Crystal form I of Aspric acid and application of crystal form I in prevention and treatment of agricultural pathogenic bacteria

Abstract

The invention relates to an Aspbic acid crystal form I and application thereof in prevention and treatment of agricultural pathogenic bacteria, the Aspbic acid crystal form I uses Cu / K alpha radiation, and an X-ray powder diffraction pattern represented by a 2 theta diffraction angle has characteristic peaks at the positions of 6.411, 10.437, 11.827, 12.830, 13.849, 15.607, 16.261, 18.081, 19.347, 20.223, 20.675, 22.207, 22.390, 24.721 and 27.395. The aspric acid crystal form I disclosed by the invention has better stability and is suitable for long-term storage; in addition, the aspric acid crystal form I disclosed by the invention is higher in purity, so that the aspric acid crystal form I has stronger bacteriostatic activity on agricultural pathogenic bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of pesticides, and particularly relates to an Aspterric acid crystal form I and its application in preventing and controlling agricultural pathogens. Background Art

[0002] Aspterric acid (AA) is a natural sesquiterpene compound mostly isolated from Aspergillus terreus. AA has the property of inhibiting pollen growth and development and is currently widely reported as a natural herbicide.

[0003]

[0004] Due to the low yield of AA from natural sources and the presence of four chiral carbon atoms in its structure, artificial synthesis is difficult, significantly hindering its commercial application. To overcome the shortcomings of the existing technology, the present invention provides a method for optimizing AA fermentation to increase its yield, as well as AA Form I and its application in controlling agricultural pathogens. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention utilizes the marine fungus Penicillium javanicum HK1-22 (abbreviated as HK1-22) to prepare AA on a large scale, and provides an AA crystal form I with good stability, high purity, and excellent anti-agricultural pathogen activity and a preparation method thereof.

[0006] The present invention provides an aspterric acid crystalline form I, characterized in that: using Cu / Kα radiation, an X-ray powder diffraction pattern expressed in 2θ diffraction angles has characteristic peaks at 6.411±0.200, 10.437±0.200, 11.827±0.200, 12.830±0.200, 13.849±0.200, 15.607±0.200, 16.261±0.200, 18.081±0.200, 19.347±0.200, 20.223±0.200, 20.675±0.200, 22.207±0.200, 22.390±0.200, 24.721±0.200, and 27.395±0.200.

[0007] Another embodiment of the present invention provides an aspterric acid crystalline form I, characterized in that: using Cu / Kα radiation, the X-ray powder diffraction pattern expressed in 2θ diffraction angles is 6.411±0.200, 10.437±0.200, 11.827±0.200, 12.830±0.200, 13.849±0.200, 15.607±0.200, 16.261±0.200, 16.548±0.200, 18.081±0.200, 18.530±0.200, 19.347±0.200. There are characteristic peaks at 0.200, 20.001±0.200, 20.223±0.200, 20.675±0.200, 20.980±0.200, 22.207±0.200, 22.390±0.200, 23.801±0.200, 24.721±0.200, 25.864±0.200, 27.395±0.200, 27.765±0.200, 30.869±0.200, and 32.892±0.200. The aspterric acid crystalline form I may also have a substantially Figure 1 The X-ray powder diffraction pattern shown represents the characteristics.

[0008] Another embodiment of the present invention provides an aspterric acid crystalline form I, characterized in that: using Cu / Kα radiation, the X-ray powder diffraction pattern of the crystalline form I is substantially as follows Figure 1 shown.

[0009] Figure 1 The X-ray powder diffraction data of mesomorph I (with a 2θ error range of ±0.200°) are summarized as follows (for details, see Figure 2 ):

[0010]

[0011] It will be understood by those skilled in the art that the relative intensities of characteristic 2θ peaks in an XRPD pattern may vary for a variety of reasons when measured on substantially the same crystalline form.

