CN110551097B - Monoacetylene compound and preparation method and application thereof - Google Patents

Monoacetylene compound and preparation method and application thereof Download PDF

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CN110551097B
CN110551097B CN201910068406.6A CN201910068406A CN110551097B CN 110551097 B CN110551097 B CN 110551097B CN 201910068406 A CN201910068406 A CN 201910068406A CN 110551097 B CN110551097 B CN 110551097B
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acetone
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武海波
石莎
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Minzu University of China
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    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
    • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
    • A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
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Abstract

The invention relates to a monoacid acetylene compound, a preparation method thereof and application of the monoacid acetylene compound in pesticides, in particular to application in preventing and treating root-knot nematodes and soil-borne pathogenic fungi. The compound has a structure represented by formula (1):

Description

Monoacetylene compound and preparation method and application thereof
Technical Field
The invention relates to a monoacid acetylene compound, a preparation method thereof and application of the monoacid acetylene compound in pesticides, in particular to application in preventing and treating root-knot nematodes and soil-borne pathogenic fungi.
Background
In recent 20 years, the development of protected areas and the cultivation of high-value-added crops greatly increase the income of farmers and realize the conversion from traditional agriculture to modern agriculture. With the development of protected areas and the successive year cultivation of crops with high added values, plant pathogens and insect eggs in soil are accumulated, destructive soil-borne diseases such as root-knot nematode disease, blight, root rot, verticillium wilt, bacterial wilt and the like occur in successive years, the disease is aggravated year by year, the yield and the quality of the crops are seriously influenced after the crops are usually planted for 3 to 5 years, the yield is generally reduced by 20 to 40 percent, the serious yield is reduced by more than 60 percent, and even the crops are completely harvested. Therefore, the root knot nematode disease and the soil-borne mycosis become prominent problems which seriously harm the production of facility vegetables, influence the income increase of farmers and restrict the sustainable development of vegetables and other important economic crops in China. At present, the prevention and treatment of soil-borne diseases mainly depends on the long-term use of a large amount of various chemically synthesized pesticides, and the pesticides generally have high toxicity and long residual period and are easy to pollute the environment and underground water sources, so that the resistance of pests is evolved, and certain negative effects are also exerted on human health and environment. The inventor researches and discovers in Shandong that local farmers use national forbidden medicaments (such as phorate, carbofuran and the like) to prevent and treat root-knot nematodes and soil-borne diseases due to no effective control method. Therefore, research and development of novel pesticides with high efficiency, low toxicity and low residue are particularly urgent.
The plant is a natural treasure house of bioactive substances, not only provides a powerful weapon for human to fight against diseases, but also provides green low-toxicity substances for the development of Chinese botanical pesticides. Artemisia absinthium L is a plant of Artemisia (Artemisia) belonging to Compositae (Compositae). It is recorded in Xinjiang Chinese herbal medicine that it has the functions of clearing heat, drying dampness, killing parasites and relieving itching, and is commonly used for joint swelling and pain, eczema and pruritus, furuncle, sore and toxin, ascariasis and inappetence. In addition, wormwood is also a main raw material for preparing wormwood wine.
According to the reports of the prior art, the composition of the extract of wormwood leaves is very complex and different active ingredients are often extracted due to different extraction methods.
