A pyrone compound, a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a pyrone compound, a preparation method and application thereof. The novel pyrone compound has a structure of formula 1, is separated into secondary metabolites of fungi Curvularia sp. ZYX-Z-4, and is found to have herbicidal effect through biological activity test evaluation. The pyrone compound has the advantages of low toxicity, high selectivity, target specificity and ecological friendliness when applied to herbicides. The herbicidal effect of the compound is evaluated by using model plants Arabidopsis thaliana. The results show that the pyrone compound has good inhibitory effect on the growth of Arabidopsis thaliana roots, can cause damage to root cytoplasmic membrane, and can cause root cell death. The pyrone compound can significantly inhibit the root length and fresh weight of three weeds, and further provides relevant theories and experimental basis for the development of biological herbicides.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a pyrone compound, a preparation method and application thereof. BACKGROUND

[0002] With the rapid growth of global population, human food consumption and demand has greatly increased. Therefore, it is urgent to expand crop production. In agricultural production, the growth of weeds often causes serious threat to the yield and quality of crops. Weeds have always affected the growth and development of crops by competing for light, space, nutrients and water, leading to a serious decline in global crop yield. In order to effectively control weeds, herbicides are widely used. Herbicides play a key role in weed management by improving crop quality and yield, reducing labor costs. However, the existing herbicides have exposed a series of significant shortcomings while playing a role in weeding. Large-scale and long-term use of synthetic herbicides has caused a series of problems, such as (1) pesticide residues, many traditional herbicides will leave chemical components in the soil, crops and surrounding environment after use. These residual pesticides can be transmitted through the food chain, potentially harming human health; (2) environmental pollution, large-scale use and improper discharge of herbicides can pollute surface water and groundwater, causing damage to aquatic organisms and the entire aquatic ecosystem. In addition, herbicide fly ash in the air can also have a negative impact on air quality; (3) evolution of herbicide resistance, due to long-term single or excessive use of a certain type of herbicide, some individuals in the weed population gradually adapt and develop resistance genes, which makes the originally effective herbicide gradually lose its effect. Farmers have to increase the dosage or use more potent herbicides, further exacerbating the problems of pesticide residues and environmental pollution.

[0003] Therefore, based on the above problems, it is urgent to develop more environmentally friendly, efficient and sustainable weeding technologies and products. Natural products have broad application prospects in the fields of pharmacy and pesticides, and the development of natural product-derived herbicides has become an important direction for the creation of green pesticides. Marine fungi have the characteristics of complex genetic information (high innovation index) and strong metabolic potential, and can produce secondary metabolites with unique structure, rich diversity and significant biological activity, which are valuable resources of lead molecules. Mining active secondary metabolites from marine fungi to create new herbicides is of great significance to weed control and agricultural development. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to provide a pyrone compound, a preparation method and application thereof. The present application provides a pyrone compound isolated from natural products, which has the advantages of low toxicity, high selectivity, target specificity and ecological friendliness when applied to herbicides.

[0005] The present application provides a pyrone compound having the structure of formula (1):

[0006] Formula (1).

[0007] The pyrone compound provided by the present application is separated from a marine fungus Curvularia sp. ZYX-Z-4 (Phytochemistry Letters, 2023, 55: 6-11), which is isolated from a South China Sea sponge sample and can metabolize to produce a series of pyrone compounds. It has low toxicity, high selectivity, target specificity and ecological friendliness when applied in herbicides.

[0008] The present application provides a preparation method of a pyrone compound, comprising the following steps:

[0009] Step 1: obtaining a fermentation product after culturing and fermenting the fungus Curvularia sp. ZYX-Z-4;

[0010] Step 2: obtaining an extract after soaking and reducing pressure concentration of the fermentation product in step 1;

[0011] Step 3: obtaining a separation phase after dissolving, extracting and separating the extract in step 2, and obtaining a total extract after concentrating the separation phase;

[0012] Step 4: obtaining a pyrone compound of formula I after separating and purifying the total extract in step 3.

