CN115245167A - Application of alkaloid compound with bactericidal activity separated from common achyranthes herb - Google Patents

Abstract

The invention relates to the field of compounds and application, in particular to application of an alkaloid compound with bactericidal activity separated from common achyranthes herb, wherein the alkaloid compound is 7-demethoxytylophorine, and can be used for preventing and treating millet white disease, potato black nevus disease, corn northern leaf blight, tomato gray mold and apple rot. The invention uses the common Bull-heart grassCynanchum komaroviiIljinski) as a raw material, a compound with better antibacterial activity is obtained by separation, and the compound is used as a main material to research out the botanical fungicide with the characteristics of high effect, no pollution and low price.

Description

Application of alkaloid compound with bactericidal activity separated from common achyranthes herb

Technical Field

The invention relates to the field of compounds and application, in particular to application of an alkaloid compound with bactericidal activity, which is separated from common achyranthes herb.

Background

Biological pesticide is one of high and new technology breakthrough for realizing sustainable development and guaranteeing food safety production of human beings, and has become the main stream direction of crop disease control. One of the important ways to create a novel biopesticide is to research natural chemical products, particularly active plant secondary metabolites, find lead compounds from the natural chemical products, then carry out lead optimization, synthesize candidate compounds, more importantly, research the action mechanism of the active compounds, explore new action targets and provide theoretical basis for the research and development of plant-derived pesticides.

In recent years, a botanical pesticide is a pesticide which is prepared by processing a natural chemical product into an active pesticide, is beneficial to supplement of the chemical pesticide, plays a role in sustainable prevention and control of diseases, pests and weeds of crops, and plays an important role in green pollution-free food production, agricultural development, ecological environment protection and sustainable development of high-efficiency, high-yield and high-quality agriculture. The botanical pesticide Buxin grass is a perennial herb, and has insecticidal activity and bacteriostatic activity, and can be used as green manure and pesticide. Researches show that the total alkaloids of the Cornus bovis Seu Bubali grass have certain inhibition effect on two pathogenic bacteria of rice blast and gray mold, experiments show that the alkaloids of the Cornus bovis Seu Bubali grass have certain inhibition effect on the growth of pathogenic bacteria of various plant diseases, but reports and the like for the determination of the bacteriostatic activity and toxicity of other chemical components in the Cornus bovis Seu Bubali grass on the pathogenic bacteria in plants are not found.

Disclosure of Invention

In order to solve the problems, the invention uses the common stonecrop herb (Cynanchum komarovii Al. Iljinski) as the raw material, obtains a compound with better antibacterial activity by separation, and researches a botanical fungicide by using the compound as the main material, and has the characteristics of high effect, no pollution and low price.

In order to realize the purpose, the invention adopts the technical scheme that:

an application of alkaloid compound with bactericidal activity separated from Cornus niveus is 7-demethoxytylophorine, and can be used for preventing and treating millet white disease, potato black nevus, corn northern leaf blight, tomato gray mold and apple rot.

Further, the preparation method can be used for preparing micro-emulsions, soluble solutions and aqueous solutions for preventing and treating the diseases of millet white, potato black nevus, corn northern leaf blight, tomato gray mold and apple rot; wherein:

the microemulsion is prepared from the following raw materials in percentage by weight:

7-demethoxytylophorine: 0.25 to 0.5%, organic solvent: 25-35%, emulsifier: 5 to 35 percent of water, and the balance of water, wherein the sum of the weight percentages of the raw materials is 100 percent;

the soluble solution is prepared from the following raw materials in percentage by weight:

7-demethoxytylophorine: 0.25 to 0.5%, surfactant: 5 to 35 percent of organic solvent, and the balance of organic solvent, wherein the sum of the weight percentages of the raw materials is 100 percent;

the water agent is prepared from the following raw materials in percentage by weight:

7-demethoxytylophorine: 0.25 to 0.5%, organic solvent: 20-35%, surfactant: 5-35%, anti-freezing agent: 4 to 10 percent of water, and the balance of water, wherein the sum of the weight percentages of the raw materials is 100 percent.

