Kasugamide composition and application thereof

Abstract

The invention provides a kasugamide composition and application thereof, and according to the total mass of the kasugamide composition, the content of kasugamide and / or salt of the kasugamide is not less than 0.01%. The kasugamide composition shows a good bacteriostatic effect and has an application prospect as a pesticide bactericide.

Description

Technical Field

[0001] The invention belongs to the technical field of agriculture, and in particular relates to a kasugalacetamide composition and application thereof. Background Art

[0002] At present, there is no relevant report on the preparation process and detection method of casulacetamide (CAS 6189-95-3 or 21256-64-4 or 38420-31-4, the structure of which is shown in Formula 1), and its efficacy and application have not been studied and developed.

[0003] Summary of the invention

[0004] In view of the problems existing in the prior art, the present application provides a preparation method and a detection method of kasugalacetamide, and finds that a composition containing kasugalacetamide and / or a salt of kasugalacetamide has a good antibacterial effect and has an application prospect as a pesticide fungicide.

[0005] In one aspect of the present invention, a cinnamonamide composition is provided, wherein the cinnamonamide composition contains cinnamonamide and / or a salt of cinnamonamide, and the content of cinnamonamide and / or a salt of cinnamonamide is not less than 0.01% based on the total mass of the composition, wherein the mass of the salt of cinnamonamide is based on the mass of the cinnamonamide contained therein.

[0006] Optionally, based on the total mass of the composition, the content of cinnamonamide and / or its salt is not less than 0.03%, or not less than 0.05%, or not less than 0.1%, or not less than 0.5%, or not less than 1%, wherein the mass of the salt of cinnamonamide is based on the mass of cinnamonamide it contains.

[0007] Considering factors such as cost, the content of cinnamonamide and / or its salt is not higher than 10%, preferably not higher than 8% or not higher than 7%, based on the total mass of the composition.

[0008] Optionally, the composition further comprises kasugamycin and / or a salt of kasugamycin, wherein the mass ratio of kasugamycin and / or a salt of kasugamycin to kasugaethanamide and / or a salt of kasugaethanamide is 10:1-200:1, wherein the mass of the salt of kasugaethanamide is calculated based on the mass of the kasugaethanamide it contains, and the mass of the salt of kasugamycin is calculated based on the mass of the kasugamycin it contains.

[0009] Optionally, the mass ratio of kasugamycin and / or a salt of kasugamycin to kasugae acetamide and / or a salt of kasugae acetamide is 13:1-130:1, or optionally, the mass ratio of kasugamycin and / or a salt of kasugamycin to kasugae acetamide and / or a salt of kasugae acetamide is 11:1, 13:1, 14:1, 15:1, 16:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 115:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 171:1, 180:1, 181:1, 182:1, 183:1, 184:1 1:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1, 110:1, 120:1, 140:1, 150:1, 160:1, 170:1, 180:1 or 190:1, or a range value between any two values, wherein the mass of the salt of kasugamycin is measured by the mass of the kasugamycin it contains, and the mass of the salt of kasugamycin is measured by the mass of the kasugamycin it contains.

[0010] Optionally, the composition is any one of a raw drug, a mother drug or a preparation.

[0011] Optionally, the dosage form of the preparation is selected from any one of powder, granules, large granules, fine granules, microgranules, microcapsule granules, wettable powder, oil-dispersible powder, water-dispersible granules, emulsions, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release particles, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, film-spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latex, dispersible liquids, pastes, concentrated gels, emulsions in water, oil emulsions, microemulsions, ointments, suspensions, microcapsule suspensions, oil suspensions, suspoemulsions, dispersible powders for seed treatment, soluble powders for seed treatment, liquid solutions for seed treatment, emulsions for seed treatment, suspensions for seed treatment, suspended seed coatings, and microcapsule suspensions for seed treatment.

[0012] Optionally, based on the total mass of the preparation, the total content of kasugamycin and / or kasugamycin salts and kasugamycin acetamide and / or kasugamycin salts in the preparation is not less than 0.01%, or not less than 0.03%, or not less than 0.1%, or not less than 1%, wherein the mass of kasugamycin salt is calculated based on the mass of kasugamycin acetamide it contains, and the mass of kasugamycin salt is calculated based on the mass of kasugamycin it contains.