[0013] In some embodiments, in step 1,

[0014] The culture is cultured in PDB medium for 2-4 days, and the culture conditions are 150-200 rpm and 25-35℃;

[0015] The fermentation is cultured in a fermentation rice medium for 20-40 days, and the fermentation temperature is 25-35℃, wherein:

[0016] The fermentation rice medium comprises rice and seawater in a mass / volume ratio of (50-150) g:(100-200) mL, and the seawater comprises sea salt and water in a mass / volume ratio of (20-50) g:(0.5-3) L

[0017] In some specific embodiments, the culture time is 3 days, and the culture conditions are 180 rpm and 28℃.

[0018] In some specific embodiments, the fermentation is cultured in a fermentation rice medium for 30 days, and the fermentation temperature is 28℃, wherein:

[0019] The fermentation rice medium includes 100 g of rice and 120 mL of seawater, wherein the seawater includes 33.3 g of sea salt and water.

[0020] In some embodiments, in step 2, the soaking reagent includes ethyl acetate, the soaking times include 2-4 times, and the volume ratio of the fermentation product to the ethyl acetate is (0.5-1):(0.5-1).

[0021] In some specific embodiments, in step 2, the soaking times include 3 times, and the volume ratio of the fermentation product to the ethyl acetate is 1:1.

[0022] In some embodiments, in step 3, the dissolving reagent includes methanol, and the extraction reagent includes petroleum ether, and the separated phase is a methanol phase.

[0023] In some specific embodiments, the dissolving uses a 90% methanol solution, and the volume ratio of the methanol solution to the petroleum ether is 1:1.

[0024] In some embodiments, the separation and purification includes:

[0025] In the first separation, the total extract is subjected to normal-phase silica gel column chromatography, gradient elution is performed using petroleum ether and ethyl acetate at a volume ratio of (10-0):1, and elution components corresponding to characteristic peaks are collected to obtain a first purification product.

[0026] In the second separation, the first purification product is subjected to reverse-phase ODS column chromatography, gradient elution is performed using a methanol aqueous solution at a concentration of 10vol%-100vol%, and elution components corresponding to characteristic peaks are collected to obtain a second purification product.

[0027] In the third separation, the second purification product is subjected to semi-preparative high-performance liquid chromatography, isocratic elution is performed using a methanol aqueous solution at a concentration of 20vol%, and the pyrone compound is obtained.

[0028] Preferably, in the first separation, gradient elution is performed using petroleum ether and ethyl acetate at a volume ratio of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:1.

[0029] More preferably, in the first separation, different eluents in the gradient elution are combined after detection by high-performance liquid chromatography and thin-layer chromatography to select elution components corresponding to characteristic peaks.

[0030] Preferably, in the second separation, the gradient elution is performed with 10vol%, 20vol%, 30vol%, 40vol%, 50vol%, 60vol%, 70vol%, 80vol%, 90vol% and 100vol% methanol aqueous solution.

[0031] More preferably, in the second separation, the different eluents in the gradient elution are combined after high performance liquid analysis detection to obtain the same components, and then the elution components corresponding to the characteristic peaks are selected.

[0032] In some embodiments,

[0033] In the first separation, the eluent of the elution component corresponding to the characteristic peak is petroleum ether-ethyl acetate with a volume ratio of 3:1.

[0034] In the second separation, the eluent of the elution component corresponding to the characteristic peak is 20vol% methanol aqueous solution.

[0035] The present application provides a pharmaceutical composition comprising the pyrone compound and / or the pyrone compound prepared by the preparation method, and a pharmaceutically acceptable excipient.

[0036] Preferably, the pharmaceutically acceptable excipient comprises at least one of talc, lignosulfonate, alkylphenol polyoxyethylene ether phosphate, and gelatin.

[0037] The present application provides at least one of the following ①~③ in the preparation of herbicides:

[0038] ①, the pyrone compound;

[0039] ②, the pyrone compound prepared by the preparation method;

[0040] ③, the pharmaceutical composition.