The major paper, "preliminary study of chemical composition and living activity of the vitamin wormwood" (major paper of Xinjiang university, una guo-touti, 2012) discloses a method for extracting volatile oil of wormwood: 1) firstly, extracting wormwood volatile oil by adopting a steam distillation method and an organic solvent extraction method, optimizing an extraction process by adopting a single factor and orthogonal experiment method, and analyzing chemical components of a prepared sample by utilizing a GC-MS method; the volatile oil extracted by steam distillation contains trans-chrysanthemumyl acetate, palmitic acid, -curcumene, caryophyllin, menthol, etc.; the volatile oil extracted with organic solvent contains more components such as dibutyl phthalate, octadeca-9, 12, 15-trienoic acid, tetratetradecane, ascorbyl dipalmitate, n-nonacosane, trans-chrysanthemumate acetate, 1, 3-glyceryl dioleate, etc.; 2. the ethanol reflux extraction method is adopted to preliminarily discuss the extraction process of the wormwood flavonoid compound; the results of single-factor and orthogonal experiments show that the optimal extraction process of the wormwood flavonoid compound comprises the following steps: the ethanol concentration is 80%, the extraction time is 5h, the extraction temperature is 80 ℃, the feed-liquid ratio is 1:25g/mL, and the content of total flavonoids is 0.5625% under the conditions; 3. extracting and separating the chemical components of Artemisia absinthium L by common extraction method and column chromatography separation technique to obtain 6 compounds, and determining the structure of the separated chemical components by using spectral techniques such as IR, 1H NMR, 13C NMR, ESI-MS, etc. The compounds are respectively absinthin lignan, quercetin, isorhamnetin-3-O-beta-D-glucoside, quercetin-3-O-beta-D-rutinoside and beta-sitosterol; 4. through hydroxyl free radical scavenging capacity determination method, DPPH scavenging capacity determination method and reducing capacity determination method, vitamin C is used as standard substance, and objective evaluation is carried out on the in vitro antioxidant activity of wormwood flavone and wormwood volatile oil crude extract; the results show that in the method for measuring the scavenging capacity of the hydroxyl free radical, the antioxidant activity is as follows: total flavone, vitamin C, crude volatile oil 2, crude volatile oil 1; the antioxidant activity of each sample in the DPPH clearance assay was weak; in the reduction capacity measurement method, the antioxidant capacity is in the order of magnitude: vitamin C, total flavone, crude volatile oil 1 and crude volatile oil 2. The in vitro antioxidant measurement result of Artemisia absinthium L shows that the flavonoid compound and volatile oil in Artemisia absinthium L have certain antioxidant effect.
In addition, the paper "extraction process of Artemisia absinthium volatile oil and chemical composition analysis" (alnus Tuliphi, Abirajiang Keyimu; chemical industry institute of Xinjiang university, 2012) discloses: from GC-MS analysis of the chemical components of Artemisia absinthium L volatile oil, it is known that the volatile oil obtained by steam distillation mainly comprises chrysanthemumene acetate, caryophyllin, palmitic acid, alpha-curcumene, menthol, 2,4, 6-trimethyl-bicyclo [4.1.0] -3-heptenylformaldehyde and the like, and the volatile oil obtained by petroleum ether extraction mainly comprises chrysanthemumene acetate, palmitic acid, octadeca-9, 12, 15-trienoic acid, n-tetradecane, n-hexadecane and the like.
The paper "experimental study of the antibacterial effect of wormwood extract" (compiled by the pharmacy management division academic annual meeting paper of the chinese medical society of 2013, liszard, li juan, wu yanchun; the university of medical, xinjiang, affiliated department of medical science, hospital for traditional Chinese medicine, guangxi institute of traditional Chinese medicine) discloses the following: decocting folium Artemisiae Argyi (leaf) with water, extracting with 70 ethanol, and reflux-extracting in water bath at 90 deg.C for 2 hr to obtain extract. The extract has inhibitory effect on certain bacteria (such as Escherichia coli, Staphylococcus albus, Bacillus subtilis, Staphylococcus aureus, and Bacillus cereus). The test proves that the wormwood water extract and the wormwood alcohol extract have the function of inhibiting bacteria in vitro.
At present, active ingredients and activity of the leaf extract of the wormwood are researched more, the wormwood is only recognized to be medical and medical hygienic activity, and no research report is found on the extract of the root of the wormwood, and no research report is found on the aspect of controlling the southern root-knot nematode and soil-borne pathogenic fungi by the monoene compound disclosed by the patent.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a monoalkyne compound, a preparation method thereof and application thereof in preventing and treating root-knot nematodes and soil-borne pathogenic fungi.
The inventors of the present application found that the extract obtained from the root of wormwood contains different components due to the difference in the extraction method. The inventor of the application finds that two specific novel monoalkenyl compounds can be extracted by adopting the extraction method disclosed by the invention, and the two compounds have good control effects on specific meloidogyne incognita and soil-borne pathogenic fungi.
According to a first aspect of the present invention, there is provided a mono-acetylenic compound having the structure shown in formula (1):
Figure BDA0001956478970000031
wherein, R is respectively selected from-C (═ O) CH3and-C (═ O) CH2CH(CH3)2。
The monoacid compounds of the invention include one or both of the following compound 1 and compound 2:
Figure BDA0001956478970000032
wherein R is selected from-C (═ O) CH3Is compound 1;
wherein R is selected from-C (═ O) CH2CH(CH3)2Is compound 2.