[0013] Another aspect of the present invention is to provide use of the above composition in controlling plant diseases.

[0014] Optionally, the plant disease is corn Curvularia leaf spot and / or rice blast.

[0015] Another aspect of the present invention is to provide use of the above composition in preparing a plant disease control formulation.

[0016] Optionally, the plant disease is corn Curvularia leaf spot and / or rice blast.

[0017] Another aspect of the present invention is to provide a method for preparing high-purity kasugalacetamide, comprising the following steps: a fermentation broth purification step and an octadecylsilane bonded silica gel column separation step.

[0018] Optionally, the fermentation broth purification step comprises the following steps:

[0019] 1) Acidifying and filtering the fermentation broth;

[0020] 2) The filtrate is adsorbed by resin and then analyzed;

[0021] 3) The analytical solution is filtered to remove impurities.

[0022] Optionally, a decolorization step is also included after the nanofiltration impurity removal.

[0023] Furthermore, the acidulant in the fermentation broth purification step 1 includes at least one of sulfuric acid, hydrochloric acid, phosphoric acid or oxalic acid.

[0024] Furthermore, the resin in the fermentation broth purification step 2 is a strongly acidic cation exchange resin.

[0025] Furthermore, in the fermentation broth purification step 2, the resin adsorption analytical agent includes at least one of ammonium chloride solution, sodium hydroxide solution or sodium chloride solution.

[0026] Optionally, in the silica gel column separation step, the eluting phase is a mixed solution of acetonitrile and sodium alkyl sulfonate;

[0027] Furthermore, the sodium alkyl sulfonate is optionally sodium hexane sulfonate or sodium dodecyl sulfonate, and acetonitrile:sodium alkyl sulfonate solution = 1%:99%-10%:90%.

[0028] The applicant unexpectedly discovered that when the ultraviolet detection wavelength of the liquid chromatography is 195nm, caesalpinia serrata has a very sensitive response. Therefore, another aspect of the present invention is to provide a method for detecting caesalpinia serrata, comprising the following steps:

[0029] The sample to be tested is dissolved in deionized water to prepare a solution with a concentration of 0.001-4 mg / mL. An appropriate amount of the solution to be tested is taken and injected into a liquid chromatograph. The peak position is used to determine whether kasugamycin acetamide is contained or the peak position is compared with that of the kasugamycin acetamide standard to determine whether kasugamycin acetamide is contained. The detection wavelength is 195 nm.

[0030] Optionally, the HPLC column is a reverse phase C18 filler;

[0031] Optionally, the mobile phase is acetonitrile and an aqueous solution of sodium alkyl sulfonate;

[0032] Optionally, running time: 10-20min;

[0033] Optionally, column temperature: 30 ± 2 °C;

[0034] Optionally, flow rate: 0.5-2 mL / min;

[0035] Optionally, signal bandwidth: 4nm-8nm;

[0036] Optionally, injection volume: 5 μL.

[0037] Furthermore, the sodium alkyl sulfonate is optionally sodium hexane sulfonate or sodium dodecyl sulfonate, and acetonitrile:sodium alkyl sulfonate solution = 1%:99%-10%:90%.

[0038] Those skilled in the art can determine whether caesium is contained by comparing the liquid phase data (spectrum) of the sample to be tested with the liquid phase data (spectrum) of the standard under the same liquid phase analysis conditions, in the presence of a standard, and the content (concentration) of caesium in the sample to be tested can be determined by the content (concentration) of the standard.

[0039] The positive improvement effect of the present invention is:

[0040] The present invention provides a composition of kasugalacetamide and / or a salt of kasugalacetamide, which has a significant inhibitory effect on plant pathogens; further, kasugalacetamide and / or a salt of kasugalacetamide and kasugamycin and / or a salt of kasugamycin are controlled in a certain ratio to show a beneficial synergistic effect, and can be better applied to the prevention and treatment of plant diseases.