[0041] The present application provides a herbicide comprising at least one of the following (1)~(3):

[0042] (1), the pyrone compound;

[0043] (2), the pyrone compound prepared by any one of the preparation methods;

[0044] (3), the pharmaceutical composition.

[0045] The present application provides a method for weeding, comprising using the herbicide for weeding.

[0046] The present application provides a pyrone compound, a preparation method and application thereof, the pyrone compound has a structure shown in formula 1, is separated into secondary metabolites of fungi Curvularia sp. ZYX-Z-4, and it is found that the compound has herbicidal effect by biological activity test evaluation, and the application in herbicide has the advantages of low toxicity, high selectivity, target specificity and ecological friendliness. The herbicidal efficiency of the compound is evaluated by using model plants Arabidopsis thaliana, and the results show that the pyrone compound has good inhibition effect on the growth of Arabidopsis thaliana roots, can cause root cytoplasmic membrane damage, root cell death, can significantly inhibit the root length and fresh weight of three kinds of weeds, and further provides related theory and experimental basis for the development of biological herbicide. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 It is a photo of Arabidopsis thaliana seeds grown on MS medium containing different concentrations of glufosinate and curvulariain A for 7 days, wherein the curvulariain A group is group 1 in the figure;

[0048] Figure 2 It is the relative fresh weight of Arabidopsis thaliana seedlings treated with glufosinate and curvulariain A, wherein the curvulariain A group is group 1 in the figure;

[0049] Figure 3 It is the relative root length of Arabidopsis thaliana seedlings treated with glufosinate and curvulariain A, wherein the curvulariain A group is group 1 in the figure;

[0050] Figure 4 It is the damage of curvulariain A treatment on Arabidopsis thaliana root cytoplasmic membrane detected by Evans Blue staining;

[0051] Figure 5 It is the effect of curvulariain A treatment on Arabidopsis thaliana root cell death detected by PI staining;

[0052] Figure 6 It is the 1 HNMR spectrum of curvulariain A;

[0053] Figure 7 It is the DEPTQ spectrum of curvulariain A;

[0054] Figure 8 It is the HSQC spectrum of curvulariain A;

[0055] Figure 9 It is the HMBC spectrum of curvulariain A;

[0056] Figure 10 is curvulariain A 1 H- 1 H COSY spectrum;

[0057] Figure 11 is a ROESY spectrum of curvulariain A;

[0058] Figure 12 is an ECD experimental and calculated curve of curvulariain A. DETAILED DESCRIPTION

[0059] The present application provides a pyrone compound, a preparation method and application thereof, and those skilled in the art can refer to the content herein, and appropriately improve the process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0060] The present application provides a pyrone compound with herbicidal effect, having the structure shown in formula 1:

[0061]

[0062] In the application, the preparation method of the pyrone compound with herbicidal effect includes the following specific steps:

[0063] A) After the Curvularia sp. ZYX-Z-4 fermentation is extracted with ethyl acetate, oil removal is performed with 90% methanol and 100% petroleum ether. Finally, the methanol phase is separated by normal phase silica gel chromatography to obtain components Fr.1~Fr.8;

[0064] B) Separate the compound having the structure of formula 1;

[0065] B1) Fr.7 is separated by reversed-phase ODS column chromatography to obtain Fr.7-1~Fr.7-6;

[0066] B2) After Fr.7-4 is detected and analyzed by high performance liquid, it is separated by isocratic elution separation using semi-preparative high performance liquid chromatography to obtain the compound curvulariain A shown in formula 1;

[0067] In the present application, the secondary metabolites of Curvularia sp. ZYX-Z-4 are used as raw materials. The secondary metabolites of Curvularia sp. are rich in natural products such as pyrones, alkaloids, terpenes, steroids, macrolides, and cyclic peptides, which have antibacterial, cytotoxic, neurotoxic, and phytotoxic biological activities. Among them, Curvularia sp. ZYX-Z-4 is a marine fungus isolated from a sponge sample collected from the Xisha Islands of the South China Sea. The Genbank number is ON386186, and it is preserved in the Institute of Tropical Bioscience and Technology, Chinese Academy of Tropical Agricultural Sciences.