[0012] The infrared spectrum of the aspterric acid crystal form I of the present invention is at about 3402±4cm -1 There is a characteristic absorption peak at about 2920±4cm -1 There is a characteristic absorption peak at about 1722±4cm -1 There is a characteristic absorption peak at the position of aspterric acid crystal form I. Figure 3 The features represented by the infrared spectrum shown.

[0013] The thermogravimetric analysis (TGA) diagram of the aspterric acid crystal form I of the present invention is substantially as follows Figure 4 shown.

[0014] The X-ray powder diffraction analysis of the aspterric acid crystal form I of the present invention was carried out at ambient temperature and humidity using a Cu / Kα source (40 kV, 40 mA, ) measurements were performed. The infrared spectroscopy analysis of the present invention was performed at ambient temperature and humidity using a Varian 670-IR+610-IR micro-infrared spectrometer using the KBr pellet method. The thermogravimetric analysis (TGA) of the present invention was performed at ambient temperature and humidity using a PerkinElmer TGA8000 thermogravimetric analyzer, using a temperature range of 30.00-800.00°C, a temperature ramp rate of 5.00°C / min, and a protective gas of nitrogen (20.0 ml / min). "Ambient temperature" is generally 0-40°C; "ambient humidity" is generally 30%-80% relative humidity.

[0015] Representative X-ray powder diffraction patterns, infrared spectra, and thermogravimetric analysis patterns of the aspterric acid form I described in the present invention are listed in the accompanying drawings of the specification. "Representative X-ray powder diffraction patterns or infrared spectra" means that the X-ray powder diffraction characteristics or infrared characteristics of the crystal form basically conform to the overall morphology shown in the spectra. It is understood that during the test process, due to the influence of various factors (such as the particle size of the test sample, the processing method of the test sample, the instrument, the test parameters, the test operation, etc.), the peak position or peak intensity of the X-ray powder diffraction pattern or infrared spectrum measured for the same crystal form will have certain differences. The 2θ value of the X-ray powder diffraction pattern may vary slightly between machines or samples, and the experimental error of its 2θ value is generally ±0.2°; the experimental error of the absorption peak in the infrared spectrum is generally ±4cm -1 The experimental error of weight loss temperature in thermogravimetric analysis is generally ±1°C, and the experimental error of weight loss percentage is generally ±2.0%.

[0016] The crystal form of crystallization is relevant with the kinetics and equilibrium condition of each crystal variation under specific conditions.Therefore, what technical staff can understand is that the crystal variation obtained depends on the kinetics and thermodynamics of crystallization process.Under some thermodynamic conditions (concentration of solvent system, temperature, pressure and The compounds of this invention), a kind of crystal variation may be more stable than another crystal variation (or in fact than any other).But the crystal variation with relatively low thermodynamic stability may be kinetics favourable.Therefore, in addition to the above conditions, kinetic factors such as time, impurity distribution, stirring, crystal seed existence or absence etc. also affect the crystal variation of crystallization.

[0017] The present invention provides a method for preparing the above-mentioned aspterric acid crystal form I, characterized in that the method includes the step of recrystallizing the crude aspterric acid product 1-3 times (preferably 2 times). The specific steps of recrystallization include crystallizing the crude aspterric acid product from a suitable solvent, separating, and drying; wherein the mass content of aspterric acid in the crude aspterric acid product is greater than 85%, the crystallization temperature is below 40°C, preferably 0-30°C, and the crystallization can be static or stirred; separation includes filtering or centrifuging to collect the precipitated solid; the drying temperature is selected from 20-50°C, and drying can be performed under normal pressure or reduced pressure; the suitable solvent is selected from one or more mixtures of dichloromethane, ethyl acetate, methanol, ethanol, petroleum ether, n-hexane, n-pentane, acetone, and water, preferably one or more mixtures of dichloromethane, ethyl acetate, petroleum ether, n-hexane, ethanol, methanol, water, and acetone. The HPLC purity of the aspterric acid crystal form I is not less than 99.0%, and more preferably not less than 99.8%.