[0041] The invention also provides a method for preparing high-purity kasugalacetamide. The obtained high-purity kasugalacetamide can be used as a standard product or applied in the detection of active ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a purity characterization diagram of caesalpinia acetamide prepared in Example 1;

[0043] Figure 2 The HPLC-DAD and total ion current (TIC) of the kauriacetamide prepared in Example 1;

[0044] Figure 3 The HPLC-MSD of caesalpinamide prepared in Example 1;

[0045] Figure 4The hydrogen nuclear magnetic spectrum (1H-NMR) of caesalpinia acetamide prepared in Example 1;

[0046] Figure 5 This is the infrared spectrum (FTIR) of kasugalacetamide prepared in Example 1;

[0047] Figure 6 It is a liquid chromatography spectrum (HPLC) of the kasugamycin aqueous composition;

[0048] Figure 7 The antibacterial effect of different concentrations of kasugae acetamide on rice blast fungus, where ck is the blank control group, a is the antibacterial effect of 0.001wt% concentration, b is the antibacterial effect of 0.01wt% concentration, c is the antibacterial effect of 0.03wt% concentration, d is the antibacterial effect of 0.05wt% concentration, and e is the antibacterial effect of 0.1wt% concentration.

[0049] For the purpose of the present invention, unless otherwise specified, the terms used in this application have the following meanings:

[0050] The term "technical product" refers to a product obtained in the production process consisting of active ingredients and related impurities, to which a small amount of additives may be added if necessary. For example, kasugamycin technical product refers to a product obtained in the production process consisting of kasugamycin and / or kasugamycin salts and related impurities, to which a small amount of additives may be added if necessary.

[0051] The term "mother drug" refers to the product obtained in the production process consisting of active ingredients and related impurities, which may contain a small amount of necessary additives and appropriate diluents. For example, kasugamycin mother drug refers to the product obtained in the production process consisting of kasugamycin and / or kasugamycin salts and related impurities, which may contain a small amount of necessary additives and appropriate diluents.

[0052] The term "formulation" refers to a stable product made from pesticide technical or concentrate and suitable adjuvants, or made by biological fermentation, plant extraction and other methods.

[0053] The term "adjuvant" refers to any single component or multiple components added to pesticide products, in addition to the active ingredient, which does not have pesticide activity and active ingredient function itself, but can or helps to enhance or improve the physical and chemical properties of the pesticide product.

[0054] The terms “around”, “about” and “substantially” refer to numerical variations within the normal experimental or measurement error range, such as “substantially” represents an error of no more than 15%, preferably no more than 10%.

[0055] The term "plant disease" refers to the phenomenon that during the growth and development of plants, due to infection by other organisms and the influence of adverse abiotic factors, the growth and development of plants is significantly hindered, and pathological changes occur in the internal and external physiology and tissue structure of the plants, resulting in morbidity or even death, resulting in reduced yield and deterioration of quality. The "plant disease" of this application specifically refers to the disease caused by the infection of plants by other organisms, including but not limited to fungi (such as corn curvature spore fungus, rice blast fungus), or bacteria (such as bacterial angular leaf spot of cucurbits).

[0056] The term "salt of kasugamycin" refers to a compound formed by combining kasugamycin with an acid ion, such as kasugamycin hydrochloride, kasugamycin sulfate, kasugamycin carboxylate, kasugamycin phosphate, kasugamycin nitrate or kasugamycin carbonate.

[0057] The term "salt of cinnamate" refers to a compound formed by the combination of cinnamate and an acid ion, such as cinnamate hydrochloride, cinnamate sulfate and the like.

[0058] In the present application, the mass of the salt of kasugamycin is measured by the mass of the kasugamycin it contains, and the mass of the salt of kasugamycin acetamide is measured by the mass of the kasugamycin it contains. For example, the composition contains 41.6g of kasugamycin hydrochloride and 1g of kasugamycin acetamide, then the mass ratio of kasugamycin hydrochloride to kasugamycin acetamide is 37.9:1 (calculation method: kasugamycin hydrochloride molecular weight is 416, kasugamycin molecular weight is 379, therefore, 41.6g of kasugamycin hydrochloride contains kasugamycin 37.9, based on the mass of kasugamycin, the mass ratio of kasugamycin hydrochloride to kasugamycin acetamide is 37.9:1). DETAILED DESCRIPTION

[0059] The reagents and solvents used in the following examples can be purchased from commercial sources or prepared by conventional methods known to those skilled in the art. All commercially available reagents and solvents were not further purified.