[0068] The extraction method of the secondary metabolites of Curvularia sp. ZYX-Z-4 is not particularly limited in the present application. Specifically, the extraction can be carried out according to the following method:

[0069] In the present application, the activated fungus Curvularia sp. ZYX-Z-4 is inoculated with mycelium into PDB medium, and cultured at 28°C, 180 rpm for 3 days to obtain the seed liquid of the strain; the fungal seed liquid is inoculated into rice solid fermentation medium and cultured at 28°C for 30 days. Among them, the fermentation rice medium is preferably 100 g of rice and 120 mL of seawater (1 L of water is mixed with 33.3 g of sea salt).

[0070] Then, all the fermentation products are taken out, and an equal volume of ethyl acetate is added and soaked for 3 times. The ethyl acetate extract is concentrated under reduced pressure to obtain the ethyl acetate extract; the ethyl acetate extract is dissolved in 90% methanol, and an equal volume of 100% petroleum ether is added for extraction of small polar oil. Finally, the methanol phase is retained and concentrated by distillation to obtain the total extract.

[0071] The total extract is separated by normal phase silica gel column chromatography, and the elution system of petroleum ether-ethyl acetate is 10:1 to 0:1 in volume ratio.

[0072] In the specific embodiment of the present application, the Curvularia sp. ZYX-Z-4 extract is separated and purified by normal phase silica gel column chromatography, and the separation elution system is petroleum ether / ethyl acetate in a volume ratio of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1 and 0:1 (V:V) respectively, gradient elution is carried out, and each part is collected, analyzed by high performance liquid chromatography and detected by thin layer chromatography, and then combined into 8 components, which are respectively marked as Fr.1~Fr.8.

[0073] Then, the Fr.7 component is separated and purified.

[0074] Among them, the compound with the structure of formula 1 can be separated from Fr.7, and the specific steps are as follows:

[0075] Fr.7 is subjected to reverse-phase ODS column chromatography to obtain Fr.7-1~Fr.7-6; wherein the elution system is preferably methanol / water, the volume ratio of the methanol and water is 10vol%~100vol%, gradient elution is carried out, and the volume of each gradient elution is 1000mL.

[0076] Fr.7-1~Fr.7-6 are obtained, Fr.7-4 is taken, subjected to high-performance liquid detection analysis, and subjected to purification and separation by using semi-preparative high-performance liquid chromatography to obtain the compound shown in formula 1, which is referred to as curvulariain A in the application.

[0077] The mobile phase of the semi-preparative high-performance liquid chromatography is preferably a methanol-water system for isocratic elution.

[0078] The application provides a pyrone compound with herbicidal activity, having the structure of formula 1. The application discloses a novel pyrone compound first discovered in secondary metabolites of Curvularia sp. ZYX-Z-4. The biological activity test evaluation shows that the compound has herbicidal activity, wherein formula 1 has inhibitory effect on the root growth of Arabidopsis thaliana, destroys the root tissue membrane integrity of Arabidopsis thaliana, causes the death of root cells, and can significantly inhibit the root length and fresh weight of three kinds of weeds.

[0079] In order to further understand the application, the influence of curvulariain A on the root length and fresh weight of Arabidopsis thaliana seedlings is measured, the membrane damage and the degree of root cell death of Arabidopsis thaliana roots after the action of curvulariain A are detected by using Evans Blue and PI staining, the activity of the pyrone compound in inhibiting the growth of Arabidopsis thaliana roots is evaluated, the herbicidal activity of the compound is measured by using a small cup method, and thus a theoretical basis is provided for the research of biological herbicides.

[0080] The test materials used in the application are all ordinary commercially available products and can be purchased in the market.

[0081] The preparation method and application of the pyrone compound with inhibitory effect on the root length of Arabidopsis thaliana provided by the application are described below in combination with examples, and the protection scope of the application is not limited by the following examples.