The inventor finds that the compound with the structure shown in the formula (1) has the advantages of better lipid solubility, permeability and the like.
According to a second aspect of the present invention, there is provided a process for the preparation of a compound as described above, comprising the steps of:
1) supercritical CO2Extracting the root of Artemisia Absinihium L to obtain supercritical CO of the root of Artemisia Absinihium L2Extracting the extract;
2) dispersing the extract in water, and sequentially extracting with petroleum ether, dichloromethane and acetone to obtain petroleum ether extract, dichloromethane extract, acetone extract and water phase extract;
3) taking the dichloromethane extract in the step 2), and carrying out gradient elution by silica gel column chromatography by using petroleum ether-acetone with the volume ratio of 15:1, 10:1, 8:1, 5:1, 3:1 and 1:1 as eluent in sequence to obtain 6 fractions which are F1-F6 in sequence;
4) taking the F2 part in the step 3), adopting silica gel column chromatography, eluting by using normal hexane-chloroform with the volume ratio of 5:1 as an eluent, detecting by using thin layer chromatography, developing, and combining elution parts with the same Rf value, wherein Rf is 0.61 and is combined into an F2-1 part, Rf is 0.48 and is combined into an F2-2 part, and Rf is 0.26 and is combined into an F2-3 part;
5) collecting F2-2 part in 4), performing Sephadex LH-20 (hydroxypropyl dextran gel) chromatography, eluting with 1:1 chloroform-methanol to obtain compound, detecting with thin layer, developing, and determining compound 1(Rf ═ 0.46) and compound 2(Rf ═ 0.39) according to Rf value.
The thin-layer chromatography color development condition of the invention is as follows: observing dark spots under an ultraviolet lamp (254nm), spraying 10% ethanol sulfate, and baking at 105 deg.C until color development.
In step 1), the solvent used for extraction is preferably ethanol.
Further, step 1) comprises pulverizing the root of Artemisia absinthium into 10-100 mesh (e.g. 20 mesh), adding into extraction kettle, extracting at 20-40MPa, preferably about 30MPa, at 30-60 deg.C, preferably about 40 deg.C, and supercritical CO2The supercritical CO is obtained at a flow rate of 10-40L, L, preferably about 20L/h, and an extraction time of 30-180min, such as 60min2And (3) extracting.
According to a third aspect of the present invention, there is also provided a pesticidal formulation comprising (a) the above-mentioned monoalkyne compound or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, and (B) an agriculturally acceptable adjuvant. In the pharmaceutical preparation, the mass percent of the component (A) is 10-25%, and the mass percent of the component (B) is 75-90%.
The mono-acetylenic compounds of the present invention include any of their crystalline forms or their stereoisomers.
In the specific application process, the monoacid acetylene compound and inorganic acid or organic acid can form physiologically acceptable salt, so that the structure of the compound is more stable, the compound is favorable for storing and preparing thread-killing and sterilizing products, and the activity of killing nematodes and inhibiting the growth of plant pathogenic fungi is exerted.
When the above-mentioned monoacetylene compounds or their salt compounds are used as agricultural nematocides or fungicides, they can be used as they are or in the form of agricultural chemical compositions.
The agriculturally and pharmaceutically acceptable auxiliary material of the present invention refers to a material contained in a dosage form in addition to an active ingredient, for example, in the case of wettable powder, it may be selected from one or more of sodium dodecyl sulfate, CMC sodium dodecyl sulfate, kaolin, attapulgite clay, a dispersant, and the like.
In one embodiment, as a wettable powder, it comprises: 15-25 wt% of compound powder, 15-25 wt% of white carbon black, 2-8 wt% of sodium dodecyl sulfate, 0.5-2 wt% of CMC sodium dodecyl sulfate, 1-6 wt% of nekal and 46-56 wt% of attapulgite.
The compounds of the present invention may also be formulated in any other suitable pesticide dosage form, including but not limited to any one of solutions, suspensions, emulsions, and the like, according to conventional methods.
According to a fourth aspect of the present invention, the present invention provides an application of the compound represented by the above formula (1), or a crystal form thereof, or a stereoisomer thereof, or an agriculturally and pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof in preparing pesticides, especially for controlling root-knot nematodes and soil-borne pathogenic fungi.
