3 mL / min, and the eluted fraction with a retention time of 29.6 min is collected to obtain compound 3 shown in formula (Ⅲ).

10. Use of Aspergillus penicillioides in the preparation of pyripyropene derivatives, wherein the structure of the pyripyropene derivatives is any one of those shown in formula (Ⅰ-Ⅲ):

Images