[0018] The method specifically comprises the following steps:

[0019] (1) Controlling crystallization below 40°C, preferably 0-30°C. The control method includes dissolving the crude aspterric acid in a relatively large amount of solvent (e.g., one or a mixture of dichloromethane, ethyl acetate, methanol, ethanol, petroleum ether, n-hexane, n-pentane, acetone, water) so that crystallization begins at below 40°C; or first dissolving the crude aspterric acid in a small amount of solvent with good solubility (e.g., one or a mixture of dichloromethane, ethyl acetate, methanol, ethanol, acetone), and then adding another solvent with poor solubility (e.g., one or a mixture of petroleum ether, n-hexane, n-pentane, water, etc.) to crystallize when the temperature is controlled below 40°C. The crystallization can be performed in a static or stirring manner.

[0020] (2) Separation, including collecting the precipitated solid by filtration or centrifugation.

[0021] (3) Drying the solid separated in step (2) at a temperature selected from 20 to 50° C., either under normal pressure or under reduced pressure.

[0022] Another embodiment of the present invention provides a method for preparing the above-mentioned aspterric acid crystalline form I, characterized in that it further comprises the following steps of preparing a crude aspterric acid product:

[0023] (1) inoculating the activated marine fungus HK1-22 into a culture medium and culturing the culture medium to obtain a seed solution;

[0024] (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product;

[0025] (3) extracting the fermented product obtained in step (2) with an organic solvent 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a crude product;

[0026] (4) The crude product obtained in step (3) was subjected to vacuum silica gel column chromatography, and petroleum ether-ethyl acetate was used for gradient elution at 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, and two column volumes were collected for each gradient. The fractions obtained by the gradient elution of 70:30, 60:40, and 50:50 were combined, concentrated, and then subjected to reverse phase silica gel column chromatography, and water-methanol was used for gradient elution at 95:5, 90:10, 80:20, 70:30, 60:40, 40:60, 30:70, 20:80, and 0:100, and two column volumes were collected for each gradient. The fractions obtained by the gradient elution of 70:30 and 60:40 were combined, concentrated, and dried to obtain the crude aspterric acid. The HPLC purity of aspterric acid in the crude aspterric acid product is above 85%.

[0027] The bacterial culture conditions and fermentation conditions (such as culture medium selection, temperature, time and other parameters) in the above-mentioned preparation method of the present invention are all routine experimental operations in this field, and those skilled in the art can make reasonable choices based on the experimental situation. In order to facilitate the understanding of the present invention, only one of the conventional operation methods is listed below: the bacterial culture medium in step (1) is a commonly used bacterial culture medium in this field, preferably PDB medium; the bacterial culture in step (1) is preferably shaker culture, the temperature is preferably room temperature to 30°C, the shaker speed is preferably 150-200r / min, and the culture time is preferably 2-4d. The fermentation medium in step (2) is a commonly used fermentation medium in this field, which can be the same as or different from the bacterial culture medium in step (1), preferably PDB medium, GPY medium, fungus No. 1 medium, rice medium, PDA medium; the inoculation amount in step (2) is preferably 1-5%, the fermentation culture temperature is room temperature to 30°C, and the culture time is 28-32d. The organic solvent in step (3) is preferably one of ethyl acetate, methanol, ethanol, dichloromethane or chloroform.

[0028] The culture medium scheme of the present invention is as follows:

[0029] (1) PDB medium (g / L): 200.0 g potato, 20.0 g glucose, 30.0 g sea salt, and dilute to 1 L with tap water.

[0030] (2) GPY medium (g / L): 20.0 g of peptone, 10.0 g of yeast extract, 20.0 g of glucose, 30.0 g of sea salt, and dilute to 1 L with tap water.

[0031] (3) Fungus medium No. 1 (g / L): sorbitol 20.0 g, maltose 20.0 g, monosodium glutamate 10.0 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.3 g, tryptophan 0.5 g, yeast extract 3.0 g, sea salt 30.0 g, and tap water to 1 L.

[0032] (4) Rice culture medium (per liter of conical flask): 40.0 g of rice and 50 mL of seawater.