[0060] 1. Preparation Example

[0061] Example 1: Preparation of caesalpinia oxycarbazone

[0062] 1) High-yield Streptomyces microaureaus strain MKL-2 was selected as the fermentation bacteria, and the seed liquid after being cultured in the seed culture medium was inoculated into a 50L fermentation tank for fermentation, wherein the culture medium in the fermentation tank was composed of: 4.0% soybean cake powder, 0.07% potassium dihydrogen phosphate, 0.3% sodium chloride, 0.72% soybean oil, 1.1% corn steep liquor powder, 0.01% polyoxypropylene polyoxyethylene glycerol ether GPE, 0.4% ammonia water, and 0.1% sodium citrate; the fermentation temperature was 30° C., the rotation speed was 60 rpm, ammonia water was dripped into the fermentation process, and the pH of the fermentation system was controlled to be 7.1±0.1 to facilitate the production of caesalpinamide, and the culture was carried out for 120 hours;

[0063] 2) taking 20 L of caesalpinia oxyphylla fermentation broth, acidifying the fermentation broth with oxalic acid, and filtering the fermentation broth with a ceramic membrane;

[0064] 3) collecting the filtrate, adsorbing it with a strong acidic cation exchange resin, and then analyzing it with an ammonium chloride solution;

[0065] 4) filtering the analyzed solution through a nanofiltration membrane and concentrating it under vacuum, and collecting the concentrated filtrate;

[0066] 5) Decolorizing the concentrated filtrate using activated carbon;

[0067] 6) adding the decolorized filtrate to an octadecylsilane bonded silica gel column and performing separation and purification using a mixture of 10% acetonitrile and 90% sodium hexanesulfonate aqueous solution as an eluent;

[0068] 7) After the separated and purified liquid is dried, about 6 g of caesalpinia lanuginosa powder is obtained.

[0069] The caesalpinia oxycarbazide powder can be directly used as the original pesticide or the mother pesticide of the pesticide.

[0070] Example 2: Preparation of caesalpinia oxyphylla aqueous solution

[0071] 100 mg of the kasugalacetamide original drug prepared in Example 1 was added to 10 mL of deionized water to prepare a 1% kasugalacetamide aqueous solution, and an appropriate amount of the 1% aqueous solution was further diluted and prepared into 4 different kasugalacetamide aqueous solutions with mass fractions of 0.01%, 0.1%, 0.3% and 0.5%.

[0072] Example 3: Preparation of caesalpinia acetamide wettable powder

[0073] The following components are available:

[0074] 1 g of the kasugalacetamide technical drug prepared in Example 1;

[0075] Sodium lauryl sulfate (wetting agent) 4g;

[0076] Sodium lignin sulfonate (dispersant) 5g;

[0077] White carbon black (filler) 10g;

[0078] Diatomaceous earth (filler) 80g;

[0079] The above components are fully mixed according to the formula weight, and crushed by an ultrafine grinder to obtain 1% caesalpinia acetamide wettable powder.

[0080] Example 4: Preparation of Kasugamycin Aqueous Solution

[0081] 85 mg of kasugamycin hydrochloride technical drug (provided by Xi'an Masidi Bioengineering Co., Ltd.) and 1.5 mg of kasugamycin acetamide technical drug prepared in Example 1 were dissolved in 50 ml of deionized water to prepare kasugamycin acetamide·kasugamycin aqueous solution.

[0082] Example 5: Preparation of kasugamycin wettable powder

[0083] The following components are available:

[0084] 1 g of the kasugalacetamide technical drug prepared in Example 1;

[0085] Kasugamycin hydrochloride technical 13 g (provided by Xi'an Masidi Bioengineering Co., Ltd.);

[0086] Sodium lauryl sulfate (wetting agent) 3g;

[0087] Sodium lignin sulfonate (dispersant) 5g;

[0088] White carbon black (filler) 10g;

[0089] Diatomaceous earth (filler) 68g;

[0090] The above components are fully mixed according to the weight of the formula, and crushed by an ultrafine grinder to obtain kasugamycin wettable powder.