Further, the organic solvent is one or a mixture of more of ethyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, acetone and N, N-dimethylformamide.

Further, the emulsifier is one or a mixture of more of alkyl benzene sulfonate, alkylphenol polyoxyethylene ether, polystyrol polyoxyethylene ether, phenethylphenol polyoxyethylene ether polyoxypropylene ether, span series, tween and agricultural anion-phosphate.

Further, the surfactant is one or a mixture of a plurality of alkyl benzene sulfonate, alkylphenol polyoxyethylene ether, phenethylphenol polyoxyethylene ether polyoxypropylene ether, an adduct of castor oil and ethylene oxide, and an aralkyl phenol polyoxyethylene ether formaldehyde condensate.

Further, the antifreezing agent is ethylene glycol or glycerol.

The invention has the following beneficial effects:

prepared from Bull-heart-shaped herbs (Cynanchum komaroviiAl. Iljinski) as raw material, separating to obtain a compound with good antibacterial activity, and using the compound as main material to research out plant source bactericide with the characteristics of high effect, no pollution and low cost.

Drawings

FIG. 1 is a schematic diagram showing the pharmacological effects of compound (4) according to the example of the present invention on Scirpus niveus.

FIG. 2 is a structural formula of compound (4) in the examples of the present invention.

FIG. 3 shows the pathogen shape of foxtail millet;

in the figure: a non-agent treated, b agent treated EC30, c agent treated EC50, d agent treated EC70.

FIG. 4 measurement of hyphal relative conductivity.

FIG. 5 shows a glycerol content calibration curve (a) and measurement of glycerol content in hyphal cells of foxtail millet bacterial blight bacteria (b);

in the figure: significance between the two sets of data was compared using student's t-test (@,p <0.05）。

FIG. 6 extracellular polysaccharide determination standard curve (a) and determination of extracellular polysaccharide content of foxtail millet breve bacteria (b);

in the figure: significance between the two sets of data was compared using student's t-test (@,p <0.05）。

FIG. 7 shows a standard curve (a) for measuring oxalic acid and a measurement (b) for measuring the amount of oxalic acid produced by Scirpus niveus;

in the figure: significance between the two sets of data was compared using student's t-test (x,p <0.05）。

Detailed Description

In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Preparation and activity analysis of extract of Bulbophyllum chinense in different polarity sections

Weighing 2.5 kg in the dry indoor environment of the Corynespora nikoense, soaking the Corynespora nikoense in industrial alcohol for 3 days, filtering the leaching liquor, concentrating the leaching liquor by a rotary evaporator to obtain a total extract 600 g (the extraction rate of the total extract is about 24%), dissolving the total extract by water, and then obtaining extracts of the Corynespora nikoense in different polarity sections by adopting different polarity section extraction methods, wherein the 4 extracts are respectively used for determining the bacteriostatic effects on 5 test strains of foxtail millet white rot pathogen, potato black nevus pathogen, maize macrophomalospora, tomato botrytis cinerea and apple canker pathogen. As shown in Table 1, the inhibition rates of the petroleum ether segment extract on foxtail millet fungus, potato black nevus, corn leaf blight, tomato botrytis cinerea and apple rot fungus are 47.1%, 46.8%, 58.0%, 60.5% and 46.5% respectively; the inhibition rate of the petroleum ether segment extract on botrytis cinerea is preferably 60.5%, the inhibition rate of the petroleum ether segment extract on apple rot pathogen is less than 46.5%, and activity tests of the petroleum ether segment extract on foxtail millet fungus, potato black nevus fungus and corn northern leaf blight fungus show that the inhibition rates of the petroleum ether segment extract on botrytis cinerea, potato black nevus fungus and corn northern leaf blight fungus are not different greatly. The inhibition rates of the trichloromethane section extractum on foxtail millet fungus, potato black nevus, corn leaf blight, tomato botrytis cinerea and apple rot fungus are 72.4%, 55.4%, 52.8%, 54.0% and 67.3% respectively; wherein the inhibition rate of the trichloromethane section extract on the foxtail millet fungus is preferably 72.4%, the inhibition rate of the trichloromethane section extract on the maculopathy bacteria is relatively low by 52.8%, and activity tests of the trichloromethane section extract on the potato black nevus, the tomato botrytis cinerea and the apple rot fungus show that the inhibition rates of the three are not very different. The n-butyl alcohol segment extractum has the inhibition rates of 100 percent, 84 percent, 76.4 percent, 80.6 percent and 92.7 percent on foxtail millet bacteria, potato black nevus bacteria, corn leaf blight bacteria, tomato botrytis cinerea and apple canker bacteria respectively; wherein the inhibition rate of the n-butyl alcohol section extract on the foxtail millet fungus is preferably 100%, and the inhibition rate of the n-butyl alcohol section extract on the sigatoka fungus is relatively low and is 76.4%. The inhibition rates of the water section extract on foxtail millet fungus, potato black nevus, corn leaf blight, tomato botrytis cinerea and apple rot fungus are respectively 22.8%, 26.6%, 33.5%, 34.9% and 24.1%; wherein the water section extract has a better inhibition rate of 34.9% on botrytis cinerea, and the water section extract has a lower inhibition rate of 22.8% on foxtail millet breve.

