Preparation of chaulmoogratree seed extract and application of chaulmoogratree seed extract in preventing and treating plant diseases

Abstract

The invention provides a botanical fungicide of a chaulmoogra seed extract, which takes sub-chaulmoogra seed oleic acid as a marker, and application of the botanical fungicide in preparation of medicines for preventing and treating plant diseases caused by pathogenic fungi and oomycetes. The hydnocarpus anthelmintica extract is obtained by extracting hydnocarpus anthelmintica seeds with a plurality of different types of organic solvents such as ethanol, petroleum ether and the like and treating the seeds by a physical method, and the hydnocarpus anthelmintica extract contains sub-hydnocarpus anthelmintica oleic acid as markers in different proportions. The extract has the effects of inhibiting conidium germination, bud tube growth or appressorium formation of fungi and oomycetes, effectively preventing pathogenic bacteria from infecting host plants and preventing and treating important plant diseases such as rice blast, mango anthracnose, potato late blight and rice false smut.

Description

[0001] This application claims priority to a prior application filed with the State Intellectual Property Office of China on April 8, 2024, with patent number 202410416539.9, entitled “Preparation of a Fructus Caryophylli Extract and Use thereof for Preventing and Controlling Plant Diseases.” The entire text of the prior application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of botanical fungicides, and particularly relates to a botanical fungicide of a cyperus rotundus extract with cyperus oleic acid as a marker and use of the botanical fungicide in preparing medicines for preventing and treating plant diseases caused by pathogenic fungi and oomycetes. Background Art

[0003] Plant fungal and oomycete diseases are numerous and account for approximately 70% of all plant diseases. A single plant can harbor several or even dozens of different fungal and oomycete diseases. For example, rice diseases such as blast, false smut, sheath blight, sesame spot, and seedling blight; wheat diseases such as head blight, powdery mildew, stripe rust, stem rust, leaf rust, root rot, and sheath blight; and corn diseases such as large leaf spot, small leaf spot, stem rot, rust, sheath blight, Curvularia leaf spot, head smut, and powdery mildew are all caused by fungi. These fungal diseases affect over 90% of the crop area affected and cause losses. In addition to fungal diseases, oomycete diseases, such as potato late blight, soybean phytophthora, pepper phytophthora, grape downy mildew, and cucumber downy mildew, are also serious threats to many crops. Statistics show that fungal and oomycete diseases cause 10%-20% of crop yield losses annually, amounting to approximately $10-20 billion.

[0004] Chemical control remains the primary method for controlling plant diseases. Commonly used synthetic chemical fungicides include tricyclazole, azoxystrobin, pyraclostrobin, boscalid, cyanobacterium-methyl, thiophanate-methyl, carbendazim, prothioconazole, and prochloraz. Reports indicate that many plant pathogenic fungi in agricultural production have developed significant resistance and cross-resistance to these agents. For example, Pyricularia oryzae, Fusarium spp., Gibberella spp., and Botrytis cinerea have shown significant resistance to fungicides such as carbendazim, prothioconazole, and boscalid, respectively. Furthermore, the long-term use of synthetic chemical fungicides has also led to a series of social problems, including environmental pollution, residual residues, and toxicity to humans and animals. As an effective alternative to synthetic chemical fungicides, botanical fungicides offer both antibacterial and fungicidal activity, along with advantages such as safety for humans and animals, environmental friendliness, resistance to drug resistance, and easy degradation.

[0005] There are 15 active ingredients of plant-derived fungicides registered in my country, namely, carvacrol (oregano extract), shiitake alcohol (Melaleuca alternifolia extract), phenylpropenone (Psoralea corylifolia seed extract), physcion (Rheum officinale rhizome extract, Polygonum cuspidatum extract), matrine (Sophora flavescens extract), pentadecene phenolic acid and tridecene phenolic acid (Ginkgo biloba fruit extract), azadirachtin (Neem seed extract), sanguinarine sulfate (Macleaya cordata extract), osthole (Cnidium monnieri extract), eugenol (extracts from plants such as cloves), catechin (tea extract), berberine (Coptis chinensis extract), allicin (extracts from bulbs of garlic and other Allium plants), and nicotine (tobacco extract). These botanical fungicides have been widely used to control various diseases of crops, fruits, and vegetables. For example, osthole is used to control powdery mildew, emodin is used to control powdery mildew, gray mold, and downy mildew, 5% carvacrol aqueous solution is effective against potato late blight, eugenol is used to control pepper blight, allicin is used to control pepper anthracnose and blight, fenpropimorph is used to control rice blast, and matrine is used to control downy mildew, powdery mildew, gray mold, and blight. Therefore, the development of botanical fungicides with diverse active ingredients, high efficacy, and a broad spectrum of control targets is an urgent need in current agricultural production. Summary of the Invention

[0006] In order to improve the deficiencies of the prior art, the present invention aims to provide a botanical fungicide of a cyperus rotundus extract with cyperus oleic acid as a marker and its use in the preparation of drugs for preventing and controlling plant diseases caused by pathogenic fungi and oomycetes.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] A preparation for preventing and treating plant diseases comprises a cyperus rotundus extract, wherein the content of cyperus rotundus oleic acid is between 10-95wt% based on the weight of the cyperus rotundus extract.

[0009] According to an embodiment of the present invention, in the Fructus Caryophylli extract, based on the weight of the Fructus Caryophylli extract, the content of sub-Fructus Caryophylli oleic acid is between 10-95wt%, for example, 11wt%, 12wt%, 13wt%, 15wt%, 16wt%, 18wt%, 19wt%, preferably between 20-95wt%, for example, 20wt%, 21wt%, 22wt%, 23wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, further preferably between 30-95wt%, for example, 33wt%, 35wt%, 36wt%, further preferably between 40-95wt%, for example, 40wt%, 45wt%, 50wt%, 55wt%, 60wt%, 65wt%, 70wt%, 75wt%, 80wt%, 85wt%, 90wt% or 95wt%.

[0010] According to an embodiment of the present invention, in the preparation, the Fructus Cynanchifolia extract is the main active ingredient, more preferably, the only active ingredient.

[0011] According to an embodiment of the present invention, the main active ingredient refers to the preparation, if it contains other active ingredients, its content does not exceed 20wt% based on the weight of the Cyperus rotundus extract, more preferably does not exceed 5wt%. For example, it is most preferred that it does not contain other active ingredients except the Cyperus rotundus extract.

[0012] According to an embodiment of the present invention, the oleic acid of the second genus is selected from the following structure A. Preferably, the oleic acid of the second genus is the structure of formula I or the structure of formula II.

[0013]

[0014] Formula I or Formula II is an isomer of Formula A. According to an embodiment of the present invention, the oleic acid comprises one or more of Formula A, Formula I or Formula II.

[0015] The molecular formula C of formula A 16 H 28 O2, molecular weight 252.39, easily soluble in organic solvents such as chloroform and ethyl acetate.

[0016] Wherein the following formula I:

[0017]

[0018]

[0019] The molecular formula C of formula I 16 H 28 O2, molecular weight 252.39, is a white solid, easily soluble in chloroform and ethyl acetate.

[0020] In some embodiments of the present invention, the Fructus Cynanchifolia extract is obtained by solvent extraction or supercritical carbon dioxide method.

[0021] In some embodiments of the present invention, the Fructus Caryophylli extract is further obtained by mechanical stirring and / or ultrasonic assisted extraction after solvent extraction.

[0022] In some embodiments of the present invention, the Fructus Cynanchifolia extract is further obtained by column chromatography separation after mechanical stirring and / or ultrasound-assisted extraction.

[0023] In some embodiments of the present invention, the solvent includes a protic solvent, an aprotic solvent, and a polar aprotic solvent.

[0024] In some embodiments of the present invention, the protic solvent is selected from one or a combination of water, methanol, ethanol, and cyclohexanol, preferably methanol or ethanol.

[0025] In some embodiments of the present invention, the aprotic solvent is selected from one or a combination of petroleum ether, n-hexane, n-pentane, dodecane, toluene, ether, dichloromethane, carbon tetrachloride, and 1,2-dichloroethane, preferably n-hexane, toluene, dichloromethane, and carbon tetrachloride.

[0026] In some embodiments of the present invention, the polar aprotic solvent is selected from one or a combination of chloroform, ethyl acetate, diethyl malonate, tetrahydrofuran, 1,4-dioxane, acetone, and acetonitrile, preferably chloroform, ethyl acetate, and acetone.

[0027] In some embodiments of the present invention, the Fructus Cynoglossi extract is obtained by extracting from the seeds, roots, stems, leaves, bark, etc. of Fructus Cynoglossi, preferably the seeds.

[0028] In some embodiments of the present invention, in the preparation, the content of the Fructus Cynanchifolia extract is 5-10 wt%, for example, 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%.

[0029] In some embodiments of the present invention, the formulation is an emulsion.

[0030] In some embodiments of the present invention, the preparation is an emulsion containing 10-15% of an emulsifier and 70-85% of an emulsifiable concentrate.

[0031] In some embodiments of the present invention, the emulsifier is selected from one or more of calcium dodecylbenzenesulfonate, tristyrylphenol polyoxyethylene polyoxypropylene ether, tristyrylphenol polyoxyethylene ether, castor oil polyoxyethylene ether, nonylphenol polyoxyethylene ether, Span80, isooctyl alcohol, and Tween 20.

[0032] In some embodiments of the present invention, the emulsifiable concentrate is selected from one or more of orange peel essential oil, rosin-based vegetable oil, eucalyptus oil, soybean oil, methyl oleate, solvent oil S-150, mineral oil C18-C24, xylene, and sec-butyl acetate.