The monoalkyne compound can be applied to the preparation of a nematicide. The research of the inventor shows that the compound has stronger insecticidal action on the meloidogyne incognita, the thread killing capability of the compound is found to be superior to that of the existing medicament abamectin sold on the market, the compound is proved to have better application prospect in the direction of killing the nematodes as a plant source pesticide, and the compound can be further prepared into a thread killing agent for preventing and treating the meloidogyne incognita.
The mono-acetylene compounds can be applied to the preparation of bactericides. The research of the inventor shows that the monoacetylene compound has obvious bacteriostatic activity on various plant soil-borne pathogenic fungi, particularly phytophthora pathogenic fungi, and the bacteriostatic ability of the monoacetylene compound is found to be stronger than that of the prior chemical medicament carbendazim on the market, so that the compound has wide bacteriostatic spectrum and obvious bacteriostatic activity, has better application prospect in the direction of inhibiting the plant pathogenic fungi as a plant source pesticide, and can be further prepared into a bactericide for preventing and treating the plant pathogenic fungi.
In the application process, the monoacid compounds can be prepared into a proper dosage form or added with one or more auxiliary materials which are conventional in the field to prepare a pharmaceutical composition for application according to the actual use requirement.
The test result shows that the compound has better effects of killing nematodes and inhibiting plant soil-borne pathogenic fungi, and provides a new choice for screening and/or preparing nematocides and bactericides in agriculture.
Obviously, according to the above-mentioned contents of the present invention, many other modifications, substitutions or changes can be made without departing from the basic technical idea of the present invention, and the modifications, substitutions or changes are also within the protection scope of the present invention.
Description of the advantages and effects of the invention
The invention has the beneficial effects that: the compound shown in the formula (1) can be obtained by extracting and separating dry roots of medicinal plants recorded in Xinjiang Chinese herbal medicine and a main raw material plant Artemisia absinthium L for brewing Artemisia absinthin wine, and is proved to be safe to human bodies; the compound shown in the formula (1) is derived from natural plants, is easy to degrade in the environment, has the characteristics of low toxicity and low residue, is safe to non-target organisms and good in environmental compatibility, and meets the requirements of green plant protection and sustainable agricultural production and development; the compound shown in the formula (1) has strong killing activity on the second-instar larvae of Meloidogyne incognita, the thread killing capability is superior to that of commercial thread killing agent abamectin, and the single alkyne compound with excellent thread killing activity can be used as a main new pesticide component or a lead compound and is suitable for controlling parasitic nematodes of agricultural plants; the compound shown in the formula (1) has stronger bacteriostatic activity on various plant soil-borne pathogenic fungi, has stronger bacteriostatic ability than a commercially available bactericide carbendazim, and can be used as a main new pesticide component or a lead compound for controlling the plant pathogenic fungi; the compound shown in the formula (1) has good comprehensive control effect on plant soil-borne pathogens (root-knot nematodes and soil-borne pathogenic bacteria), and contributes to green development of protected areas in China and sustainable cultivation of crops with high added value by reducing the frequency and the dosage of the respective use of the conventional chemical pesticide nematocide and bactericide.
Drawings
FIG. 1 is a HRESIMS plot of Compound 1 prepared in example 1.
FIG. 2 is a drawing of Compound 1 prepared in example 11HNMR spectrogram.
FIG. 3 is a drawing of Compound 1 prepared in example 113C NMR spectrum.
Fig. 4 is a HMBC spectrum of compound 1 prepared in example 1.
FIG. 5 is a HRESIMS plot of Compound 2 prepared in example 1.
FIG. 6 is a drawing of Compound 2 prepared in example 11H NMR spectrum.
FIG. 7 is a drawing of Compound 2 prepared in example 113C NMR spectrum.
Fig. 8 is a HMBC spectrum of compound 2 prepared in example 1.
Detailed Description
Example 1: preparation of monoalkyne compounds
1. Experimental materials:
1) medicinal materials
The root of Artemisia absinthium L.was collected from Xinjiang province in 2016, and was identified as dry root of Artemisia absinthium L.of Artemisia of Compositae by the inventor of the present invention, Wuhaibo.