[0082] Example 1: Separation and purification of the pyrone compound curvulariain A

[0083] The present application inoculates the activated fungal Curvularia sp. ZYX-Z-4 mycelium into PDB medium, and is cultured at 28 DEG C and 180 rpm for 3 days to obtain a seed liquid of the strain; the seed liquid is inoculated into a rice solid fermentation medium and is cultured at 28 DEG C for 30 days. Then, all the fermentation products are extracted with ethyl acetate for 3 times to obtain an ethyl acetate extract; the ethyl acetate extract is subjected to oil removal treatment to obtain a total extract. The rice solid fermentation medium is preferably 100 g of rice and 120 mL of seawater (1 L of water is mixed with 33.3 g of sea salt).

[0084] The Curvularia sp. ZYX-Z-4 total extract is separated by normal phase silica gel column chromatography, and is subjected to gradient elution using a petroleum ether and ethyl acetate system with a specific volume ratio of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1 and 0:1 (V:V), and is collected in sections, and is combined after thin layer chromatography and high performance liquid detection analysis to obtain 8 components Fr.1-Fr.8.

[0085] Fr.7 is separated by ODS column chromatography, and is subjected to gradient elution using a 10%-100% methanol aqueous solution, and is subjected to high performance liquid analysis and is combined after the same component part is combined to obtain 6 components Fr.7-1-Fr.7-6. Fr.7-4 is subjected to semi-preparative high performance liquid chromatography, and is subjected to isocratic elution (V:V=20:80) using a methanol-water system as a mobile phase at a flow rate of 4 mL / min to separate and purify to obtain compound curvulariain A (8.2 mg).

[0086] Example 2 Structure identification of compound curvulariain A

[0087] All the separated compounds curvulariain A are subjected to nuclear magnetic resonance, infrared, mass spectrometry detection and other modern spectroscopic techniques and are identified by chemical methods. It is identified that the compound curvulariain A is a new pyrone compound.

[0088] The structural formula of curvulariain A (formula 1) is as follows:

[0089]

[0090] Curvulariain A is a colorless oil, and HRESIMS at m / z 263.0881 shows an [M + Na]+ peak, indicating that the molecular formula is C 12 H 16 NaO5, and the unsaturation degree is 5. The one-dimensional 1 H NMR spectrum of curvulariain A is analyzed Figure 6), indicating the presence of an olefinic proton shift at δ H 6.03 (s, H-5), two methyl groups, shifts at δ H 1.31 (d, J = 6.2 Hz, H3-13), 0.99 (t, J = 7.4 Hz, H3-10). DEPTQ spectrum (Figure 2) Figure 7 It can be seen that the compound has 12 carbon signals, of which 2 are carbonyl carbon signals, 4 are double bond carbon signals, 2 are methyl carbon signals, 3 are sp 3 hybridized CH2signals and one oxidized CH signal. Integrating HSQC, HMBC, 1 H- 1 H COSY and ROESY (Figures 3 Figure 8 , Figure 9 , Figure 10 , Figure 11 ), a dibutanol fragment was found from COSY correlation signals H3-13 (δ H 1.31) / H-12 (δ H 4.27) / H2-11 (δ H 2.60) and the key HMBC signals H2-11 with C-13 (δ C 23.7), C-6 (δ C 169.3); H-12 with C-6; and H-5 (δ H 6.03) with C-11 (δ C 43.9) were determined and connected at position C-6. A 2-pentanone fragment was determined from successive COSY correlation signals H3-10 (δ H 0.99) / H2-9 (δ H 1.69) / H2-8 (δ H 3.03) and the key HMBC signals H3-10 with C-9 (δ C 17.5), C-8 (δ C 43.7); H2-9 with C-7 (δ C 208.0) and H2-8 with C-7 were determined. The carbonyl group at position 7 was determined by chemical shift δ C 208.0. The double bond C at position 4 was determined by shift δ C 181.3. The shift at position 12, δ C 65.4, also determined that an OH was connected here. The stereochemical configuration at C-12 was determined to be R (R Figure 12Based on the above, the structure of the compound curvulariain A (Formula 1) was identified and it was found to be a new pyrone compound. 13 C NMR (125 MHz) and 1 The H NMR (500 MHz) data are shown in Table 1 below. The solvent was CDCl3.