[0033] (5) PDA culture medium (g / L): 200.0 g potato, 20.0 g glucose, 15.0-20.0 g agar, 30.0 g sea salt, and dilute to 1 L with tap water.

[0034] Another embodiment of the present invention provides a composition characterized in that the composition contains the aspterric acid crystalline form I or a pesticide-acceptable salt thereof as an active ingredient. The composition is used to prevent and control diseases caused by agricultural pathogens.

[0035] The above composition may also include other pesticide active ingredients and pesticide-acceptable adjuvants. Its dosage form may be solid preparation, liquid preparation or semi-solid preparation.

[0036] Another embodiment of the present invention provides use of the above composition in preventing and controlling diseases caused by agricultural pathogens.

[0037] Another embodiment of the present invention provides the use of the aspterric acid crystalline form I or a pesticide-acceptable salt thereof in preventing and controlling diseases caused by agricultural pathogens.

[0038] Another embodiment of the present invention provides use of the aspterric acid crystalline form I or a pesticide-acceptable salt thereof in the preparation of a medicament for preventing and controlling diseases caused by agricultural pathogens.

[0039] The agricultural pathogens of the present invention are selected from agricultural pathogenic fungi and / or agricultural pathogenic bacteria. The agricultural pathogenic fungi are selected from one or more of the following: strawberry gray mold pathogen, grape anthracnose pathogen, peach brown rot pathogen, tobacco brown spot pathogen, rice seedling pathogen, rice sheath blight pathogen, rice blast pathogen, wheat take-all pathogen, wheat head blight pathogen, wheat sheath blight pathogen, and cotton wilt pathogen. The agricultural pathogenic bacteria are selected from one or more of the following: cabbage black rot pathogen, potato black shank pathogen, and rice bacterial leaf blight pathogen.

[0040] The marine fungus Penicillium sp. HK1-22 described in the present invention has the following deposit information: Depository Name: General Microbiology Center, China Culture Collection Administration; Deposit Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit Date: June 11, 2018; Deposit Number: CGMCC No. 15865; Taxonomic Nomenclature: Penicillium sp. This species is described in Chinese patent applications (Application Numbers: 201910448403.5 and 201910448312.1).

[0041] Compared with the prior art, the advantage of the present invention is that compared with commercially available aspterric acid (solid) or aspterric acid crystals previously prepared by the inventors (literature product, Bioorganic Chemistry 93 (2019) 103331), the aspterric acid crystalline form I of the present invention has better stability and is suitable for long-term storage; in addition, the aspterric acid crystalline form I of the present invention has higher purity (HPLC purity greater than 99%); the characteristics of good stability and high purity make it more antibacterial activity against agricultural pathogens (fungi and bacteria).

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The X-ray powder diffraction pattern of the aspterric acid crystal form I of the present invention is shown in FIG.

[0044] Figure 2 This is a diffraction peak report diagram of the aspterric acid crystal form I of the present invention by X-ray powder diffraction.

[0045] Figure 3 This is the infrared spectrum of aspterric acid crystal form I of the present invention.

[0046] Figure 4 This is a thermogravimetric analysis diagram of aspterric acid Form I of the present invention.

[0047] Figure 5 TLC traces of crude products from different culture systems, with vanillin as the colorant (left) and 254 nm (right). Note: The developing solvent ratio used was CH₂Cl₂:CH₃OH = 10:1. The culture medium order was: 1. PDB static, 2. Fungus No. 1 shaker, 3. GPY shaker, 4. Rice methanol extraction, 5. GPY static, 6. Rice ethyl acetate extraction, 7. PDA, 8. PDB shaker, 9. Fungus No. 1 static. 65 represents pure AA; unless otherwise noted, crude products were extracted with ethyl acetate.

[0048] Figure 6 It's aspterric acid 1 H NMR spectrum.

[0049] Figure 7 It's aspterric acid 13 C NMR spectrum. DETAILED DESCRIPTION

[0050] In order to facilitate further understanding of the present invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the invention and are not intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following.