2) Reagents and fillers
Thin layer chromatography silica gel GF254 (chemical purity) purchased from Qingdao ocean silica gel desiccant factory; column chromatography silica gel of 200-300 mesh (reagent grade) purchased from Qingdao ocean silica gel desiccant factory; a GF254 silica gel preparation thin layer purchased from Yangtze river friend silica gel development Co., Ltd; sephadex LH-20 sepharose, available from Amersham, Sweden; analytically pure reagents such as petroleum ether, n-hexane, trichloromethane, ethyl acetate, acetone, methanol and the like are purchased from Beijing chemical plants.
3) Laboratory apparatus
Bruker-AVIIIHD-600 NMR (Bruker, Switzerland); BP211D one tenth ten million electronic balance (Sartorius, switzerland); nicolet 5700 Infrared Spectroscopy (Thermo corporation, USA); model DZG-6050 vacuum drying oven (shanghai semen); r-210 Rotary evaporator (BUCHI, Switzerland).
2. Separation and purification of components:
1) pulverizing 500g of common wormwood root into 20 meshes, adding into an extraction kettle, extracting at 30MPa and 40 ℃ under supercritical CO2The flow rate is 20L/h, the extraction time is 60min, and the supercritical CO is obtained2Extracting the extract;
2) supercritical CO2Dispersing the extract in water, and sequentially extracting with petroleum ether, dichloromethane and acetone to obtain petroleum ether extract, dichloromethane extract, acetone extract and water phase extract;
3) taking the dichloromethane extract obtained in the step 2), performing gradient elution by silica gel column chromatography sequentially by using petroleum ether-acetone as an eluent with the volume ratio of 15:1, 10:1, 8:1, 5:1, 3:1 and 1:1, detecting by thin-layer chromatography, developing, combining the same elution parts to obtain 6 fractions which are F1-F6 sequentially; wherein F1 is a petroleum ether-acetone volume ratio 15:1 fraction, and F2 is a petroleum ether-acetone volume ratio 10:1 fraction; f3 is petroleum ether-acetone volume ratio 8:1 fraction; f4 is petroleum ether-acetone volume ratio 5:1 fraction; f5 is petroleum ether-acetone volume ratio 3:1 fraction; f6 is petroleum ether-acetone volume ratio of 1:1 fraction, total 6 fractions.
4) Taking the F2 part in the step 3), adopting silica gel column chromatography, eluting by using normal hexane-chloroform with the volume ratio of 5:1 as an eluent, detecting by thin layer chromatography, developing, and combining elution parts with the same Rf value, wherein Rf is 0.61 and is combined into an F2-1 part, Rf is 0.48 and is combined into an F2-2 part, and Rf is 0.26 and is combined into an F2-3 part;
5) taking the F2-2 part obtained in the step 4), carrying out Sephadex LH-20 (hydroxypropyl Sephadex) and eluting with 1:1 chloroform-methanol to obtain a compound, carrying out thin layer detection and color development on the compound, and determining that 11.5mg of the compound 1(Rf & lten & gt 0.46) and 3.6mg of the compound 2(Rf & lten & gt 0.39) according to Rf values;
6) the thin-layer chromatography color development condition of the invention is as follows: observing dark spots under an ultraviolet lamp (254nm), spraying 10% ethanol sulfate, and baking at 105 deg.C until color development.
3. Compound identification:
after separating to obtain each compound, identifying its physicochemical properties and molecular structure to obtain compound 1 and compound 2 as new compounds, the molecular structure of compound 1-2 is shown as formula (2), and its specific physicochemical properties and spectral data are shown in table 1 below:
TABLE 1 Nuclear magnetic data for Compounds 1 and 2
Figure BDA0001956478970000071
Compound 1: a yellow oil;
Figure BDA0001956478970000072
HRESIMS m/z,299.0176[M+Na]+,calcd for C14H12NaO2S2299.0175; HRESIMS pattern of Compound 1,1H NMR chart and13the C NMR charts are shown in FIGS. 1 to 3, respectively.
Compound 2: a yellow oil;
Figure BDA0001956478970000073
HRESIMS m/z,341.0640[M+Na]+,calcd for C17H18NaO2S2341.0646; HRESIMS pattern of Compound 2,1H NMR chart and13the C NMR charts are shown in FIGS. 5 to 7, respectively.