[0091] Table 1

[0092]

[0093] Curvulariain A (1) is a colorless oil, soluble in CDCl3; [α]20 D -63 (c 0.1, MeOH); UV(MeOH) λ max (log ε): 226 (3.75), 260 (3.09), 310 (3.93) nm; ECD (MeOH) λ max 247 (-0.42), 309 (-0.99) nm; 1 H (CDCl3, 500 MHz) and 13 C NMR (CDCl3, 125 MHz) data are shown in Table 1; IR (KBr) ν max (cm -1 ): 3865, 3408, 2973, 1689, 1645, 1562, 1412, 1119, 1001; HRESIMS m / z 263.0881 [M + Na] + (Theoretical value C 12 H 16 NaO5, 263.0890).

[0094] Example 3

[0095] The pharmaceutical composition composed of the compound of the present invention and agricultural adjuvants has the activity of inhibiting the root growth of Arabidopsis thaliana.

[0096] 3-1) Experimental Methods for Root Growth Inhibition in Arabidopsis

[0097] 3-1.1) Root growth inhibition activity assay in Arabidopsis thaliana

[0098] Arabidopsis seeds were transferred into sterilized centrifuge tubes, sterilized with 75% ethanol for 3-5 min, and then washed with sterile water for 8 times. After being placed in a 4°C refrigerator for 3 days, the Arabidopsis seeds were sowed on a 3.5 cm diameter culture dish. The compound was prepared as a 40 mM stock solution with DMSO, and the positive drug was glyphosate. 20 μL of the compound and 2 mL of MS medium were added to each culture dish, mixed well, and allowed to solidify. The concentration gradient was 200, 100, 50, 25, 12.5, and 0 μM. DMSO was used as a blank control.

[0099] After the MS medium was completely cooled and solidified, Arabidopsis seeds were sowed in the culture dishes, about 10 seeds per dish, and then the culture dishes were sealed with sealing film and placed vertically in a 22 ± 1°C, 16h / 8h (L:D) light incubator. After 7 days, the growth of Arabidopsis roots was observed. Then the seedlings were carefully removed with tweezers, washed with clean water to remove the surface agar, and then the surface water was absorbed with filter paper. The fresh weight was accurately measured using a 100,000th scale, and the root length of Arabidopsis seedlings was measured.

[0100] 3-1.2) Analysis of root plasma membrane integrity by Evans Blue staining

[0101] Evans Blue can stain cells with poor cell membrane integrity and inactivation to blue, and is a tissue staining method for detecting plant root activity. The deeper the blue color, the stronger the cell membrane permeability and the more serious the cell membrane damage. After 5 days of culture, the Arabidopsis seedlings were carefully transferred to MS plates containing different concentrations of compounds and continued to grow for 3 days. Then the Arabidopsis seedlings were carefully removed with tweezers, washed to remove the surface agar, and then the water was absorbed with filter paper. The roots were stained with 0.125% Evans Blue dye for 15 min. After staining, the surface dye was washed thoroughly. Then the roots were observed under a body microscope.

[0102] 3-1.3) Analysis of root cell activity by propidium iodide (PI) staining

[0103] Cell death or cell membrane rupture are stained red by the fluorescent probe propidium iodide (PI). PI was used as a fluorescent probe to monitor the extent of cell death in Arabidopsis root tips by measuring red fluorescence density. Five-day-old Arabidopsis seedlings were carefully transplanted into MS medium containing 200 μM of the compound. Samples were taken after 9, 12, and 24 hours of treatment. A blank control Arabidopsis seedling was also taken, washed with ultrapure water, and dried with filter paper. 1 mL of 10 μg / mL PI solution was added and staining was carried out in the dark for 1 minute. The solution was then aspirated and the seedlings were washed twice with ultrapure water. Cotyledons were carefully removed with forceps, and Arabidopsis roots were prepared for sectioning. Root sections were observed using a laser confocal microscope with an excitation wavelength of 488 nm and a detection wavelength of 605–675 nm. Root cells were photographed at different times of compound treatment.