[0033] The present invention also provides a method for preparing a plant disease prevention and control preparation, wherein the plant disease prevention and control preparation comprises an extract of the cyperus rotundus. The method for preparing the cyperus rotundus extract comprises the following steps:

[0034] 1) Grinding and crushing the seeds of the Herba Cynanchifoliae, soaking them in a solvent, and performing mechanical stirring or ultrasonic-assisted extraction to obtain an extract;

[0035] 2) adding solvent to the residue, ultrasonic extraction, and then mechanical immersion extraction at room temperature to obtain an extract;

[0036] 3) combining the two extracts and concentrating the extracts into an extract to obtain the Fructus Caryophylli var.

[0037] According to an embodiment of the present invention, in the step 1) of preparing the cyperus rotundus extract, the solvent is selected from one or more of water, methanol, ethanol, cyclohexanol, petroleum ether, n-hexane, n-pentane, dodecane, toluene, ether, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, chloroform, ethyl acetate, diethyl malonate, tetrahydrofuran, 1,4-dioxane, acetone, and acetonitrile, and the ethanol concentration is 70-95%, preferably 95% ethanol.

[0038] According to an embodiment of the present invention, in the preparation step 1) of the Fructus Caryophylli extract, the Fructus Caryophylli seeds are ground into coarse powder, the ultrasonic extraction time in steps 1) and 2) is 0.5-2h, preferably 1h, the mechanical soaking time is 1-5d, preferably 2d, and the content of sub-Fructus Caryophylli oleic acid in the Fructus Caryophylli extract obtained in step 3) is 13-40wt%.

[0039] In some embodiments of the present invention, in step 3) of preparing the Fructus Caryophylli extract, the extract is extracted 3-5 times, preferably 3 times, with petroleum ether (60-90° C.) and water. The organic phases are combined, the solvent is removed by rotary evaporation, and the Fructus Caryophylli extract is separated by column chromatography, wherein the Fructus Caryophylli oleic acid content is 67-95% by weight. The solvent used in the column chromatography is petroleum ether (60-90° C.): ethyl acetate in a ratio of 20:1, the chromatography column is filled with 200-300 mesh silica gel, and the mass ratio of sample to silica gel is 10:1.

[0040] In some embodiments of the present invention, the preparation step of the cyperus rotundus extract is 1) after ultrasonic extraction or mechanical stirring and soaking at room temperature, steam distillation is used for extraction for 2-6 hours, preferably 4 hours, the oil layer material is recovered, and water is removed by anhydrous sodium sulfate to obtain the cyperus rotundus extract, wherein the cyperus rotundus oleic acid content is 15-20wt%.

[0041] In some embodiments of the present invention, the method for preparing the plant disease prevention and control preparation further comprises the step of mixing the prepared Fructus Cynanchifolia extract with other auxiliary materials or carriers.

[0042] The present invention also provides a method for preparing the cyperus rotundus extract, comprising crushing cyperus rotundus seeds into coarse powder, soaking the powder in a solvent, and performing Soxhlet extraction to obtain an extract; further adding the solvent to the medicinal residue, performing Soxhlet extraction to obtain an extract; combining the two extracts, and concentrating the extract into an extract to obtain the cyperus rotundus extract, wherein the oleic acid content of the cyperus rotundus is 18-40wt%.

[0043] The present invention also provides a method for preparing a Fructus Cynanchifolia extract, the method comprising:

[0044] 1) Grinding and crushing the seeds of the Herba Cynanchifoliae, soaking them in a solvent, and performing mechanical stirring or ultrasonic-assisted extraction to obtain an extract;

[0045] 2) adding solvent to the residue, ultrasonic extraction, and then mechanical immersion extraction at room temperature to obtain an extract;

[0046] 3) combining the two extracts and concentrating the extracts into an extract to obtain the Fructus Caryophylli var.

[0047] The present invention also provides a method for preparing a cyperus rotundus extract, comprising crushing cyperus rotundus seeds into coarse powder, adding the powder into a material bottle of a supercritical carbon dioxide device, setting a pressure of 10-30 MPa, preferably 20 MPa, an extraction temperature of 30-50° C., preferably 45° C., and an extraction time of 1-3 h, preferably 1.5 h, to obtain a cyperus rotundus extract, wherein the cyperus rotundus oleic acid content is 35-40 wt%.

[0048] The present invention also provides a use of the cyperus rotundus extract in preparing a medicament for preventing and treating plant diseases, which can be used for preventing and treating plant diseases caused by pathogenic fungi and oomycetes.

[0049] According to an embodiment of the present invention, in the use, the content of oleic acid in the Fructus Caryophylli extract is 13-95 wt %, preferably 25-95 wt %, more preferably 40-95 wt %, further preferably 95 wt %.

[0050] According to an embodiment of the present invention, in the use, the concentration of the Fructus Cynanchifolia extract is 50-1200 mg / L.

[0051] According to an embodiment of the present invention, in the use, the plant diseases caused by fungi and oomycetes are selected from the following: rice blast, millet blast, mango anthracnose, banana anthracnose, pepper anthracnose, cucumber anthracnose, rubber anthracnose, litchi anthracnose, betel nut anthracnose, potato late blight, tomato late blight, pepper late blight, rice false smut, rice sheath blight, strawberry gray mold, tomato gray mold, grape gray mold, sugarcane whip smut, ginseng rust, ginseng sclerotinia, etc.

[0052] According to an embodiment of the present invention, in the use, the Fructus Cynanchifolia extract has the effect of inhibiting the conidia germination, germ tube growth or appressorium formation of fungi and oomycetes, and effectively preventing pathogens from infecting host plants.

[0053] According to an embodiment of the present invention, in the use, the extract of the cyperus rotundus has no obvious inhibitory effect on the growth of vegetative hyphae of fungi and oomycetes, and the resistance risk of the extract of the cyperus rotundus for disease prevention and control is low.

[0054] The present invention also provides a fungicide composition for preventing and controlling agricultural, forestry and horticultural plant diseases, comprising a cyperus rotundus extract and another botanical fungicide, wherein the cyperus rotundus extract has a oleic acid content of 10-95 wt%, preferably 25-95 wt%, more preferably 40-95 wt%, and further preferably 95 wt%.

[0055] According to an embodiment of the present invention, the additional botanical fungicide is selected from one or more combinations of carvacrol, shiitake mushroom alcohol, phenylpropenone, physcion, matrine, pentadecene phenolic acid and tridecene phenolic acid, azadirachtin, sanguinarine sulfate, osthole, eugenol, catechin, berberine, allicin, and nicotine.

[0056] According to an embodiment of the present invention, in the fungicide composition, the ratio of the Fructus Cynanchifolia extract to the other botanical fungicide is 1:10-10:1.

[0057] According to an embodiment of the present invention, the fungicidal composition can be used to prevent and control plant diseases caused by pathogenic fungi and oomycetes. The plant diseases caused by fungi and oomycetes are selected from the following: rice blast, millet blast, mango anthracnose, banana anthracnose, pepper anthracnose, cucumber anthracnose, rubber anthracnose, litchi anthracnose, betel nut anthracnose, potato late blight, tomato late blight, pepper late blight, rice false smut, rice sheath blight, strawberry gray mold, tomato gray mold, grape gray mold, sugarcane whip smut, ginseng rust, ginseng sclerotinia, etc.

[0058] The present invention also provides a use of oleic acid in preparing medicines for preventing and treating plant diseases caused by pathogenic fungi and oomycetes.

[0059] The present invention also provides a method for preparing oleic acid of cyclopentene, which comprises the following steps: obtaining a pure product of 2-cyclopentene-1-acetic acid through eight-step chemical synthesis and one-step chiral resolution.

[0060]

[0061] 1) 2-cyclopentenyl-1-acetic acid was placed in a reaction flask, ether was added thereto, stirring was started, and solid lithium aluminum hydride was slowly added under an ice-water bath and stirred at room temperature for 3 h. TLC detection reaction was completed, water was added under an ice-water bath to quench the reaction, stirred at room temperature for 30 min, the solid was filtered, the solid was washed with ether, the liquid was combined and separated, the organic phase was washed 3 times with saturated brine, the aqueous phase was washed 3 times with ether, the organic layers were combined, and dried over anhydrous sodium sulfate. The organic layer was concentrated to obtain compound DF2-P2-1.

[0062] 2) Compound DF2-P2-1 was placed in a reaction flask, ether was added, and the mixture was stirred. Phosphorus tribromide was added under an ice-water bath and stirred at room temperature for 2 h. The reaction was detected by TLC. Saturated sodium bicarbonate solution was added under an ice-water bath to quench the reaction. Water and ether were added for extraction. The organic phase was washed with saturated brine, the organic layers were combined, and dried over anhydrous sodium sulfate. The organic layer was concentrated and separated by column chromatography to obtain compound DF2-P2-2.

[0063] 3) 9-Bromoundecanol was placed in a reaction flask, dichloromethane was added, and stirring was initiated. Tert-butyldiphenylsilyl chloride and imidazole were added at room temperature, and the mixture was stirred at room temperature for 2 h. The reaction was confirmed by TLC. The insoluble solid was filtered and washed three times with dichloromethane. The organic phases were combined, concentrated, and separated by column chromatography to obtain compound DF2-P2-4.