[0051] Example 1 Optimal Fermentation Medium Optimization Screening

[0052] The culture medium scheme is as follows:

[0053] ①PDB medium (g / L): 200.0 g potato, 20.0 g glucose, 30.0 g sea salt, and dilute to 1 L with tap water.

[0054] ②GPY medium (g / L): 20.0 g of peptone, 10.0 g of yeast extract, 20.0 g of glucose, 30.0 g of sea salt, and dilute to 1 L with tap water.

[0055] ③Fungus medium No. 1 (g / L): sorbitol 20.0 g, maltose 20.0 g, MSG 10.0 g, dipotassium hydrogen phosphate 0.5 g, magnesium sulfate heptahydrate 0.3 g, tryptophan 0.5 g, yeast extract 3.0 g, sea salt 30.0 g, and dilute to 1 L with tap water.

[0056] ④ Rice culture medium (per liter of conical flask): 40.0 g of rice, 50 mL of seawater.

[0057] ⑤PDA culture medium (g / L): 200.0 g potato, 20.0 g glucose, 15.0-20.0 g agar, 30.0 g sea salt, and dilute to 1 L with tap water.

[0058] (1) The activated marine fungus HK1-22 was inoculated into PDB and cultured in a constant temperature shaker at 28°C and 160 rpm for 3 days to obtain seed solution;

[0059] (2) inoculating the seed solution obtained in step (1) into PDB medium, GPY medium, and fungus medium No. 1, respectively, and performing shaking fermentation culture (28°C, 160r / min culture for 28 days) and static fermentation culture (28°C culture for 28 days); and performing static culture (28°C culture for 28 days) on rice medium and PDA medium to obtain fermentation products;

[0060] (3) extracting the fermentation product obtained in step (2) with an organic solvent (ethyl acetate, methanol, ethanol, dichloromethane or chloroform) 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a crude product;

[0061] (4) Multi-culture medium screening: The crude products obtained from different culture media were compared with AA standards by TLC analysis to select the optimal culture medium (see Figure 5 ).

[0062] Figure 5Thin-layer chromatography (TLC) results showed that low-concentration pure AA formed characteristic pink spots upon vanillin staining, but showed no significant absorption at 254 nm. Comparative analysis of cultures 1-9 with AA revealed that the total AA yield in culture systems 1 (system 1) using static PDB medium was relatively high, with prominent pink spots. AA spots also appeared in system 6 (rice medium), but the yield was lower than that of system 1. Pink spots or bands also appeared in systems 2, 3, and 5, but their polarity was lower than that of AA, significantly different from AA. These are presumably derivatives formed by dehydration of the hydroxyl group of AA. No other systems showed obvious AA-related spots. Therefore, static fermentation using PDB medium is beneficial for obtaining higher yields of AA products. Therefore, the optimal fermentation conditions were determined to be: PDB medium, static culture, incubation temperature: 28°C, and incubation time: 28 days.

[0063] Example 2

[0064] (1) The activated marine fungus HK1-22 was inoculated into PDB and cultured in a constant temperature shaker at 28°C and 160 rpm for 3 days to obtain seed solution;

[0065] (2) The seed solution obtained in step (1) was inoculated into PDB medium (10 mL of seed solution per bottle, a total of 100 bottles, and a fermentation volume of 30 L), and the culture was statically cultured at 28° C. for 28 days to obtain a fermentation product;

[0066] (3) extracting the fermented product obtained in step (2) with ethyl acetate three times, combining the extracts and concentrating under reduced pressure to obtain a crude product;

[0067] (4) The crude product obtained in step (3) was subjected to vacuum silica gel column chromatography, and petroleum ether-ethyl acetate was used as the gradient elution at 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, and two column volumes were collected for each gradient. The fractions obtained by the 70:30, 60:40, and 50:50 gradient elutions were combined, concentrated, and then subjected to reverse phase silica gel column chromatography, and water-methanol was used as the gradient elution at 95:5, 90:10, 80:20, 70:30, 60:40, 40:60, 30:70, 20:80, and 0:100, and two column volumes were collected for each gradient. The fractions obtained by the 70:30 and 60:40 gradient elutions were combined, concentrated, and dried to obtain the crude aspterric acid (HPLC purity 85.6%).