[0091] 2. Identification and Characterization

[0092] 1. Purity determination

[0093] 1.1 Preparation of the test solution: Accurately weigh 38 mg of the caesium acetamide prepared in Example 1, dissolve it in water and dilute it to 25 mL to prepare a caesium acetamide standard stock solution; accurately transfer 1.25 mL of the above standard stock solution to a 25 mL volumetric flask, dilute it to the scale with water and mix it evenly to complete the preparation of the test solution.

[0094] 1.2 The analysis conditions are as follows:

[0095] Instrument: Agilent HPLC 1100;

[0096] HPLC column: Agilent aichrombond-AQ C18 4.6mm*250mm*5μm;

[0097] Mobile phase: acetonitrile: 20 MM sodium hexanesulfonate aqueous solution (H 3 PO 4 Adjust pH to 3) = 5%: 95% (v / v);

[0098] Running time: 13min;

[0099] Column temperature: 30°C;

[0100] Injection volume: 5 μL;

[0101] Detection wavelength: 195nm;

[0102] Flow rate: 1.0 mL / min;

[0103] Signal bandwidth: 4nm.

[0104] The results are as follows Figure 1 As shown, the retention time of caesalpinia cerifera is 10.469 min and the purity factor is 999.843, indicating that the method described in Example 1 of the present invention can prepare caesalpinia cerifera with high purity. The high-purity caesalpinia cerifera ...

[0105] 2. Liquid chromatography-mass spectrometry analysis (HPLC-MSD)

[0106] Accurately weigh 25.06 mg of the standard prepared in Example 1 into a 25 mL volumetric flask, dissolve it in water, make up to volume, and mix well to obtain a standard stock solution.

[0107] Accurately transfer 0.50 mL of the above standard stock solution into a 10 mL volumetric flask, then dilute to the mark with water and mix well. The analysis conditions are as follows:

[0108] Liquid phase conditions:

[0109] Instrument: Agilent 1100 / 1200 HPLC;

[0110] HPLC column: Waters CORTECS HILIC, 150 x 4.6 mm, 2.7 μm;

[0111] Mobile phase: acetonitrile: 50 mmol ammonium formate solution = 75:25 (V / V);

[0112] Column temperature: 30°C;

[0113] Injection volume: 5 μL;

[0114] Running time: 12.00min;

[0115] Flow rate: 1.00 mL / min;

[0116] Detection wavelength: 205nm;

[0117] Signal bandwidth: 4nm;

[0118] Reference wavelength: off;

[0119] Reference bandwidth: off;

[0120] Spectral range: All (190nm-400nm).

[0121] MS conditions:

[0122] MS source: API-ES;

[0123] Polarity: positive;

[0124] Collision voltage: 70V;

[0125] Drying gas temperature: 350℃;

[0126] Drying gas flow rate: 12.0L / min;

[0127] Cap voltage: 3000V;

[0128] Acquisition mode: Scan;

[0129] Scanning range: 100-1000amu.

[0130] result:

[0131] UV chromatogram (DAD) and total ion current (TIC) Figure 2 As shown, the DAD retention time is about 6.8min, and the TIC retention time is about 7.0min; the mass spectrum is as shown Figure 3 As shown, the main fragment ions m / z are 171, 351, 373, and 701, which are consistent with the structural characteristics of caesalpinia oxyphylla.

[0132] 3. Nuclear magnetic analysis (1H-NMR)

[0133] The analysis method is as follows:

[0134] 7 mg of solid sample was dissolved in heavy water solvent and subjected to nuclear magnetic resonance analysis on Bruker AVANCE NEO 400 (with BBO probe, Bruker, GER).

[0135] NMR data such as Figure 4 As shown, the relevant features are consistent with the chemical structure characteristics of kaempferol acetamide.