[0104] 3-2) Analysis of Arabidopsis root growth inhibition results

[0105] 3-2.1) Root growth inhibitory activity of curvulariain A in Arabidopsis

[0106] Arabidopsis thaliana was treated with curvulariain A and its root growth inhibitory effect was observed after 7 days of culture. After 7 days of culture, the seedlings were weighed, the root length was measured, and photos were taken. Figure 1 、 Figure 2 、 Figure 3 As shown in Figure 3, curvulariain A treatment significantly inhibited the root length and fresh weight of Arabidopsis thaliana.

[0107] 3-2.2) Effects of curvulariain A treatment on the plasma membrane permeability of Arabidopsis seedling root tip cells

[0108] After Arabidopsis seedlings were treated with curvulariain A, the Evans blue staining of their root tips was observed. Figure 4 ) showed that after three days of culture of Arabidopsis seedlings on culture medium containing curvulariain A, the control (0 μM) showed virtually no color. At a concentration of 50 μM, curvulariain A already damaged the roots, staining the entire root light blue. In the 100 μM and 200 μM treatment groups, the roots were completely stained dark blue. This suggests that higher treatment concentrations lead to greater damage to the plasma membrane.

[0109] 3-2.3) Arabidopsis root cell death under curvulariain A treatment

[0110] PI can penetrate the broken cell membrane, but cannot penetrate the intact cell membrane, so when the cell membrane is broken or the cell is dead, it can be dyed red by PI. After the Arabidopsis seedlings were treated with curvulariain A for different times (9 h, 12 h, 24 h), the PI staining of the root tips was observed by laser confocal microscopy. As shown in FIG. 8, the PI staining of the root tips of the control group was relatively light, the root growth was normal, and the number of dead cells was small. With the increase of the treatment time of curvulariain A, the PI staining of the root tips was deepened. The root tip cells of Arabidopsis were damaged after being treated with curvulariain A for 9 h, and the red fluorescence was very obvious after 24 h, indicating that the root cell membrane was damaged seriously after being treated with curvulariain A, and the number of dead cells in the root tip was large. Figure 5

[0111] Example 4

[0112] The inhibitory effect of the pharmaceutical composition of the compound of the present application and the agricultural adjuvant on the growth of weeds.

[0113] 4-1) Herbicidal activity determination of curvulariain A on weeds

[0114] The herbicidal activity of curvulariain A on weeds (Eupatorium odoratum, Bidens pilosa, Alternanthera philoxeroides) was determined by the small cup method. The seeds of weeds with the same size and fullness were selected, disinfected with 2% NaClO for 20 min, and then washed with water for 2-3 times. The seeds were placed in a culture dish covered with two layers of filter paper, and an appropriate amount of deionized water was added to the seeds until they did not float. The culture dish was placed in a constant temperature incubator at 26 ± 2°C under dark conditions until the seeds germinated. 2-3 layers of small glass beads were added to the bottom of a disposable small plastic cup, and an appropriate amount of Hoagland nutrient solution containing curvulariain A (200 μM) was added to make the liquid level of the glass beads flush with the liquid level, and a filter paper sheet was placed on it. 10 seeds of just germinated seeds were placed in each small cup, sealed with plastic wrap and pierced, and no drug group was set as a blank control group. The experiment was placed in a light incubator at 22 ± 1°C, 16h / 8h (L:D). Glyphosate was used as a positive control for comparison experiments, and the experimental conditions and methods were consistent with curvulariain A. After 7 days of culture, the root growth and overall condition of the plants were observed, the fresh weight was accurately weighed using a hundredth scale, and the root length of the weed seedlings was measured.