[0064] 4) Magnesium chips were placed in a reaction flask, ether and iodine pellets were added thereto, nitrogen was replaced, a small amount of ether solution of compound DF2-P2-2 was added dropwise, and the system was boiled with a hair dryer under stirring until the iodine faded. The ether solution of compound DF2-P2-2 was slowly added dropwise. After the addition was completed, the mixture was refluxed for 1 hour to obtain an ether solution containing compound DF2-P2-3 without separation.

[0065] 5) Compound DF2-P2-4 was placed in a reaction flask, tetrahydrofuran was added thereto, stirring was started, nitrogen was replaced, and the negative

[0066] A 0.1M solution of dilithium tetrachlorocuprate in tetrahydrofuran was added at 78°C, followed by a dropwise addition of a freshly prepared solution of compound DF2-P2-3 in diethyl ether. After complete addition, the mixture was stirred at room temperature for 8 hours. After TLC analysis of the reaction, aqueous ammonium chloride was added to quench the reaction under an ice-water bath. The mixture was extracted with water and diethyl ether, and the organic phase was washed with saturated brine. The combined organic layers were then dried over anhydrous sodium sulfate. The organic layer was concentrated and separated by column chromatography to obtain compound DF2-P2-5.

[0067] 6) Compound DF2-P2-5 was placed in a reaction flask, tetrahydrofuran was added thereto, stirring was started, and a tetrabutylammonium fluoride solution in tetrahydrofuran was added at room temperature. The reaction was detected to be complete by TLC, and ice water was added to quench the reaction. The liquid was extracted and separated, and the organic layer was concentrated and separated by column chromatography to obtain compound DF2-P2-6.

[0068] 7) Compound DF2-P2-6 was placed in a reaction flask, dichloromethane was added thereto, and stirring was started. Dess-Martin periodinane was added at room temperature. The reaction was detected by TLC to be complete. The reaction was quenched by adding a 1:1 mixture of aqueous sodium bicarbonate solution and aqueous sodium thiosulfate solution. The liquids were separated by extraction, and the organic layer was concentrated to obtain compound DF2-P2-7.

[0069] 8) Compound DF2-P2-7 was placed in a reaction flask, tert-butanol was added thereto, stirring was started, and a freshly prepared mixed solution of sodium chlorite and sodium dihydrogen phosphate was added dropwise at room temperature. The reaction was detected to be complete by TLC, and ethyl acetate was added to quench the reaction. The liquid was extracted and separated, and the organic layer was concentrated and separated by column chromatography to obtain compound DF2-P2-8.

[0070] 9) Compound DF2-P2-8 was separated by chiral preparative HPLC to obtain DF2-P2.

[0071] Beneficial effects of the present invention:

[0072] 1) The extract of the radix schonophylli with oleic acid as a marker (the oleic acid is preferably a compound of formula A, more preferably a compound of formula I or formula II) has the effect of inhibiting the conidia germination, germ tube growth or appressorium formation of fungi and oomycetes, effectively preventing pathogens from infecting host plants.

[0073] 2) The effectiveness of the extract of Fructus Caryophylli against plant diseases was verified by inoculation experiments on detached plant leaves or fruits, indoor pot experiments, and field plot experiments.

[0074] 3) The extract of the radix schoenopsis had no obvious inhibitory effect on the growth of vegetative hyphae of fungi and oomycetes. Therefore, it can be seen that the resistance risk of the extract of the radix schoenopsis for disease prevention and control is low.

[0075] 4) A cyperus rotundus extract with oleic acid as a marker (the oleic acid is preferably a compound of formula A, more preferably a compound of formula I or formula II) can be used to prevent and control plant diseases caused by pathogenic fungi and oomycetes. The plant diseases caused by fungi and oomycetes are selected from the following: rice blast, millet blast, mango anthracnose, banana anthracnose, pepper anthracnose, cucumber anthracnose, rubber anthracnose, litchi anthracnose, betel nut anthracnose, potato late blight, tomato late blight, pepper late blight, rice false smut, rice sheath blight, strawberry gray mold, tomato gray mold, grape gray mold, sugarcane whip smut, ginseng rust, ginseng sclerotinia rot, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Gas chromatography-mass spectrometry of the Fructus Cynanchifolia extract DF2-P1.

[0077] Figure 2 Gas chromatography-mass spectrometry of DF2-A3 of Fructus Cynanchifolia extract.

[0078] Figure 3 Gas chromatography-mass spectrum of pure marker DF2-P2-hypopigineic acid.

[0079] Figure 4Inhibitory effects of different concentrations of Fructus dasyphylla extract DF2-A3 on conidia germination and appressorium formation of Magnaporthe grisea.

[0080] Figure 5 Inhibitory effects of different concentrations of DF2-P1 extract from Fructus oryzae on conidia germination and appressorium formation of Magnaporthe grisea.

[0081] Figure 6 Inhibitory effects of different concentrations of Fructus Caryophylli extract DF2-A3 on conidia germination and appressorium formation of Colletotrichum thunbergii.

[0082] Figure 7 Inhibitory effects of different concentrations of Fructus Caryophylli extract DF2-P1 on conidia germination and appressorium formation of Colletotrichum thunbergii.

[0083] Figure 8 The control effect of different concentrations of DF2-A3 extract from Fructus oryzae against blast fungus infection of detached barley leaves in vitro.

[0084] Figure 9 The control effect of different concentrations of DF2-P1 extract from Fructus oryzae against Magnaporthe grisea infection of detached barley leaves in vitro.

[0085] Figure 10 The control effect of DF2-A3 (50 mg / L) of Fructus Caryophylli extract on blast fungus infection of detached rice leaves in indoor environment.

[0086] Figure 11 The preventive effect of different concentrations of DF2-A3 extract from Fructus Caryophylli on the infection of anthracnose in detached mango leaves in vitro.

[0087] Figure 12 The preventive effect of DF2-A3 (200 mg / L) of Fructus oryzae extract on rice blast infection in potted rice.

[0088] Figure 13 The preventive effect of DF2-A3 (200 mg / L) of Fructus Caryophylli extract on late blight infection of potted potatoes.

[0089] Figure 14 The preventive effect of 1200 mg / L cyperus rotundus extract DF2-A3 on gray mold infection in strawberry fruit.

[0090] Figure 15 The control effect of DF2-A3 (500 mg / L) of Fructus Caryophylli extract on anthracnose infection in mango fruit.

[0091] Figure 16 The control effect of DF2-A3 (1200 mg / L) of Fructus Caryophylli extract on rice false smut in the field. DETAILED DESCRIPTION

[0092] The present invention will be described in further detail below with reference to specific examples. It should be understood that the following examples are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0093] Example 1: Determination of the content of the marker oleic acid in the extract of the radix schizonepetae

[0094] The present invention provides a method for determining the content of a marker, oleic acid, in a cyperus rotundus extract by gas chromatography-mass spectrometry (GC-MS). The specific method is as follows: (1) Program settings: HP-5MS 5% PhenylMethylSiloxane elastic quartz capillary column (250 μm×30 m, 0.25 μm), injection volume 1 μL, vaporization chamber temperature 300°C, column temperature 60°C, then heating to 280°C at 10°C / min and holding for 2 minutes, column head pressure 8.2317 psi, carrier gas flow rate 1.0 mL / min, carrier gas is high-purity helium (99.999%), split ratio 10:1. Mass spectrometry conditions: ion source EI source, ion source temperature 230°C, quadrupole temperature 150°C, emission current 35.0μA, multiplier voltage 983V, electron energy 70eV, mass range m / z 35-300, interface temperature 280°C (2) Establishment of standard curve: The standard samples (14.5, 19, 23.75, 28.5, 47.5 mg / L) were determined by the above method, with concentration as the horizontal axis and the corresponding peak area as the vertical axis to establish a standard curve. The content of the marker oleic acid in the extract of the radix schizonepetae was determined by the above method. The reference substance of the marker was synthesized by the applicant himself and has the structure of formula I.

[0095]

[0096] For the specific synthesis method of the reference substance Formula I, see Example 3.

[0097] Example 2: Preparation of Fructus Caryophylli Extract

[0098] The present invention provides a general method for extracting the Fructus Caryophylli, including sample grinding and crushing, and various extraction methods and combinations thereof, such as solvent extraction (ethanol, methanol, petroleum ether, n-hexane, ethyl acetate, chloroform, ether, leaching, Soxhlet extraction, etc.), mechanical stirring or ultrasonic-assisted extraction, steam distillation, or supercritical carbon dioxide extraction. The Fructus Caryophylli extract can be obtained by any of the following steps.

[0099] 1) Preparation of Fructus Caryophylli Extract DF2-A1

[0100] The seeds of the genus Fructus were ground into a coarse powder, soaked in water, and ultrasonically extracted for 1 hour. The mixture was then mechanically stirred and soaked at room temperature for 1 day. The extract was then steam distilled for 4 hours. The oil layer was recovered and dehydrated with anhydrous sodium sulfate to obtain the Fructus Fructus extract DF2-A1. GC-MS analysis revealed that the content of the marker oleic acid in the Fructus Fructus Fructus obtained using this method was 18 wt%.

[0101] 2) Preparation of Fructus Caryophylli Extract DF2-A2

[0102] The seeds of the genus Fructus were ground into a coarse powder, soaked in 95% ethanol, ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The residue was then further added to 95% ethanol and ultrasonically extracted for 1 hour, followed by mechanical soaking at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-A2. GC-MS analysis showed that the content of the marker oleic acid in the genus Fructus Fructus Fructus obtained using this method was 36% by weight.