[0068] (5) Primary recrystallization: At room temperature, the crude aspterric acid was dissolved in a suitable amount of a mixed solvent of petroleum ether and ethyl acetate (volume ratio 3:1), and the mixture was allowed to stand for 24-48 hours. The solvent evaporated naturally, and after crystals precipitated, the mixture was filtered and the solid was collected. The solid was rinsed with a mixed solvent of petroleum ether and ethyl acetate (volume ratio 3:1), and dried to obtain aspterric acid crystals (HPLC purity 98.0%). Secondary recrystallization: At room temperature, the obtained aspterric acid crystals were dissolved again in a mixed solvent of petroleum ether and ethyl acetate (volume ratio 3:1), and the mixture was allowed to stand for 24-48 hours. The solvent evaporated naturally, and after crystals precipitated, the mixture was filtered and the solid was collected. The solid was rinsed with a mixed solvent of petroleum ether and ethyl acetate (volume ratio 3:1), and dried to obtain the aspterric acid crystal form I (HPLC purity 99.80%, melting point 168±1°C), XRPD pattern, IR pattern, TGA pattern (as shown in FIG. Figure 1-4 shown).

[0069] Example 3

[0070] Step (5) in Example 2 can be replaced by the following operation: controlling the temperature below 40°C, dissolving the crude aspterric acid in an appropriate amount of ethyl acetate, adding petroleum ether dropwise with stirring until turbidity appears, maintaining the temperature below 40°C overnight, filtering, collecting the solid, and repeating the above operation once to obtain the aspterric acid crystalline form I (HPLC purity 99.83%, XRPD, IR, and TGA data consistent with Example 2).

[0071] Ethyl acetate can be replaced by methanol, ethanol, acetone, etc., and petroleum ether can be replaced by n-hexane, n-pentane, water, etc.

[0072] Example 4

[0073] The commercially available aspterric acid (HPLC purity of commercial product 1 is 96.0%, and the HPLC purity of commercial product 2 is 95.8%) and the aspterric acid crystals prepared by the inventors previously (HPLC purity is 97.3%, referred to as the literature product, Bioorganic Chemistry 93(2019)103331), the results showed that commercially available products 1 and 2 had no obvious 2θ diffraction peaks and were both amorphous; while the XRPD pattern of the product in the literature was different from the crystalline form I described in the present invention. The product in the literature used Cu / Kα radiation, and the X-ray powder diffraction pattern expressed in 2θ diffraction angles had no characteristic peaks at 10.437±0.200, 11.827±0.200, 12.830±0.200, 16.261±0.200, 22.390±0.200, 23.801±0.200, 24.721±0.200, etc.

[0074] Example 5 Storage stability test

[0075] Aspterric acid Form I of the present invention, aspterric acid from the prior art (commercially available product 1), and a reference product were stored at 40°C and 75% relative humidity (results are shown in Table 1), and their storage stability after 30 days was tested. Regarding storage stability, the purity of each test compound was measured initially and after 30 days of storage (day 31), and the results were compared (for specific methods, see the method described in WO2009128421A1).

[0076] Storage stability test at 40°C and 75% relative humidity

[0077]

[0078] It can be seen from the test results in the above table that the aspterric acid crystalline form I of the present invention has extremely excellent storage stability and is convenient for long-term storage.