[0136] 4. Infrared test (FTIR)

[0137] Weigh about 2 g of spectrally pure KBr, grind it and dry it at 105°C for 2 h, then transfer the KBr to a desiccator and cool it to room temperature. Weigh 10 mg of kauriacetamide analytical standard, dry it at 105°C for 2 h, then transfer it to a desiccator and cool it to room temperature.

[0138] About 200 mg of anhydrous KBr powder was weighed into an agate mortar, ground and pressed into tablets by an infrared spectroscopy tablet press. The potassium bromide salt tablets were scanned by an infrared spectrometer.

[0139] Weigh about 2 mg of dry kauriacetamide analytical standard into about 200 mg of KBr, mix and grind evenly. Use an infrared spectroscopy tableting device to compress the mixture into a semi-transparent tablet and then scan it with an infrared spectrometer.

[0140] FTIR results are as follows Figure 5 As shown, the relevant infrared characteristic peaks are consistent with the absorption peak positions of the corresponding groups in the chemical structure of caesalpinia acetamide.

[0141] 5. Detection of Kasugamycin-Kasugamycin Aqueous Composition

[0142] The kasugamycin aqueous solution in Example 4 was analyzed by HPLC, and the analysis conditions were as follows:

[0143] Instrument: Agilent HPLC 1100;

[0144] HPLC column: Agilent aichrombond-AQ C18 4.6mm*250mm*5μm;

[0145] Mobile phase: acetonitrile: 20 MM sodium hexanesulfonate aqueous solution (H 3 PO 4 Adjust pH to 3) = 5%: 95% (v / v);

[0146] Running time: 13min;

[0147] Column temperature: 30°C;

[0148] Detection wavelength: 195nm;

[0149] Flow rate: 1.0 mL / min;

[0150] Signal bandwidth: 4nm;

[0151] Injection volume: 5 μL.

[0152] The results are as follows Figure 6 As shown, the retention times of kasugamycin and kasugamycin amide are 8.462 min and 10.464 min, respectively, indicating that the detection method of the present invention has good separation and sensitivity response to kasugamycin and kasugamycin amide with similar structures, and the detection method of the present invention can be used to effectively identify kasugamycin and kasugamycin amide with similar structures.

[0153] Without being limited by the examples in the embodiments of the present invention, routine operations performed by those skilled in the art based on the specific scheme described in the present invention, such as adjustment of separation and detection parameters according to different analytical instruments, also fall within the scope of protection requested by the present invention.

[0154] 3. Effect Experiment

[0155] Example 6: Antibacterial test of caesalpinia cerevisiae

[0156] Raw materials: Prepare caesalpinia acetamide technical drug according to the method of Example 1.

[0157] Pathogenic bacteria: Curvularia zeae was collected from diseased corn leaves in a farm in Weinan City, isolated and purified by tissue separation method, and then identified; Rice blast was collected from diseased rice in the field, isolated, purified and cultivated.

[0158] Take 1 mL of the caesium acetamide aqueous solution with mass fractions of 0.01%, 0.1%, 0.3%, 0.5% and 1% in Example 2, mix them evenly with PSA culture medium in a ratio of 1 mL to 9 mL, and pour them into culture dishes to prepare drug-containing plates with final concentrations of 0.001%, 0.01%, 0.03%, 0.05% and 0.1%, respectively, and use PSA culture medium with an equal amount of sterile water as a blank control.

[0159] The cultured pathogens were punched to prepare a 5 mm diameter bacterial cake, placed in the above plate, and cultured in an incubator at 28 ± 1 ° C. When the edge of the colony in the control group was close to the wall of the culture dish, the colony diameter of each treatment group was measured by the cross method, and the relative inhibition percentage was calculated. The calculation formula is as follows:

[0160] Inhibition rate (%) = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%;

[0161] An inhibition rate of 1-24% was considered effective (△), 25%-39% was considered effective (▲), and ≥40% was considered markedly effective (▲▲).

[0162] The results of inhibition rate determination and antibacterial test are shown in Table 1 and Figure 7 shown.