[0103] 3) Preparation of Fructus Caryophylli Extract DF2-P1

[0104] The seeds of the plant were ground into a coarse powder, soaked in 95% ethanol, ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The residue was further added to 95% ethanol and ultrasonically extracted for 1 hour, followed by mechanical soaking at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated to form an extract. The extract was extracted three times with petroleum ether (60-90°C) and water. The organic phases were combined, the solvent was removed by rotary evaporation, and the compound DF2-P1 was isolated by column chromatography. GC-MS analysis showed that the content of the marker oleic acid in the plant extract obtained using this method was 95% by weight.

[0105] 4) Preparation of Fructus Caryophylli Extract DF2-A3

[0106] The seeds of the genus Fructus were ground into a coarse powder, soaked in 95% ethanol, and Soxhlet extracted for 1 hour to obtain an extract. The residue was then further added to 95% ethanol and Soxhlet extracted for 1 hour to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-A3. GC-MS analysis showed that the genus Fructus Fructus extract obtained using this method contained 40% by weight of the marker oleic acid.

[0107] 5) Preparation of Fructus Caryophylli Extract DF2-A4

[0108] The seeds of the genus Fructus were ground into a coarse powder, soaked in methanol, and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The residue was further added to the dregs of the dregs and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-A4. GC-MS analysis revealed that the genus Fructus Fructus extract obtained using this method contained 30 wt% of the marker oleic acid.

[0109] 6) Preparation of Fructus Caryophylli Extract DF2-A5

[0110] The seeds of the genus Fructus were ground into a coarse powder, soaked in cyclohexanol, and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The residue was then further added to the cyclohexanol and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-A5. GC-MS analysis revealed that the genus Fructus Fructus extract obtained using this method contained 22 wt% of the marker oleic acid.

[0111] 7) Preparation of Fructus Caryophylli Extract DF2-B1

[0112] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in petroleum ether (60-90°C), ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The residue was further added to petroleum ether (60-90°C), ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-B1. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 23% by weight.

[0113] 8) Preparation of Fructus Caryophylli Extract DF2-B2

[0114] The seeds of the radix sylvestris were ground into a coarse powder, soaked in petroleum ether (60-90°C), and Soxhlet extracted for 1 hour to obtain an extract. The residue was then added to petroleum ether (60-90°C) and Soxhlet extracted for 1 hour to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix sylvestris extract DF2-B2. GC-MS analysis showed that the content of the marker oleic acid in the radix sylvestris extract obtained using this method was 23 wt%.

[0115] 9) Preparation of Fructus Caryophylli Extract DF2-B3

[0116] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in n-hexane, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added to n-hexane and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-B3. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 25 wt%.

[0117] 10) Preparation of Fructus Caryophylli Extract DF2-B4

[0118] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in n-pentane, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added to n-pentane and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-B4. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 20 wt%.

[0119] 11) Preparation of Fructus Caryophylli Extract DF2-B5

[0120] The seeds of the genus Fructus were ground into a coarse powder, soaked in dodecane, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added with dodecane and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-B5. GC-MS analysis showed that the content of the marker oleic acid in the genus Fructus Fructus Fructus obtained using this method was 19 wt%.

[0121] 12) Fructus Caryophylli Extract DF2-B6

[0122] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in toluene, and ultrasonically extracted for 1 hour. The extract was then mechanically stirred and soaked at room temperature for 2 days to obtain an extract. The residue was then further added toluene and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract concentrate to obtain the radix dasyphyllae extract DF2-B6. GC-MS analysis revealed that the radix dasyphyllae extract obtained using this method contained 28 wt% of the marker oleic acid.

[0123] 13) Preparation of Fructus Caryophylli Extract DF2-B7

[0124] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in dichloromethane, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added to dichloromethane and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-B7. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 25 wt%.

[0125] 14) Preparation of Fructus Caryophylli Extract DF2-B8

[0126] The seeds of the genus Fructus were ground into a coarse powder, soaked in dichloromethane, and Soxhlet extracted for 1 hour to obtain an extract. The residue was then added to the dichloromethane and Soxhlet extracted for 1 hour to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-B8. GC-MS analysis showed that the genus Fructus Fructus extract obtained using this method contained 26% by weight of the marker oleic acid.

[0127] 15) Preparation of Fructus Caryophylli Extract DF2-B9

[0128] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in carbon tetrachloride, and ultrasonically extracted for 1 hour, followed by mechanical extraction at room temperature for 2 days to obtain an extract. The residue was further subjected to carbon tetrachloride and ultrasonically extracted for 1 hour, followed by mechanical extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-B9. GC-MS analysis revealed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 27 wt%.

[0129] 16) Preparation of Fructus Caryophylli Extract DF2-B10

[0130] The seeds of the genus Fructus were ground into a coarse powder, soaked in 1,2-dichloroethane, and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The residue was then further added to the genus Fructus 1,2-dichloroethane and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The two extracts were combined and concentrated into an extract to obtain the genus Fructus 1,2-B10 extract. GC-MS analysis revealed that the content of the marker oleic acid in the genus Fructus 1,2-B10 extract obtained using this method was 23 wt%.

[0131] 17) Preparation of Fructus Caryophylli Extract DF2-C1

[0132] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in chloroform, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added to chloroform and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-C1. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 28 wt%.

[0133] 18) Preparation of Fructus Caryophylli Extract DF2-C2

[0134] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in ethyl acetate, and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The residue was further added to ethyl acetate and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain a solution. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-C2. GC-MS analysis revealed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 33 wt%.

[0135] 19) Preparation of Fructus Caryophylli Extract DF2-C3

[0136] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in ethyl acetate, and Soxhlet extracted for 1 hour to obtain an extract. The residue was then added to ethyl acetate and Soxhlet extracted for 1 hour to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-C3. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 33 wt%.

[0137] 20) Preparation of Fructus Caryophylli Extract DF2-C4

[0138] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in diethyl malonate, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added with diethyl malonate, ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-C4. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 21 wt%.

[0139] 21) Preparation of Fructus Caryophylli Extract DF2-C5

[0140] The seeds of the radix dasyphylla were ground into a coarse powder, soaked in ether, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added to ether and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphylla extract DF2-C5. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphylla extract obtained using this method was 18 wt%.

[0141] 22) Preparation of Fructus Caryophylli Extract DF2-C6

[0142] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in ether, and Soxhlet extracted for 1 hour to obtain an extract. The residue was then added to ether and Soxhlet extracted for 1 hour to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-C6. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 18 wt%.

[0143] 23) Preparation of Fructus Caryophylli Extract DF2-C7

[0144] The seeds of the radix dasyphyllae were ground into a coarse powder, soaked in tetrahydrofuran, and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The residue was further added to tetrahydrofuran and ultrasonically extracted for 1 hour, followed by mechanical soaking and extraction at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the radix dasyphyllae extract DF2-C7. GC-MS analysis showed that the content of the marker oleic acid in the radix dasyphyllae extract obtained using this method was 15 wt%.

[0145] 24) Preparation of Fructus Caryophylli Extract DF2-C8

[0146] The seeds of the genus Fructus Caryophylli were ground into a coarse powder, soaked in 1,4-dioxane, ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The residue was further added with 1,4-dioxane, ultrasonically extracted for 1 hour, and then mechanically soaked at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Caryophylli extract DF2-C8. GC-MS analysis showed that the content of the marker oleic acid in the genus Fructus Caryophylli extract obtained using this method was 16 wt%.

[0147] 25) Preparation of Fructus Caryophylli Extract DF2-C9

[0148] The seeds of the genus Fructus were ground into a coarse powder, soaked in acetone, and ultrasonically extracted for 1 hour. The extract was then mechanically stirred and soaked at room temperature for 2 days to obtain an extract. The dregs of the dregs were then further ultrasonically extracted for 1 hour, followed by mechanical soaking at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-C9. GC-MS analysis revealed that the genus Fructus Fructus extract obtained using this method contained 35% by weight of the marker oleic acid.

[0149] 26) Preparation of Fructus Caryophylli Extract DF2-C10

[0150] The seeds of the genus Fructus were ground into a coarse powder, soaked in acetonitrile, and ultrasonically extracted for 1 hour. The extract was then mechanically stirred and soaked at room temperature for 2 days to obtain an extract. The dregs of the dregs were then added to acetonitrile and ultrasonically extracted for 1 hour. The extract was then mechanically soaked at room temperature for 2 days to obtain an extract. The two extracts were combined and concentrated into an extract to obtain the genus Fructus Fructus extract DF2-C10. GC-MS analysis revealed that the genus Fructus Fructus extract obtained using this method contained 13 wt% of the marker oleic acid.

[0151] 27) Preparation of Fructus Caryophylli Extract DF2-D1

[0152] The seeds of the radix sylvestris were ground into a coarse powder and added to the feed bottle of a supercritical carbon dioxide apparatus. The pressure was set at 20 MPa and the extraction temperature was set at 45°C for 1.5 hours to obtain the radix sylvestris extract DF2-D1. GC-MS analysis showed that the content of the marker oleic acid in the radix sylvestris extract obtained using this method was 40 wt%.

[0153] In Example 2, the dichotoma extracts DF2-A1, DF2-A2, DF2-A4, DF2-A5, DF2-B1, DF2-B2, DF2-B3, DF2-B4, DF2-B5, DF2-B6, DF2-B7, DF2-B8, DF2-B9, DF2-B10, DF2-C1, DF2-C2, DF2-C3, DF2-C4, DF2-C5, DF2-C6, DF2-C7, DF2-C8, DF2-C9, and DF2-C10 can be further purified by methods such as extraction and chromatography to increase the content of the marker dichotoma oleic acid in the dichotoma extracts to more than 40 wt%.