[0115] Root length inhibition rate = (average root length of blank control group - average root length of experimental group) / average root length of blank control group × 100%

[0116] Fresh weight inhibition rate = (average fresh weight of blank control group - average fresh weight of experimental group) / average fresh weight of blank control group × 100%​

[0117] 4-2) Curvularia in A herbicidal activity results on weeds

[0118] After 7 days of treatment with 200 μM curvulariain A and positive control group (200 μM glyphosate) on E. crus-galli, B. pilosa and A. philoxeroides, the root growth and fresh weight of the three weeds in the treatment groups showed obvious inhibition compared with the blank control group. The root length and fresh weight inhibition rates of curvulariain A group are shown in Table 2, and the root length and fresh weight inhibition rates of glyphosate group are shown in Table 3. By comparing Table 3, it can be seen that the activity of curvulariain A on the three weeds is better than that of glyphosate.

[0119] Table 2 Growth determination results of curvulariain A on three weeds

[0120]

[0121] Table 3 Growth determination results of glyphosate on three weeds

[0122]

[0123] The above are only preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A pyrone compound, characterized in that It has the structure of formula (1):

2. A method for preparing a pyrone compound, characterized in that: The steps include: Step 1: Cultivating and fermenting the fungus Curvularia sp. ZYX-Z-4 to obtain a fermentation product; Step 2: soaking the fermentation product in step 1 and concentrating under reduced pressure to obtain an extract; Step 3, dissolving, extracting, and separating the extract in step 2 to obtain a separated phase, and concentrating the separated phase to obtain a total extract; Step 4: Take the total extract described in step 3 and separate and purify it to obtain the pyrone compound of formula 1.

3. The preparation method according to claim 2, characterized in that In the step 1, The culture is carried out using PDB medium for 2 to 4 days under the following conditions: 150 to 200 rpm, 25 to 35° C. The fermentation is carried out using a fermentation rice culture medium for 20 to 40 days at a temperature of 25 to 35° C., wherein: The fermented rice culture medium comprises rice and seawater in a mass-to-volume ratio of (50-150) g:(100-200) mL, and the seawater comprises sea salt and water in a mass-to-volume ratio of (20-50) g:(0.5-3) L.

4. The preparation method according to claim 2 or 3, characterized in that In step 2, the soaking reagent includes ethyl acetate, the soaking times include 2 to 4 times, and the volume ratio of the fermentation product to the ethyl acetate is (0.5 to 1): (0.5 to 1).

5. The preparation method according to claim 2, characterized in that In step 3, the dissolving reagent includes methanol, the extracting reagent includes petroleum ether, and the separation phase is a methanol phase.

6. The preparation method according to claim 2, characterized in that In step 4, the separation and purification includes: The total extract is first separated by normal phase silica gel column chromatography, and gradient eluted with petroleum ether and ethyl acetate in a volume ratio of (10-0):1, and elution fractions corresponding to characteristic peaks are collected to obtain a first purified product; The first purified product was subjected to a second separation on a reverse phase ODS column, eluted with a gradient of 10 vol% to 100 vol% methanol-water solution, and the eluted fractions corresponding to the characteristic peaks were collected to obtain a second purified product; The second purified product was subjected to a third separation on a semi-preparative high performance liquid chromatography column and isocratically eluted with a 20 vol% methanol aqueous solution to obtain the pyrone compound.

7. The preparation method according to claim 6, characterized in that In the first separation, the eluent for the elution component corresponding to the characteristic peak is petroleum ether-ethyl acetate in a volume ratio of 3:1; In the second separation, the eluent for the elution component corresponding to the characteristic peak is a 20 vol% methanol aqueous solution.

8. A pharmaceutical composition, characterized in that The invention comprises the pyrone compound according to claim 1 and / or the pyrone compound prepared by the preparation method according to any one of claims 2 to 7, and pharmaceutically acceptable excipients.

9. Use of at least one of the following ① to ③ in the preparation of a herbicide:

1. The pyrone compound according to claim 1; ②. A pyrone compound obtained by the preparation method according to any one of claims 2 to 7; ③. The pharmaceutical composition according to claim 8.

10. A herbicide, characterized in that Include at least one of the following (1) to (3): (1) The pyrone compound according to claim 1; (2) A pyrone compound obtained by the preparation method according to any one of claims 2 to 7; (3) The pharmaceutical composition according to claim 8.

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