TABLE 1 bacteriostatic effect of Anemone cordifolia extractum with different polarity segments on 5 test strains

Obtaining monomer compound by n-butanol section column chromatography

Dissolving the n-butanol extract with methanol, adding 1.2 times of silica gel (200-300 mesh), removing excessive methanol with rotary evaporator, and oven drying the mixture at 70 deg.C. At the same time, column packing was performed (silica gel was dissolved with chloroform: methanol = 1:1); the reaction was continued with chloroform: methanol 1:1 was run through the column until the silica gel surface was not dropping. The ratio of eluent was changed to chloroform: methanol 50, elution was continued for 3 column volumes and loading was started. Grinding the mixture of n-butanol fraction and silica gel to fine powder, loading into a chromatographic column, and continuously eluting with chloroform: methanol 50.

Eluent: in the process of n-butanol section extract chromatography, gradient elution is used, and an eluent is chloroform: methanol was gradually increased in polarity from the first 50: methanol 50.

Effect of Compounds on Graham's disease

The 6 separated compounds are prepared into mother liquor of 5 mg/mL, the mother liquor is diluted to a required concentration, then the mother liquor is added into a PDA culture medium, the mixture is uniformly mixed, and the mixture is poured into a sterilized culture dish, so that the concentration of the compounds contained in the culture dish is 0, 10, 20, 40, 60, 80 and 160 mu g/mL. Cooling the ultraviolet sterilization standing culture medium, beating bacterial dishes with the diameter of 5 mm at the colony edges of the foxtail millet white hair bacteria after continuous transfer culture of 72 h, and respectively inoculating the bacterial dishes into the center of a PDA plate culture medium containing medicaments. Each treatment requires 3 repeated experiments, the experiments are repeated for 3 times, then the obtained product is placed in a constant temperature incubator and is cultured at 25 ℃ for 72 h, the diameters of a control group and a treated colony are measured by a cross method, and the diameters of the control group and the treated colony are measured by a cross method. The inhibition rate (%) under different concentrations is calculated by using a formula, and a virulence regression equation and an EC50 value are solved by using DPS data processing software.

The results show that the compound (1) has EC on white hair germ of millet ₅₀ 120.0802 μ g/mL; EC of compound (2) against foxtail millet fungus ₅₀ 67.2009 μ g/mL; EC of compound (3) against foxtail millet fungus ₅₀ 100.4090 μ g/mL; EC of compound (4) against foxtail millet fungus ₅₀ 4.6637 μ g/mL; EC of compound (5) against foxtail millet fungus ₅₀ 75.1693 μ g/mL; EC of Compound (6) against Scirpus oryzae ₅₀ 37.9202 μ g/mL; the result shows that the compound (4) has better inhibition effect on the white hair germ of millet, and EC ₅₀ It was 4.6637. Mu.g/mL.