[0079] Example 6 Anti-plant pathogenic fungi activity test

[0080] Two aspterric acid samples (aspterric acid crystal form I of Example 1 of the present invention and literature product) were evaluated for their anti-plant pathogenic fungal activity using the poisonous culture medium method. The 11 agricultural pathogen test strains included strawberry gray mold pathogen, grape anthracnose pathogen, peach brown rot pathogen, tobacco brown star pathogen, rice seedling pathogen, rice sheath blight pathogen, rice blast pathogen, wheat take-all pathogen, wheat head blight pathogen, wheat sheath blight pathogen, and cotton wilt pathogen; Sample pretreatment: The two aspterric acid samples were dissolved separately with DMSO to 1 mg / mL. Appropriate amounts of sample solutions were added to the PDA culture medium that had been sterilized at high temperature and shaken well, so that the concentration of the culture medium in each conical flask was 50 μg / mL and 10 μg / mL, respectively. The culture medium containing the samples was poured into a plate for standby use. DMSO was added to a blank PDA culture medium and poured into a plate as a control. Activity Test: 10mm diameter colonies of 10 agricultural pathogens were plated onto the center of a pre-prepared solid plate containing two aspterric acid samples. Three replicates were performed for each concentration of each fungus. A blank PDA plate containing DMSO was used as a control. The plates were incubated at 37°C. When the control group nearly filled the plate, the diameter of the colonies was measured. This was used to calculate the inhibitory rate of the sample against the mycelial growth of the pathogen at a specific concentration.

[0081] Mycelial growth inhibition rate (%) = [(control colony growth diameter - treated colony growth diameter) / control colony growth diameter] × 100%

[0082] The test results are shown in the table below. The results show that the aspterric acid crystal form I of the present invention has an inhibitory effect on 11 agricultural pathogens at concentrations of 10 and 50 μg / mL (both greater than 28%). In particular, when the concentration is 50 μg / mL, the inhibition rate against all 11 agricultural pathogens is greater than 46%, showing plant protection function. In addition, the inhibitory activity of the aspterric acid crystal form I of the present invention against 10 agricultural pathogens is stronger than that of the product in the literature (Bioorganic Chemistry 93 (2019) 103331). This may be due to the high purity and good stability of the aspterric acid crystal form I of the present invention, which enables it to exert better biological activity.

[0083]

[0084] Example 7 Test of activity against plant pathogenic bacteria

[0085] The aspterric acid crystal form I of the present invention was tested for its activity against plant pathogens using the filter paper method. The test strains included: cabbage black rot pathogen, potato black shank pathogen, and rice bacterial blight pathogen. Filter paper activity test: 200 μL of bacterial solution was pipetted onto an LB plate and evenly spread across the entire surface of the plate using a sterile cotton swab. The sample to be tested is dissolved into a methanol solution with a concentration of 50μg / μL. Use a pipette to accurately draw 0.5 and 1μL (i.e., the test concentrations are 25 and 50μg per piece, respectively) of the solution and drop them onto filter paper. After the filter paper is completely dry, stick it on a plate coated with indicator bacteria. Penicillin is used as a positive control drug. The amount of drug on each filter paper is 5μg. Three parallels are made for each group. The samples are placed in a 28℃ incubator for culture, usually for 18-24 hours. Pay attention to the formation of inhibition zones. The diameter of the inhibition zones also needs to be measured as an experimental record (inhibition zone diameter = blank circle diameter - filter paper diameter, unit: mm). The test results are shown in the table below.

[0086]

[0087] All documents mentioned in this application are incorporated herein by reference, just as if each document were individually incorporated by reference. The Chinese and English abbreviations, code names, etc. used in this application can be found in the references or technical manuals, textbooks, and reference books of the prior art. Furthermore, it should be understood that after reading the foregoing disclosure, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents are also within the scope of the claims appended hereto.

Claims

1. An aspterric acid crystalline form I, characterized in that: Using Cu / Kα radiation, the X-ray powder diffraction pattern expressed in 2θ diffraction angles has characteristic peaks at 6.411±0.200, 10.437±0.200, 11.827±0.200, 12.830±0.200, 13.849±0.200, 15.607±0.200, 16.261±0.200, 18.081±0.200, 19.347±0.200, 20.223±0.200, 20.675±0.200, 22.207±0.200, 22.390±0.200, 24.721±0.200, and 27.395±0.

200.