[0154] Example 3: Preparation of pure marker DF2-P2-hypopigine oleic acid

[0155] The pure marker DF2-P2 was obtained from the commercially available drug 2-cyclopentene-1-acetic acid via an eight-step chemical synthesis followed by a one-step chiral resolution. GC-MS analysis revealed that the content of oleic acid in the obtained product was 99.99%.

[0156]

[0157] first step:

[0158]

[0159] 20g of 2-cyclopentenyl-1-acetic acid was placed in a reaction flask, 300mL of ether was added thereto, stirring was started, 18g of lithium aluminum hydride solid was slowly added under an ice-water bath, and the mixture was stirred at room temperature for 3h. TLC detection reaction was completed, 350mL of water was added under an ice-water bath to quench the reaction, stirred at room temperature for 30min, the solid was filtered, the solid was washed with ether, the liquid was combined and the liquid was separated, the organic phase was washed 3 times with saturated brine, the aqueous phase was washed 3 times with ether, the organic layers were combined, and the mixture was dried over anhydrous sodium sulfate. The organic layer was concentrated to obtain 13g of compound DF2-P2-1.

[0160] Step 2:

[0161]

[0162] 13g of compound DF2-P2-1 was placed in a reaction flask, 200mL of ether was added thereto, and the mixture was stirred. 5.4mL of phosphorus tribromide was added under an ice-water bath, and the mixture was stirred at room temperature for 2h. After TLC detection, the reaction was quenched by adding saturated sodium bicarbonate aqueous solution under an ice-water bath. Water and ether were added for extraction, and the organic phase was washed with saturated brine. The organic layers were combined and dried over anhydrous sodium sulfate. The organic layer was concentrated and separated by column chromatography to obtain 8.8g of compound DF2-P2-2.

[0163] Step 3:

[0164]

[0165] 7 g of 9-bromodecanol was placed in a reaction flask, 100 mL of dichloromethane was added, and stirring was initiated. 10 mL of tert-butyldiphenylsilyl chloride and 3.3 g of imidazole were added at room temperature, and the mixture was stirred at room temperature for 2 h. After TLC analysis of the reaction, the insoluble solid was filtered and washed three times with dichloromethane. The organic phases were combined, concentrated, and separated by column chromatography to obtain 14 g of compound DF2-P2-4.

[0166] Step 4 and Step 5:

[0167]

[0168] Place 2.8 g of magnesium chips in a reaction flask, add 20 mL of ether and iodine pellets, replace nitrogen, add a small amount of ether solution of compound DF2-P2-2 dropwise, boil the system with a hair dryer while stirring until the iodine fades, slowly add 50 mL of ether solution containing 5 g of compound DF2-P2-2 dropwise, reflux for 1 hour after the addition is completed, and obtain an ether solution containing compound DF2-P2-3 without separation.

[0169] 6g of compound DF2-P2-4 was placed in a reaction flask, tetrahydrofuran was added, stirring was initiated, the nitrogen atmosphere was purged, and 5.8mL of dilithium tetrachlorocuprate was added at -78°C. A fresh ether solution of compound DF2-P2-3 was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 8 hours. TLC confirmed the reaction was complete. The reaction was quenched by adding aqueous ammonium chloride in an ice-water bath. Water and ether were added for extraction. The organic phase was washed with saturated brine, the combined organic layers were dried over anhydrous sodium sulfate, and the organic layers were concentrated and separated by column chromatography to obtain 2g of compound DF2-P2-5.

[0170] Step 6:

[0171]

[0172] 2 g of compound DF2-P2-5 was placed in a reaction flask, tetrahydrofuran was added thereto, and stirring was started. 4.3 mL of 1 M tetrabutylammonium fluoride tetrahydrofuran solution was added at room temperature. The reaction was detected to be complete by TLC. The reaction was quenched with ice water, and the liquid was extracted and separated. The organic layer was concentrated and separated by column chromatography to obtain 982 mg of compound DF2-P2-6.

[0173] Step 7:

[0174]

[0175] 982 mg of compound DF2-P2-6 was placed in a reaction flask, dichloromethane was added thereto, and stirring was started. 3.5 g of Dess-Martin periodinane was added at room temperature. The reaction was detected to be complete by TLC. A mixed solution of sodium bicarbonate aqueous solution: sodium thiosulfate aqueous solution = 1:1 was added to quench the reaction. The liquid was extracted and separated, and the organic layer was concentrated to obtain 970 mg of compound DF2-P2-7.

[0176] Step 8:

[0177]

[0178] Compound DF2-P2-7 (970 mg) was placed in a reaction flask, tert-butanol was added, and stirring was started. A mixed solution of freshly prepared 3.3 g of sodium chlorite and 4.4 g of sodium dihydrogen phosphate was added dropwise at room temperature. The reaction was detected to be complete by TLC. Ethyl acetate was added to quench the reaction, and the liquid was separated by extraction. The organic layer was concentrated and separated by column chromatography to obtain 683 mg of compound DF2-P2-8.

[0179] Step 9:

[0180]

[0181] Compound DF2-P2-8 was separated by chiral preparative high performance liquid chromatography to obtain 100 mg of compound DF2-P2.

[0182] Compound structure characterization data:

[0183] In the description hereinafter, "NMR" means nuclear magnetic resonance spectroscopy, and the following abbreviations are used: s = singlet, br = broad, d = doublet, dd = doublet of doublets, t = triplet, td = triplet of doublets, q = quartet, m = multiplet.

[0184] 1 HNMR(500MHz, CDCl3):5.68(m,2H),2.60(m,1H),2.34(t,2H),2.30(m,1H),2. 22(m,1H),2.00(m,1H),1.38(m,1H),1.62(quint,J=7.5Hz,2H),1.26(m,16H). 13 C NMR (125MHz, CDCl3): 179.2, 135.5, 130.0, 45.6, 36.2, 33.9, 32.0, 29.9, 29.6-28.0, 24.2.

[0185] Example 4: Preparation of formulation

[0186] F1.1 to F1.18: Emulsion

[0187]

[0188] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0189]

[0190] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0191]

[0192] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0193]

[0194] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0195]

[0196] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0197]

[0198] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0199]

[0200]

[0201] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0202]

[0203] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0204]

[0205] Emulsions of the desired concentration for use can be prepared by diluting the concentrate with water or other dispersants.

[0206] Example 5: Biological Experiment

[0207] (1) Inhibitory effect of cyperus rotundus extract on conidia germination and appressorium formation of rice blast fungus

[0208] Experimental methods:

[0209] (1) Production of rice blast conidia: The rice blast fungus (Magnaporthe oryzae) strain P131 to be tested was spotted onto a tomato oat agar plate (OTA) and cultured in a constant temperature and light incubator at 28°C. After 3-5 days, the rice blast fungus hyphae on the OTA were fully broken, and then evenly spread onto a new OTA plate and cultured in a constant temperature and light incubator at 28°C. When new hyphae were visible growing out of the culture medium surface, they were gently broken with a cotton swab, rinsed with sterile water, and dried. The culture dish was covered with a single layer of gauze and cultured at 28°C for 48 hours to produce a large number of conidia on the OTA surface.

[0210] (2) Preparation of rice blast fungus conidia suspension: Elute the culture on OTA with sterile water and filter with three layers of lens paper. The filtrate is the conidia suspension. Use a hemocytometer to adjust the conidia concentration in the conidia suspension to 2×10 5 pieces / mL.

[0211] (3) The test compound was added to the conidia suspension at different concentrations, resulting in working concentrations of 200 mg / L and 50 mg / L. The solution was then spotted onto hydrophobic glass slides. Four spots were placed on each slide and the slide was kept in the dark and moisturized. 12 h after inoculation, the conidia germination rate and appressorium formation rate were observed and counted under a microscope.

[0212] (4) Statistics and Analysis: Count three inoculation points on each hydrophobic slide. Count the number of germinated conidia and the number of appressorium formed in the center of each inoculation point. Calculate the average of the three data sets to determine the conidia germination rate and appressorium formation rate. Use the conidia germination rate and appressorium formation rate obtained from a parallel experiment with the solvent as a control to calculate the inhibitory effect of the test compound on conidia germination and appressorium formation.

[0213] Table 1 shows the biological data of some of the extracts of Fructus Caryophylli: "+" represents that the inhibitory effect at 200 mg / L is less than 40%, "++" represents that the inhibitory effect at 50 mg / L is greater than 40%, and "+++" represents that the inhibitory effect at 50 mg / L is greater than 80%.

[0214] Table 1

[0215]

[0216]

[0217] (II) Inhibitory effect of cyperus rotundus extract on conidia germination and appressorium formation of Colletotrichum thunbergii

[0218] Experimental methods:

[0219] (1) Production of Anthracnose Conidia: The strain SC2-1 of the mango anthracnose fungus (Colletotrichum gloeosporioides) to be tested was spotted onto a potato culture medium (PDA) plate and cultured in a constant temperature and light incubator at 28°C. After 3-5 days, 1-2 small pieces of mycelium were picked up with a toothpick and placed in potato dextrose broth (PDB) medium. The culture was shaken at 28°C and 160 rpm for 3-5 days to produce a large number of conidia in the PDB. The conidia were filtered and centrifuged at 5000 rpm for 5 minutes to obtain the conidia.