TABLE 2 Effect of the 6 Compounds on the bacterial pathogen of Gouzolzia

Drug effect of compound (4) on onset of millet leukoderma

And selecting the millet leaves with consistent growth vigor and the same position, and performing pesticide effect measurement indoors. Scrubbing the leaves with 70% ethanol, respectively, putting into gauze soaked with sterilized water, and inoculating foxtail millet germs on the leaves; 7 days after inoculation, the leaves were treated with 10 ppm of compound (4); the change of the leaves was observed after 7 days, and as shown in fig. 1, the leaves after spraying had better effect than those without spraying.

By testing the nuclear magnetism one-dimensional spectrogram ¹ H-NMR、 ¹³ C-NMR, DEPT135 and nuclear magnetic two-dimensional spectrum COSY, HMBC, HSQC and High Resolution Mass Spectrum (HRMS), and the molecular weight are obtained by consulting literature and the likeFormula (iv), results: the compound (4) is 7-demethoxytylophorine; the molecular formula is as follows: C23H25NO3; molecular weight: 363.45, the structural formula is shown in figure 2.

Influence of 7-demethoxytylophorine on growth morphology of foxtail millet fungus

The morphology of the hyphae of the gray hair germs on the culture medium after the culture medium is treated by adding 7-demethoxylated tylophora ovata alkali with different concentrations and cultured by 48 h is observed by an optical microscope, and the hyphae mycelium treated by the medicament is mostly bent, has more top branches and is seriously twisted and can not grow normally. Without treatment with the agent, the mycelium was more straight and had fewer apical branches. As can be seen from the change of the hypha growth form, the 7-demethoxytylophorine has great inhibition effect on the hypha growth of the foxtail millet fungus.

Influence of 7-demethoxytylophorine on hyphal cell membrane permeability of foxtail millet white hair germ

As can be seen from the standard graph (fig. 4), the relative conductivity of the drug treated or non-treated samples increased with time. Treating foxtail millet fungus with 7-demethoxytylophorine for 5 min, and EC ₃₀ 、EC ₅₀ 、EC ₇₀ The conductivity is 23.87%, 23.36% and 22.78% respectively, the conductivity of CK is 21.63%, the conductivity is increased by 2.24%, 1.73% and 1.15% respectively, the conductivity is 29.05%, 31.73% and 28.10% respectively after 80 min, the conductivity of CK is 27.96%, the conductivity is increased by 1.09%, 3.77% and 0.14% respectively, the conductivity is 34.34%, 37.31% and 34.48% respectively after 180 min, the conductivity of CK is 32.90%, and the conductivity is increased by 1.44%, 4.41% and 1.58% respectively. The increase in conductivity indicates that the electrolyte in the hyphae is extravasated and the permeability of the cell membrane is increased.

Influence of 7-demethoxytylophorine on cell glycerol content of foxtail millet fungus

Looking at the standard curve (fig. 5 a), it can be seen that as the glycerol concentration increases, the absorbance value of the glycerol solution at 630 nm also increases. Through test optimization, the content of glycerol in the hyphae of the foxtail millet bacterial is calculated by using a regression equation in a standard curve, the content of the glycerol in untreated hyphae cells is 0.557 mg/gEC in drug-treated hyphal cells ₃₀ 、EC ₅₀ 、EC ₇₀ The glycerol contents of 0.920 mg/g, 1.320 mg/g and 1.948 mg/g are respectively increased by 1.7 times, 2.4 times and 3.5 times, so that the glycerol content of the hyphae treated by 7-demethoxy tylophorine is obviously increased (fig. 5 b).