[0163] Table 1 Inhibition rate of caesalpinia acetamide on two pathogens

[0164]

[0165]

[0166] Through Table 1 and Figure 7 It can be seen that the inhibitory effect of kasugamycin on rice blast fungus is similar to that of kasugamycin, both of which have high sensitivity, 0.01% concentration shows effectiveness effect, 0.03% concentration shows significant effect, and the inhibition rate reaches 41.6%. At the same time, kasugamycin also has a significant inhibitory effect on corn curvature fungus that is insensitive to kasugamycin. At a concentration of 0.01%, kasugamycin is effective against corn curvature fungus, and at a concentration of 0.05%, it is effective, and the inhibition rate reaches 42.2%. It can be seen from the above experimental results that kasugamycin and kasugamycin have similarities in the control effect of certain pathogens, which may be related to their similar chemical structures. However, for some pathogens, their control effects have great differences, which may indicate that the two have great differences in mechanism of action and / or effect.

[0167] Example 7: Indoor toxicity test of kasugamycin combination against rice blast

[0168] 1. Inhibition of medium concentration determination (EC50)

[0169] Raw materials: kasugamycin technical (prepared according to the method of Example 1); kasugamycin technical (kasugamycin hydrochloride, provided by Xi'an Masidi Bioengineering Co., Ltd.); rice blast was collected from diseased rice in the field, separated, purified and cultivated to obtain.

[0170] Test method: Refer to "Agricultural Industry Standard NY / Tll54.7-2006", potting method. Select rice seedlings at the three-leaf stage with the same growth potential, 2 seedlings per pot, and 5 pots of test rice seedlings for each treatment. Culture the rice blast fungus on tomato oatmeal agar medium, wash the spores with sterile water after spore production, and make 1×10 5 The suspension of spores / mL was evenly sprayed on the test rice seedlings, and then covered with black plastic bags for moisturizing and cultured for 24 hours. 24 hours after inoculation, the pesticides were treated, and 5 concentration gradients were set for each pesticide. The Potter spray tower was used to spray at a pressure of 50Psi, and about 5mL was used for each pot. After spraying, the rice seedlings were cultured at 28℃ and 92% relative humidity. After 8 days, the disease index of the whole leaf was investigated according to the grading standard of rice blast, and the control effect was calculated.

[0171] Disease index = {[∑(number of diseased leaves at each level of each treatment × corresponding level value)] / total number of leaves investigated × 9} × 100;

[0172] Control effect (%) = [(Disease index of blank control - Disease index of medicament treatment) / Disease index of blank control group] × 100.

[0173] Convert the control effect into probit value (y), convert the liquid medicine concentration (ug / ml) into logarithm value (x), and calculate the toxicity equation and median inhibitory concentration EC50 by the least square method.

[0174] 2. Co-toxicity experiment of春雷乙酰胺·春雷霉素composition

[0175] Calculate the toxicity index and co-toxicity coefficient (CTC) of the春雷乙酰胺·春雷霉素composition according to Sun Yunpei method.

[0176] Toxicity index of single agent = (EC50 of standard medicament / EC50 of tested single agent) × 100;

[0177] Actual toxicity index (ATI) = (EC50 of standard medicament / EC50 of tested medicament) × 100;

[0178] Theoretical toxicity index (TTI) of mixture = Toxicity index of agent A × Percentage content of A in mixture + Toxicity index of agent B × Percentage content of B in mixture;

[0179] Co-toxicity coefficient (CTC) = [Actual toxicity index (ATI) of mixture / Theoretical toxicity index (TTI) of mixture] × 100;

[0180] When CTC ≤ 80, the composition shows antagonistic effect; when 80 < CTC < 120, the composition shows additive effect; when CTC ≥ 120, the composition shows synergistic effect.

[0181] The results of the co-toxicity experiment are shown in Table 2.

[0182] Table 2 Co-toxicity experiment data

[0183]

[0184]

[0185] From the co-toxicity experiment of the春雷乙酰胺·春雷霉素composition, it can be seen that in the range of mass ratio 13:1 - 130:1,春雷霉素and春雷乙酰胺show a synergistic effect. After mixing them in a certain proportion, they have a good synergistic effect and can improve the control effect on rice blast.