[0220] (2) Preparation of anthrax conidia suspension: Elute the conidia obtained by centrifugation with sterile water to obtain conidia liquid. Use a hemocytometer to adjust the conidia concentration in the conidia liquid to 2×10 5 pieces / mL.

[0221] (3) The test compound was added to the conidia suspension at varying concentrations, resulting in solutions with working concentrations of 200 mg / L and 50 mg / L. The solutions were then spotted sequentially on hydrophobic glass slides. Four spots were placed on each slide, and the slides were kept in the dark and moisturized. Twelve hours after inoculation, the conidia germination rate and appressorium formation rate were observed and counted under a microscope.

[0222] (4) Statistics and Analysis: Count three inoculation points on each hydrophobic slide. Count the number of germinated conidia and the number of appressorium formed in the center of each inoculation point. Calculate the average of the three data sets to determine the conidia germination rate and appressorium formation rate. Use the conidia germination rate and appressorium formation rate obtained from a parallel experiment with the solvent as a control to calculate the inhibitory effect of the test compound on conidia germination and appressorium formation.

[0223] Table 2 shows the biological data of some of the extracts of Fructus Caryophylli: "+" represents that the inhibitory effect at 200 mg / L is less than 40%, "++" represents that the inhibitory effect at 50 mg / L is greater than 40%, and "+++" represents that the inhibitory effect at 50 mg / L is greater than 80%.

[0224] Table 2

[0225]

[0226]

[0227] (III) Inhibitory effect of koji extract on conidia germination of Aspergillus oryzae

[0228] Experimental methods:

[0229] (1) Production of conidia of rice spores: The strain PJ52 of rice spores to be tested was inoculated onto potato dextrose medium (PDA) and cultured in a constant temperature and light incubator at 28°C. After 7-10 days, the pellets were punched and placed into potato sucrose liquid medium (PSB) and incubated at 28°C and 180 rpm for 7-10 days to produce a large number of conidia.

[0230] (2) Preparation of conidia suspension of Aspergillus oryzae: Filter the shake culture product with three layers of lens paper. The filtrate is the conidia suspension. Use a hemocytometer to adjust the conidia concentration in the conidia suspension to 2×10 5 pieces / mL.

[0231] (3) The test compound was added to the conidia suspension at different concentration gradients to prepare working concentrations of 200 mg / L and 50 mg / L. The solution was then spotted onto a hydrophobic glass slide. Four spots were placed on each slide and kept in the dark to maintain moisture. 12 h after inoculation, the conidia germination rate was observed and counted under a microscope.

[0232] (4) Statistics and Analysis: Count three inoculation points on each hydrophobic slide, and count the number of germinated conidia from the center of each inoculation point. Calculate the average of the three data sets to determine the conidia germination rate. Use the conidia germination rate obtained from a parallel experiment with the solvent as a control to calculate the inhibitory effect of the test compound on conidia germination.

[0233] Table 3 shows the biological data of some of the extracts of Fructus Caryophylli: "+" represents that the inhibitory effect at 200 mg / L is less than 40%, "++" represents that the inhibitory effect at 50 mg / L is greater than 40%, and "+++" represents that the inhibitory effect at 50 mg / L is greater than 80%.

[0234] Table 3

[0235]

[0236]

[0237] (IV) The preventive effect of cyperus rotundus extract on blast fungus infection of detached barley leaves

[0238] Experimental methods:

[0239] (1) Preparation of barley leaves: Take barley leaves with one leaf and one heart from Emai No. 9 and place them in an inoculation box for later use.

[0240] (2) The production of conidia of the test rice blast fungus strain P131 is the same as that of biological experiment (I).

[0241] (3) Preparation of Rice Blast Conidia Suspension: Elute the culture on the OTA with sterile water and filter through three layers of lens paper. The filtrate is the conidia suspension. Centrifuge at 5000 rpm for 5 min at room temperature. Suspend the precipitated conidia in gelatin and adjust the conidia concentration to 1 × 10 using a hemocytometer. 5 pieces / mL.

[0242] (4) Preparation of test compound solution: The mother solution of the test extract was added to the prepared rice blast fungus conidia solution and diluted to a working concentration of 200 mg / L and 50 mg / L.

[0243] (5) Leaf spotting: For the evaluation of preventive effect, 4 μL of the extract of R. oryzae was spot-inoculated on barley leaves in advance, 3 spots per leaf, for a total of 3 leaves; 12 hours later, 4 μL of the conidia solution of Pyricularia oryzae was spot-inoculated on the droplets. For the evaluation of therapeutic effect, 4 μL of the conidia solution of Pyricularia oryzae was spot-inoculated on barley leaves, 3 spots per leaf, for a total of 3 leaves; 12 hours later, 4 μL of the working solution of the extract of R. oryzae was spot-inoculated on the droplets. The leaves were cultured in the dark under moisturizing conditions for 24 hours, followed by normal culture. Five days after inoculation, the control effect of the test compound on the indoor infection of barley leaves by Pyricularia oryzae was evaluated.

[0244] (6) Statistics and Analysis: Three leaves were counted for each treatment, and three inoculation points were counted for each leaf. The lesion area at each inoculation point was calculated, and the average of the three data sets was calculated to obtain the lesion area. The lesion area obtained from the parallel experiment with the solvent was used as a control to calculate the inhibitory effect of the test compound on the infection of blast fungus in detached barley leaves.

[0245] Table 4 shows the biological data of some of the extracts of Fructus Caryophylli: "+" represents that the inhibitory effect at 200 mg / L is less than 40%, "++" represents that the inhibitory effect at 50 mg / L is greater than 40%, and "+++" represents that the inhibitory effect at 50 mg / L is greater than 80%.

[0246] Table 4

[0247]

[0248]

[0249] (V) The preventive effect of cyperus rotundus extract on rice blast fungus infection in detached rice leaves

[0250] Experimental methods:

[0251] (1) Preparation of rice leaves: Take the leaves with four leaves and one heart from the susceptible varieties CO39 and Xiangwanxian 11 and place them in an inoculation box for use.

[0252] (2) Production of conidia of the test rice blast fungus strain P131: Preparation of rice blast fungus conidia suspension, same as biological experiment (IV).

[0253] (3) Preparation of test compound solution: The mother solution of the test extract was added to the prepared rice blast fungus conidia solution and diluted to a working concentration of 200 mg / L and 50 mg / L.

[0254] (4) Leaf spotting: For the evaluation of preventive effect, 4 μL of the extract of R. oryzae was spot-inoculated on rice leaves in advance, 3 spots per leaf, for a total of 3 leaves; 24 hours later, 4 μL of the conidia solution of Pyricularia oryzae was spot-inoculated on the droplets. For the evaluation of therapeutic effect, 4 μL of the conidia solution of Pyricularia oryzae was spot-inoculated on rice leaves, 3 spots per leaf, for a total of 3 leaves; 24 hours later, 4 μL of the working solution of the extract of R. oryzae was spot-inoculated on the droplets. The leaves were cultured in the dark and moisturized for 24 hours, followed by normal culture. Seven days after inoculation, the control effect of the test compound on rice leaves infected by Pyricularia oryzae was evaluated.

[0255] (5) Statistics and Analysis: For each treatment, three leaves were counted, and three inoculation points were counted on each leaf. The lesion area at each inoculation point was calculated, and the average of the three data sets was calculated to obtain the lesion area. The lesion area obtained from the parallel experiment with the solvent was used as a control to calculate the inhibitory effect of the test compound on the infection of blast fungus in detached rice leaves.

[0256] Table 5 shows the biological data of some of the extracts of Fructus Caryophylli: "+" represents that the inhibitory effect at 200 mg / L is less than 40%, "++" represents that the inhibitory effect at 50 mg / L is greater than 40%, and "+++" represents that the inhibitory effect at 50 mg / L is greater than 80%.

[0257] Table 5

[0258]

[0259]

[0260] (VI) The preventive effect of the extract of Fructus Caryophylli on anthracnose infection of detached mango leaves

[0261] Experimental methods:

[0262] (1) Preparation of mango leaves: Take the young leaves of Guifei mango that have just turned green and place them in the inoculation box for use.

[0263] (2) The production of conidia of the mango Siamese anthracnose fungus strain SC2-1 is the same as that of biological experiment (II).

[0264] (3) Preparation of anthrax conidia suspension: Elute the culture on the OTA with sterile water and filter with three layers of lens paper. The filtrate is the conidia suspension. Use a hemocytometer to adjust the conidia concentration in the conidia suspension to 1×10 6 pieces / mL.

[0265] (4) Preparation of test compound solution: The test compound was added to the anthrax spore suspension to prepare solutions with working concentrations of 1200 mg / L and 600 mg / L.

[0266] (5) Mixed inoculation: 10 μL of drug-treated Colletotrichum conidia solution was applied to three mango leaves, two spots per leaf. The leaves were incubated in the dark for 24 h, followed by normal inoculation. Seven days after inoculation, the efficacy of the test extract against Colletotrichum infection of mango leaves was evaluated.

[0267] (6) Statistics and Analysis: For each treatment, three leaves were counted, and 1-2 inoculation points were counted on each leaf. The lesion area at each inoculation point was calculated, and the average of the three data sets was calculated to obtain the lesion area. The lesion area obtained from the parallel experiment with the solvent was used as a control to calculate the inhibitory effect of the test compound on anthracnose infection of mango leaves.

[0268] Table 6 shows the biological data of some of the extracts of Fructus Caryophylli: "+" represents that the inhibitory effect at 1200 mg / L is less than 40%, "++" represents that the inhibitory effect at 600 mg / L is greater than 40%, and "+++" represents that the inhibitory effect at 600 mg / L is greater than 80%.