Example 2: preparation of pesticidal formulations
20% of powder of the compound 1 obtained in example 1, 20% of white carbon black, 5% of sodium dodecyl sulfate, 1% of CMC sodium dodecyl sulfate, 3% of nekal and the balance of attapulgite, wherein the% are based on the total weight of the pesticide preparation. Mixing the above materials uniformly, controlling processing temperature at 0-50 deg.C, pulverizing in jet mill or other high mesh pulverizer to more than 150 mesh, controlling water content at 6-8% (by mass), and controlling pH at 7-7.5. So as to prepare 20 percent of wettable powder of the compound shown in the formula (1). The water dispersivity, the diluent stability and the cold and hot storage stability of the preparation meet the requirements of commercial pesticide preparations.
Example 3: preparation of pesticidal formulations
20% of compound 2 powder obtained in example 1, 20% of white carbon black, 5% of sodium dodecyl sulfate, 1% of CMC sodium dodecyl sulfate, 3% of nekal, and the balance of attapulgite. Mixing the above materials uniformly, controlling processing temperature at 0-50 deg.C, pulverizing in jet mill or other high mesh pulverizer to more than 150 mesh, controlling water content at 6-8% (by mass), and controlling pH at 7-7.5. So as to prepare 20 percent of wettable powder of the compound shown in the formula (1). The water dispersivity, the diluent stability and the cold and hot storage stability of the preparation meet the requirements of commercial pesticide preparations.
Test 1: virulence determination for second instar larvae of meloidogyne incognita
1) Sample preparation:
the compound to be tested and the commercial nematocide abamectin are dissolved by acetone to prepare the solution with the concentration of 104mg/L of mother liquor. And (3) diluting the compound to be detected by using a 0.1% (V/V) Tween-80 aqueous solution to ensure that the content of the organic solvent in the final compound to be detected is not more than 1% (V/V).
2) Preparing second-instar larvae of Meloidogyne incognita:
cucumber roots infected by meloidogyne incognita (M.incognitia) are collected, tomato roots are washed lightly with sterilized distilled water, egg masses are gently peeled off by tweezers under a stereoscopic microscope, the egg masses are placed in a culture dish containing a small amount of distilled water, and the culture dish is incubated for 3-5 days at 28 ℃. The hatched 2-year-old root-knot nematodes can be used as target organisms for bioassays.
3) And (3) biological activity determination:
collecting 96-well biochemical test plate, collecting 100 μ L nematode suspension, keeping the number of second-instar nematode in each well 80-100, recording the number of live nematode, slowly adding 100 μ L test solution along the edge of the culture plate to avoid adverse effect on the nematode, and covering the plate to prevent water evaporationAnd (4) sending. The 96-well plate was placed in an incubator at 28 ℃ in the dark. Each treatment was repeated 4 times. Survival and mortality of second instar larvae of Meloidogyne incognita were checked 24 hours after treatment, and mortality and corrected mortality were calculated. And calculating LC of the medicament by SAS software50. Mortality and corrected mortality were calculated as follows:
Figure BDA0001956478970000081
Figure BDA0001956478970000082
4) and (4) the result of bioassay:
TABLE 2 nematicidal virulence (LC) of two monoalkenyl compounds against Meloidogyne incognita50，mg/L)
Figure BDA0001956478970000083
LC of abamectin as positive control for root-knot nematode50LC of 9.47mg/L, novel Compounds 1 and 2502.69 and 4.17mg/L, respectively. From the above experimental data, it can be seen that the monoacetylene compound obtained by the invention has excellent nematicidal activity.
Test 2: determination of bacteriostatic activity against plant soil-borne pathogenic fungi
1) Sample preparation:
the compounds 1 and 2 and carbendazim are prepared by DMSO, and the concentration of the mother liquor is 51200mg/L
2) Preparing a bacterial liquid:
fusarium oxysporum (Fusarium oxysporum), Fusarium solani (Fusarium solani), Phytophthora infestans (Phytophthora infestans) and Phytophthora capsici (Phytophthora capsicii) were cultured in PDA medium at 28 ℃ for 7 days, rinsed with sterile water to obtain spore suspension, observed under a microscope, and diluted to 1 × 106CFU/mL, spare.