[0186] From the above experimental results, it can be seen that although the chemical structures of kasugamycin and kasugamycin are similar, they have obvious differences in the prevention and treatment effects of pathogenic bacteria, and there is a certain complementarity and synergy between the two. Although the internal mechanism is still unclear, it is speculated that it may be because kasugamycin has better lipophilicity than kasugamycin, which improves the permeability of the substance on the cell wall of pathogenic bacteria.

[0187] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0188] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

Claims

1. A caesalpinia acetamide composition, characterized in that The kasugalacetamide composition contains kasugalacetamide and / or a salt of kasugalacetamide; Based on the total mass of the composition, the content of casulacetamide and / or the salt of casulacetamide is not less than 0.01%; preferably, the content of casulacetamide and / or the salt of casulacetamide is not less than 0.03%; The mass of the salt of caesalpinia acetamide is calculated based on the mass of caesalpinia acetamide it contains.

2. The composition according to claim 1, characterized in that The composition further comprises kasugamycin and / or a salt of kasugamycin.

3. The composition according to claim 2, characterized in that The mass ratio of kasugamycin and / or the salt of kasugamycin to kasugae acetamide and / or the salt of kasugae acetamide is 10:1-200:1; preferably, the mass ratio of kasugamycin and / or the salt of kasugamycin to kasugae acetamide and / or the salt of kasugae acetamide is 13:1-130:1; The mass of the kasugamycin salt is calculated based on the mass of kasugamycin contained therein.

4. A composition according to any one of claims 1 to 3, characterized in that The composition is any one of a raw drug, a mother drug or a preparation.

5. The composition of claim 4, characterized in that the dosage form of the preparation is selected from any one of powders, granules, large granules, fine granules, microcapsule granules, wettable powders, oil-dispersible powders, water-dispersible granules, emulsifiable granules, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release particles, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, film-spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latex, dispersible liquids, pastes, concentrated gels, emulsions in water, oil emulsions, microemulsions, ointments, suspensions, microcapsule suspensions, oil suspensions, suspoemulsions, dispersible powders for seed treatment, soluble powders for seed treatment, seed treatment liquids, seed treatment emulsions, seed treatment suspensions, suspended seed coatings, and microcapsule suspensions for seed treatment.

6. Use of the composition according to any one of claims 1 to 5 in preventing and controlling plant diseases; preferably, the plant disease is corn Curvularia leaf spot and / or rice blast.

7. Use of the composition according to any one of claims 1 to 5 in the preparation of a plant disease control formulation; preferably, the plant disease is corn Curvularia leaf spot and / or rice blast.

8. A method for preparing high-purity caesalpinamide, characterized in that: The method comprises the following steps: a fermentation broth purification step and an octadecylsilane bonded silica gel column separation step. Optionally, the fermentation broth purification step comprises the following steps: 1) Acidifying and filtering the fermentation broth; 2) The filtrate is adsorbed by resin and then analyzed; 3) The analytical solution is filtered to remove impurities; Optionally, a decolorization step is also included after the nanofiltration impurity removal.

9. The method according to claim 8, characterized in that The resin in the fermentation broth purification step 2 is a strongly acidic cation exchange resin; the resin adsorption analytical agent includes at least one of ammonium chloride solution, sodium hydroxide solution or sodium chloride solution.

10. Use of casulacetamide prepared by the method according to claim 8 or 9 as a standard or in the detection of active ingredients.

11. A method for analyzing and detecting caesalpinia serrata, characterized in that: The composition containing cinnabar acetamide is analyzed and detected by liquid chromatography or mass spectrometry, wherein the liquid chromatography detection wavelength is 195 nm; the fragment ions of cinnabar acetamide in the mass spectrometry analysis include one or more fragment ions with a mass-to-charge ratio (m / z) of 171, 351, 373 or 701, wherein the mass spectrometry analysis mode is a positive ion mode.

12. The detection method according to claim 11, characterized in that: The liquid chromatography column is a reverse phase C18 filler, and optionally, the liquid chromatography mobile phase flow rate is 0.5 to 2 mL / min; The fragment ions of kasugalacetamide in mass spectrometry analysis include two or more fragment ions with a mass-to-charge ratio (m / z) of 171, 351, 373 or 701.

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