2. The aspterric acid crystalline form I according to claim 1, characterized in that: The X-ray powder diffraction pattern expressed in 2θ diffraction angles using Cu / Kα radiation was 6.411±0.200, 10.437±0.200, 11.827±0.200, 12.830±0.200, 13.849±0.200, 15.607±0.200, 16.261±0.200, 16.548±0.200, 18.081±0.200, 18.530±0.200, 19.347±0.200, 2 There are characteristic peaks at 0.001±0.200, 20.223±0.200, 20.675±0.200, 20.980±0.200, 22.207±0.200, 22.390±0.200, 23.801±0.200, 24.721±0.200, 25.864±0.200, 27.395±0.200, 27.765±0.200, 30.869±0.200, and 32.892±0.

200.

3. The aspterric acid crystalline form I according to any one of claims 1 to 2, characterized in that The infrared spectrum of the crystal form I is at about 3402 and 1722 cm -1 There is a characteristic absorption peak at the position with an error range of ±4cm -1 .

4. The method for preparing the aspterric acid crystal form I according to any one of claims 1 to 3, characterized in that The method comprises the steps of recrystallizing a crude aspterric acid product 1-3 times (preferably 2 times); the mass content of aspterric acid in the crude aspterric acid product is above 85%.

5. The preparation method according to claim 4, characterized in that The specific steps of the recrystallization include crystallizing the crude aspterric acid from a suitable solvent, separating, and drying; the crystallization temperature is below 40°C, preferably 0-30°C, and the crystallization can be static or stirred; separation includes filtering or centrifuging to collect the precipitated solid; the drying temperature is selected from 20-50°C, and drying can be done at normal pressure or under reduced pressure; the suitable solvent is selected from one or a mixture of dichloromethane, ethyl acetate, methanol, ethanol, petroleum ether, n-hexane, n-pentane, acetone, and water, preferably one or a mixture of dichloromethane, ethyl acetate, petroleum ether, n-hexane, ethanol, methanol, water, and acetone. The HPLC purity of the aspterric acid crystal form I is not less than 99.0%.

6. The preparation method according to any one of claims 4 to 5, characterized in that Also included is the step of preparing crude aspterric acid: (1) inoculating the activated marine fungus HK1-22 into a culture medium and culturing the culture medium to obtain a seed solution; (2) inoculating the seed solution obtained in step (1) into a fermentation medium for fermentation to obtain a fermentation product; (3) extracting the fermented product obtained in step (2) with an organic solvent 2 to 4 times, combining the extracts and concentrating under reduced pressure to obtain a crude product; (4) The crude product obtained in step (3) was subjected to vacuum silica gel column chromatography, and petroleum ether-ethyl acetate was used for gradient elution at 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, and two column volumes were collected for each gradient. The fractions obtained by the gradient elution of 70:30, 60:40, and 50:50 were combined, concentrated, and then subjected to reverse phase silica gel column chromatography, and water-methanol was used for gradient elution at 95:5, 90:10, 80:20, 70:30, 60:40, 40:60, 30:70, 20:80, and 0:100, and two column volumes were collected for each gradient. The fractions obtained by the gradient elution of 70:30 and 60:40 were combined, concentrated, and dried to obtain the crude aspterric acid.

7. A composition characterized in that The composition comprises the aspterric acid crystalline form I or a pesticide-acceptable salt thereof according to any one of claims 1 to 3 as an active ingredient and is used for preventing and controlling diseases caused by agricultural pathogens.

8. The composition of claim 7, characterized in that The composition may also include other pesticide active ingredients and pesticide-acceptable adjuvants. The dosage form can be a solid preparation, a liquid preparation or a semisolid preparation.

9. Use of the aspterric acid crystalline form I or a pesticide-acceptable salt thereof according to any one of claims 1 to 3, or the composition according to any one of claims 7 to 8, in preventing and controlling diseases caused by agricultural pathogens, or in the preparation of a medicament for preventing and controlling diseases caused by agricultural pathogens.

10. The use according to claim 9, characterized in that The agricultural pathogens are selected from agricultural pathogenic fungi and / or agricultural pathogenic bacteria.

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