3) Biological activity assay (MIC method):
the method is carried out by using a 96-well micropore dilution plate by adopting a microdilution method. And adding 10 mu L of liquid medicine into 1-10 holes of each row of 12 holes in each row, diluting the liquid medicine into a series of liquid medicines by using 90 mu L of bacteria-carrying potato glucose culture solution, adding 100 mu L of bacteria-carrying potato glucose culture solution into the 11 th hole as a blank control, and adding 10 mu L of DMSO and 90 mu L of bacteria-carrying potato glucose culture solution into the 12 th hole as a solvent control. The concentration ratio of the liquid medicine in the 1 st to 10 th holes is decreased progressively. After the completion of the operation, the mixture was shaken and mixed, and then cultured at 28 ℃ for 48 hours, taken out, and the result was observed. The lowest concentration of drug contained in the sterile growth well was the MIC of the sample.
4) And (4) the result of bioassay:
TABLE 3 MIC (mg/L) of two monoalkenyl Compounds against four plant soil-borne pathogenic fungi
Figure BDA0001956478970000091
The experimental result of the activity of inhibiting the plant soil-borne fungi proves that the bacteriostatic activity of the compound 1 and the compound 2 to the phytophthora infestans and the phytophthora capsici is obviously superior to that of the existing commercially available bactericide carbendazim.
The compound of the formula (1) obtained by separation is extracted from a medicinal and edible dual-purpose plant Artemisia absinthium L, is easy to decompose under natural conditions, does not cause a biological enrichment phenomenon, is safe to people and livestock, and can be used as a natural pesticide for a long time. The root-knot nematode and the phytophthora commonly infect the same crop, such as cucumber, pepper and the like, and the compound of the formula (1) obtained by the invention not only can reduce the amount and reduce the application of chemical pesticides, but also can avoid the negative influence caused by the application of the chemical pesticides, and is beneficial to the sustainable agricultural development of China.

Claims (6)

1. A mono-acetylenic compound having the structure shown in formula (1):
Figure FDA0002575098700000011
wherein R is selected from-C (═ O) CH3and-C (═ O) CH2CH(CH3)2。
2. A process for the preparation of a compound according to claim 1, comprising the steps of:
1) supercritical CO2Extracting the root of Artemisia Absinihium L to obtain supercritical CO of the root of Artemisia Absinihium L2Extracting the extract;
2) supercritical CO2Dispersing the extract in water, and sequentially extracting with petroleum ether, dichloromethane and acetone to obtain petroleum ether extract, dichloromethane extract, acetone extract and water phase extract;
3) taking the dichloromethane extract in the step 2), and carrying out gradient elution by silica gel column chromatography by using petroleum ether-acetone with the volume ratio of 15:1, 10:1, 8:1, 5:1, 3:1 and 1:1 as eluent in sequence to obtain 6 fractions which are F1-F6 in sequence;
4) taking the F2 part in the step 3), adopting silica gel column chromatography, eluting by using normal hexane-chloroform with the volume ratio of 5:1 as an eluent, detecting by using thin layer chromatography, developing, and combining elution parts with the same Rf value, wherein Rf is 0.61 and is combined into an F2-1 part, Rf is 0.48 and is combined into an F2-2 part, and Rf is 0.26 and is combined into an F2-3 part;
5) taking the F2-2 part in the step 4), carrying out Sephadex LH-20 hydroxypropyl dextran gel chromatography, eluting with 1:1 chloroform-methanol to obtain a compound, detecting and developing by thin layer chromatography, and determining a compound 1 with Rf being 0.46 and a compound 2 with Rf being 0.39 according to Rf values:
Figure FDA0002575098700000012
3. a pesticide formulation comprising component a: a mono-acetylenic compound according to claim 1 or a pharmaceutically acceptable salt thereof and component B: an agriculturally and pharmaceutically acceptable auxiliary material.
4. The pesticide preparation according to claim 3, wherein the mass percent of the component A is 10-25%, and the mass percent of the component B is 75-90%.
5. Use of the mono-acetylenic compound according to claim 1 or a stereoisomer thereof or an agriculturally pharmaceutically acceptable salt thereof for controlling root-knot nematodes.
6. Use of the monoalkyne compound according to claim 1, or a stereoisomer thereof, or an agriculturally pharmaceutically acceptable salt thereof, for controlling soil-borne pathogenic fungi.
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