[0269] Table 6

[0270]

[0271]

[0272] (VII) Preventive effect of cyperus rotundus extract on rice blast infection in potted rice

[0273] Experimental methods:

[0274] (1) Preparation of rice: Prepare susceptible varieties CO39 and Xiangwanxian 11, and place rice seedlings with four leaves and one heart in an inoculation box for use.

[0275] (2) The production of conidia of the test rice blast fungus strain P131 is the same as that of biological experiment (I).

[0276] (3) Preparation of Rice Blast Conidia Suspension: Elute the culture on the OTA with sterile water and filter through three layers of lens paper. The filtrate is the conidia suspension. Centrifuge at 5000 rpm for 5 min at room temperature. Suspend the precipitated conidia in gelatin and adjust the conidia concentration to 5 × 10 using a hemocytometer. 4 pieces / mL.

[0277] (4) Preparation of test compound solution: The mother solution of the test extract was added to the prepared rice blast fungus conidia solution and diluted to a working concentration of 200 mg / L.

[0278] (5) Spray inoculation: First, evenly spray 10 mL of the Fructus oryzae extract onto the rice plants. 24 hours later, spray 10 mL of the conidia of the blast fungus. After inoculation, incubate the plants in the dark and moisturize for 24 hours. Then, incubate them at 28°C as normal. Seven days after inoculation, evaluate the preventive effect of the Fructus oryzae extract against rice blast.

[0279] Table 7 shows the biological data of the Fructus Cynanchifolia extract of the present invention. The rice blast disease survey was conducted in accordance with the agricultural industry standard "Technical Procedures for Field Monitoring of Rice Blast Resistance" (NYT3685-2020). The specific standards are as follows: Level 0: The entire leaf is disease-free; Level 1: There are needle-tip-sized brown necrotic spots on the leaves; Level 2: There are larger (1-2 mm in diameter) brown necrotic spots on the leaves, but no typical lesions; Level 3: There are typical rice blast lesions, and the lesion area is <2%; Level 4: There are typical rice blast lesions, 2%≤lesion area<5%; Level 5: There are typical rice blast lesions, 5%≤lesion area<10%; Level 6: There are typical rice blast lesions, 10%≤lesion area<25%; Level 7: There are typical rice blast lesions, 25%≤lesion area<50%; Level 8: There are typical rice blast lesions, 50%≤lesion area<75%; Level 9: There are typical rice blast lesions, and the lesion area is ≥75%.

[0280] Table 7

[0281]

[0282]

[0283] (8) Preventive effect of cyperus rotundus extract on late blight infection of potted potatoes

[0284] Experimental methods:

[0285] (1) Preparation of potatoes: After normal cultivation, the potato variety "Desiree" susceptible to late blight is placed in an inoculation box for use.

[0286] (2) Preparation of zoosporangia of potato late blight pathogen: Select the medium-strong strain "MZ" and culture it on a suitable culture medium. After the zoosporangia are produced, wash them with sterile water and filter them with double-layer gauze to prepare a zoosporangium suspension. Place it at a low temperature of 4°C in the dark for 3 hours and store it for future use.

[0287] (3) Preparation of zoosporangium suspension: Prepare the zoosporangium suspension by adjusting the treated zoosporangium solution with 4°C sterile water to a concentration of 4×10 4 / mL of suspension.

[0288] (4) Preparation of spray working solution: Dilute the mother solution of the extract to be tested with water to a working concentration of 200 mg / L.

[0289] (5) Spray inoculation: Before inoculation, the plants to be treated were placed in an artificial climate greenhouse at 20°C for 4 hours to allow them to grow slowly. After the growth was complete, the working solution was evenly sprayed on both sides of the leaves of the treated plants. After 24 hours, the spore suspension was evenly sprayed. Two plants were treated for each treatment, and each plant was sprayed with 60 mL. After spraying the spore suspension, the plants were kept in the dark for 24 hours and then cultured under normal light (20°C, 18 hours of light / 6 hours of darkness). After 7 days, the preventive effect of the tested cyperus extract on potato late blight was evaluated.

[0290] Table 8 shows the biological data of the Fructus Caryophylli extract of the present invention. Potato late blight disease was investigated according to the grading standard, which is as follows: Grade 0: no lesions; Grade 1: lesion area accounts for less than 5% of the total leaf area; Grade 3: lesion area accounts for 5-10% of the total leaf area; Grade 5: lesion area accounts for 10.1-20% of the total leaf area; Grade 7: lesion area accounts for 20.1-50% of the total leaf area; Grade 9: lesion area accounts for more than 50% of the total leaf area.

[0291] Table 8

[0292]

[0293]

[0294] (IX) The preventive effect of cycad extract on gray mold infection of strawberry fruit

[0295] Experimental methods:

[0296] (1) Preparation of strawberry fruits: The fruits of the strawberry variety "Hongyan" are disinfected with alcohol and placed in an inoculation box for use.

[0297] (2) Preparation of conidia suspension of Botrytis cinerea: Elute the culture grown on PDA for 7 days with sterile water and filter it with three layers of lens paper. The filtrate is the conidia suspension. Use a hemocytometer to adjust the conidia concentration to 1×10 5 pieces / mL.

[0298] (3) Preparation of inoculation working solution: Add the mother solution of each extract of the tested Fructus Botrytis cinerea to the prepared conidia solution of Botrytis cinerea and dilute it to a working concentration of 1200 mg / L.

[0299] (4) Inoculation: A small hole 3 mm in diameter and 10 mm deep was made on the shoulder of a strawberry fruit. 6 μL of the working solution was injected into each treatment of 5 strawberries. The inoculated treatments were then incubated at 21°C under a 12 h / 12 ​​h light / dark cycle. The control efficacy of the test compound was evaluated 5 days after inoculation.

[0300] Table 9 shows the biological data of the Fructus sylvestris extract of the present invention. Strawberry gray mold disease survey was conducted according to the grading standard, which is as follows: Grade 0: disease-free; Grade 1: lesion area accounts for less than 5% of the fruit area; Grade 3: lesion area accounts for 5-10% of the fruit area; Grade 5: lesion area accounts for 10.1-25% of the fruit area; Grade 7: lesion area accounts for 25.1-50% of the fruit area; Grade 9: lesion area accounts for more than 50.1% of the fruit area.

[0301] Table 9

[0302] Extract number Disease occurrence level DF2-A1 7 DF2-A3 5 DF2-B1 7 DF2-B7 7 DF2-C3 5 DF2-C8 7 DF2-D1 5 DF2-P1 3 DF2-P2 3

[0303] (10) The preventive effect of Ficus microcarpa extract on anthrax infection in mango fruits

[0304] Experimental methods:

[0305] (1) Preparation of mango fruits: Wash the "Tainong" mango fruits of uniform size and maturity three times with sterile water, dry them and place them in an inoculated fresh-keeping box for use.

[0306] (2) Production of conidia of the mango Siamese anthrax strain SC2-1. Preparation of anthrax conidia suspension, same as in biological experiment (VI).

[0307] (3) Preparation of inoculation working solution: Add the mother solution of the cyperus rotundus extract to be tested into the prepared anthrax spore solution / sterile water and dilute to a working concentration of 1000 mg / L and 500 mg / L.

[0308] (4) Natural disease inoculation: soak the fruits in the corresponding concentration of drugs for 1 minute, place them in a fresh-keeping box, and incubate them at 28℃ for 7-10 days to observe the disease. Each treatment is 6 fruits, and the treatment is repeated 4 times. The control effect of the test compound is evaluated.

[0309] Table 10 shows the biological data of the Ficus microcarpa extract of the present invention. A disease survey of mango anthracnose was conducted according to the grading standard, which is as follows: Grade 0: disease-free; Grade 1: lesion area accounts for less than 5% of the fruit area; Grade 3: lesion area accounts for 5-10% of the fruit area; Grade 5: lesion area accounts for 10.1-25% of the fruit area; Grade 7: lesion area accounts for 25.1-50% of the fruit area; Grade 9: lesion area accounts for more than 50% of the fruit area.

[0310] Table 10

[0311] Extract number Disease occurrence level DF2-A1 7 DF2-A3 3 DF2-B1 5 DF2-B7 5 DF2-C3 3 DF2-C8 7 DF2-D1 3 DF2-P1 3 DF2-P2 3

[0312] (11) The preventive and control effect of radix dasyphylla extract on rice blast in the field

[0313] Experimental methods:

[0314] (1) Rice planting: The variety “Yanfeng 47” is planted in plots, with each plot covering 20 square meters.

[0315] (2) Application plan: Spray once at the heading stage and once at the full heading stage of rice, with a working solution concentration of 1200 mg / L and a spraying volume of 2.7 L per plot.

[0316] Table 11 shows the biological data of the cyperus rotundus extract of the present invention. Referring to the agricultural industry standard "Technical Procedure for Field Monitoring of Rice Blast Resistance" (NYT3685-2020), a rice blast disease survey was conducted 15 days after the second spraying of the cyperus rotundus extract. The specific standards are as follows: Level 0: All leaves are disease-free; Level 1: There are needle-sized brown necrotic spots on the leaves; Level 2: There are larger (diameter 1-2mm) brown necrotic spots on the leaves, but no typical lesions; Level 3: There are typical rice blast lesions, and the lesion area is less than 2%; Level 4: Typical rice blast lesions present, 2% ≤ lesion area < 5%; Level 5: Typical rice blast lesions present, 5% ≤ lesion area < 10%; Level 6: Typical rice blast lesions present, 10% ≤ lesion area < 25%; Level 7: Typical rice blast lesions present, 25% ≤ lesion area < 50%; Level 8: Typical rice blast lesions present, 50% ≤ lesion area < 75%; Level 9: Typical rice blast lesions present, lesion area ≥ 75%. The disease index is calculated based on the disease severity level.

[0317] Table 11

[0318]

[0319]

[0320] (12) The preventive and control effect of cyperus rotundus extract on rice false smut in the field

[0321] Experimental methods:

[0322] (1) Rice planting: The variety “Liaoxing No. 1” is planted in plots, with each plot being 60 square meters.

[0323] (2) Application plan: Spray once at the heading stage and once at the full heading stage of rice, with a working solution concentration of 1200 mg / L and a spraying volume of 2.7 L per plot.

[0324] Table 12 shows the biological data of the pyracantha extract of the present invention. Fifteen days after the second spraying of the extract, a survey of rice ear blast disease was conducted, using the following criteria: Grade 0: healthy rice ear, no blast-affected grains; Grade 1: one blast-affected grain in the ear; Grade 3: two blast-affected grains in the ear; Grade 5: three to five blast-affected grains in the ear; Grade 7: six to nine blast-affected grains in the ear; Grade 8: typical rice blast lesions present, with lesion area 50% or less and less than 75%; Grade 9: ten or more blast-affected grains in the ear. Disease indexes were calculated based on the disease grade.

[0325] Table 12

[0326] Extract number Disease occurrence level DF2-A3 1 DF2-B1 3 DF2-C8 5 DF2-D1 1 DF2-P1 1

[0327] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A preparation for preventing and treating plant diseases, characterized in that: The invention comprises a cyperus rotundus extract, wherein the cyperus rotundus extract has a cyperus oleic acid content of 10-95 wt %, preferably 20-95 wt %, further preferably 25-95 wt %, and even more preferably 40-95 wt %, based on the weight of the cyperus rotundus extract.

2. The preparation according to claim 1, characterized in that The cyperus rotundus extract is obtained by solvent extraction or supercritical carbon dioxide method; preferably, the cyperus rotundus extract is further obtained by mechanical stirring immersion or ultrasonic assisted extraction; more preferably, the cyperus rotundus extract is further obtained by petroleum ether-water extraction and column chromatography separation.

3. The preparation according to claim 2, characterized in that The solvent includes a protic solvent, an aprotic solvent, and a polar aprotic solvent. Preferably, the protic solvent is selected from one or more combinations of water, methanol, ethanol, and cyclohexanol. Preferably, the aprotic solvent is selected from one or more combinations of petroleum ether, n-hexane, n-pentane, dodecane, toluene, ether, dichloromethane, carbon tetrachloride, and 1,2-dichloroethane. Preferably, the polar aprotic solvent is selected from one or more combinations of chloroform, ethyl acetate, diethyl malonate, tetrahydrofuran, 1,4-dioxane, acetone, and acetonitrile. Further preferably, the solvent is selected from n-hexane, dichloromethane, carbon tetrachloride, chloroform, toluene, methanol, ethyl acetate, acetone, and ethanol.

4. The preparation according to claim 1, characterized in that The Fructus cyrtonema extract is obtained by extracting the seeds, roots, stems, leaves, bark, etc. of Fructus cyrtonema, preferably the seeds; preferably, the preparation is an emulsion.

5. A method for preparing the preparation according to claim 1, characterized in that: The preparation method of the Fructus Cynanchifolia extract comprises the following steps: 1) Grinding and crushing the seeds of the Herba Cynanchifoliae, soaking them in a solvent, and performing mechanical stirring or ultrasonic-assisted extraction to obtain an extract; 2) adding solvent to the residue, ultrasonic extraction, and then mechanical immersion extraction at room temperature to obtain an extract; 3) combining the two extracts and concentrating the extracts into an extract to obtain a cyperus rotundus extract; Preferably, in the step 1) of preparing the cyperus rotundus extract, the solvent is selected from one or more of water, methanol, ethanol, cyclohexanol, petroleum ether, n-hexane, n-pentane, dodecane, toluene, ether, dichloromethane, carbon tetrachloride, 1,2-dichloroethane, chloroform, ethyl acetate, diethyl malonate, tetrahydrofuran, 1,4-dioxane, acetone, and acetonitrile, and the ethanol concentration is 70-95%, preferably 95% ethanol; preferably, in the step 1) of preparing the cyperus rotundus extract, the cyperus rotundus seeds are ground into coarse powder, the ultrasonic extraction time in steps 1) and 2) is 0.5-2 h, preferably 1 h, and the mechanical soaking time is 1-5 d, preferably 2 d; Preferably, in the step 3) of preparing the Fructus Caryophylli extract, the extract is further extracted with petroleum ether (60-90° C.) and water for 3-5 times, preferably 3 times, the organic phases are combined, a large amount of solvent is removed by rotary evaporation, and the Fructus Caryophylli extract is separated by column chromatography; Preferably, the preparation step of the cyperus rotundus extract is 1) after ultrasonic extraction and mechanical stirring and soaking at room temperature, steam distillation is used for extraction for 2-6 hours, preferably 4 hours, the oil layer material is recovered, and water is removed by anhydrous sodium sulfate to obtain the cyperus rotundus extract.

6. A method for preparing a Fructus Cynanchifolia extract, characterized in that: The method is selected from the following methods: 1) comprising crushing the seeds of the radix sylvestris into coarse powder, soaking the powder in a solvent, and performing Soxhlet extraction to obtain an extract; further adding the solvent to the residue, performing Soxhlet extraction to obtain an extract; combining the two extracts, and concentrating the extracts into an extract to obtain the radix sylvestris extract; or 2) The method comprises grinding the seeds of the radix truncatum into coarse powder, adding the powder into a material bottle of a supercritical carbon dioxide device, setting the pressure to 10-30 MPa, preferably 20 MPa, the extraction temperature to 30-50° C., preferably 45° C., and the extraction time to 1-3 h, preferably 1.5 h, to obtain the radix truncatum extract.

7. Use of a cyperus rotundus extract in preparing a medicament for preventing and treating plant diseases, characterized in that: In the cyperus rotundus extract, based on the weight of the cyperus rotundus extract, the content of oleic acid is between 10-95wt%, preferably between 20-95wt%, further preferably between 25-95wt%, and further preferably between 40-95wt%. The plant diseases are caused by pathogenic fungi and oomycetes; preferably, the plant diseases caused by fungi and oomycetes are selected from rice blast, millet blast, mango anthracnose, banana anthracnose, pepper anthracnose, cucumber anthracnose, rubber anthracnose, litchi anthracnose, betel nut anthracnose. disease, potato late blight, tomato late blight, pepper late blight, rice false smut, rice sheath blight, strawberry gray mold, tomato gray mold, grape gray mold, sugarcane whip smut, ginseng rust, ginseng sclerotinia, etc.; preferably, the cyperus rot extract has the effect of inhibiting the conidia germination, germ tube growth or appressorium formation of fungi and oomycetes, and effectively prevents pathogens from infecting host plants; preferably, the cyperus rot extract has no inhibitory effect on the growth of vegetative hyphae of fungi and oomycetes, and the resistance risk of the cyperus rot extract for disease prevention and control is low.

8. A fungicidal composition for preventing and controlling agricultural, forestry and horticultural plant diseases, characterized in that: The invention comprises a compound of a cyperus rotundus extract and another botanical fungicide, wherein the content of cyperus rotundus oleic acid in the cyperus rotundus extract is 25-95%, preferably 40-95%; preferably, the active ingredient of the botanical fungicide is selected from one or more combinations of carvacrol, shiitake alcohol, phenylpropenone, physcion, matrine, pentadecene phenolic acid and tridecene phenolic acid, azadirachtin, sanguinarine sulfate, osthole, eugenol, catechin, berberine, allicin, and nicotine; preferably, the usage ratio of the cyperus rotundus extract to the other botanical fungicide is 1: 10-10:1; preferably, the fungicidal composition can be used for preventing and controlling plant diseases caused by pathogenic fungi and oomycetes; preferably, the plant diseases caused by fungi and oomycetes are selected from the following rice blast, millet blast, mango anthracnose, banana anthracnose, pepper anthracnose, cucumber anthracnose, rubber anthracnose, litchi anthracnose, betel nut anthracnose, potato late blight, tomato late blight, pepper late blight, rice false smut, rice sheath blight, strawberry gray mold, tomato gray mold, grape gray mold, sugarcane whip smut, ginseng rust, ginseng sclerotinia, etc.

9. A use of oleic acid from hyoscyamine in the preparation of a medicament for preventing and treating plant diseases caused by pathogenic fungi and oomycetes, characterized in that: The plant diseases caused by fungi and oomycetes are selected from the following rice blast, millet blast, mango anthracnose, banana anthracnose, pepper anthracnose, cucumber anthracnose, rubber anthracnose, litchi anthracnose, betel nut anthracnose, potato late blight, tomato late blight, pepper late blight, rice false smut, rice sheath blight, strawberry gray mold, tomato gray mold, grape gray mold, sugarcane whip smut, ginseng rust rot, ginseng sclerotinia rot, etc.

10. A method for preparing oleic acid of schizonepeta tenuifolia, characterized in that: The method comprises the following steps: obtaining a pure product of 2-cyclopentene-1-acetic acid through eight-step chemical synthesis and one-step chiral resolution.

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