Influence of 7-demethoxytylophorine on exopolysaccharide content of foxtail millet fungus

The exopolysaccharide can increase the drought and cold resistance of the foxtail millet fungus and improve the survival ability of the foxtail millet fungus. According to the exopolysaccharide calibration curve (FIG. 6 a), the exopolysaccharide content in the hypha of the bacterial strain of the gray mold of millet is calculated, the exopolysaccharide content is 1.1 mu g/mL without being treated by the medicament, and the EC is calculated after the treatment of the medicament ₃₀ 、EC ₅₀ 、EC ₇₀ The extracellular polysaccharide content in the hyphae treated by 7-demethoxytylophorine was significantly increased by 4.5 times, 8 times and 12 times as compared with 5.0. Mu.g/mL, 8.9. Mu.g/mL and 12.8. Mu.g/mL, respectively, as can be analyzed by data (FIG. 6 b).

Influence of 7-demethoxytylophorine on oxalate content of foxtail millet fungus

The oxalic acid content was calculated according to the standard curve for oxalic acid assay (FIG. 7 a), and it can be seen that the oxalic acid content in mycelia of Sclerotinia graminis (FIG. 7 b) was reduced after 7-demethoxytylophorine treatment, the oxalic acid content in Sclerotinia graminis (EC) without drug treatment was 13 μ g/mL, and the concentration of oxalic acid in the mycelia of Sclerotinia graminis (EC) was 13 μ g/mL ₃₀ 、EC ₅₀ 、EC ₇₀ After the treatment, the ratio of the concentration of the compound was reduced by 1.7 times, 5.6 times and 2.9 times, respectively, to 7.4. Mu.g/mL, 5.6. Mu.g/mL and 4.5. Mu.g/mL.

The experimental research finds that the 7-demethoxytylophorine has higher bactericidal activity on the foxtail millet fungus. In the test for determining the sensitivity of the foxtail millet white disease to 7-demethoxytylophorine, the EC of 7-demethoxytylophorine to foxtail millet white disease germ ₃₀ 、EC ₅₀ 、EC ₇₀ The values are 1.6505. Mu.g/mL, 4.6637. Mu.g/mL, 13.1776. Mu.g/mL, EC ₇₀ The value of the bacterial strain for the white hair of millet is far higher than that of other two concentrations, and the sensitivity of the bacterial strain is lower than that of other concentrations. EC of 0.1 mg/mL and 0.2 mg/mL ₃₀ 、EC ₅₀ 、EC ₇₀ After treatment, the shape of the hyphae of the foxtail millet bacterial is obviously changed, the hyphae is distorted, the top branches are increased, and the shape of the untreated hyphae under an electron microscope is not obviously changed, which shows that the 7-demethoxytylophorine after treatment has certain influence on the shape of the foxtail millet bacterial. Treating foxtail millet fungus with 7-demethoxytylophorine for 5 min, and EC ₃₀ 、EC ₅₀ 、EC ₇₀ The conductivity is 23.87%, 23.36% and 22.78% respectively, the conductivity of CK is 21.63%, the conductivity is increased by 2.24%, 1.73% and 1.15% respectively, the conductivity is 29.05%, 31.73% and 28.10% respectively after 80 min, the conductivity of CK is 27.96%, the conductivity is increased by 1.09%, 3.77% and 0.14% respectively, the conductivity is 34.34%, 37.31% and 34.48% respectively after 180 min, the conductivity of CK is 32.90%, and the conductivity is increased by 1.44%, 4.41% and 1.58% respectively. The conductivity is increased, which reflects the exosmosis of electrolyte in the hypha cells of the foxtail millet breve, so that the permeability of cell membranes is increased. The content of glycerol in untreated hyphal cell is 0.557 mg/g, EC in hyphal cell treated with the agent ₃₀ 、EC ₅₀ 、EC ₇₀ The glycerol contents of 0.920 mg/g, 1.320 mg/g and 1.948 mg/g are respectively increased by 1.7 times, 2.4 times and 3.5 times, the content without medicament treatment is 1.1 mu g/mL, and the EC after medicament treatment is ₃₀ 、EC ₅₀ 、EC ₇₀ Respectively 5.0 mu g/mL, 8.9 mu g/mL and 12.8 mu g/mL, which are increased by 4.5 times, 8 times and 12 times, the exopolysaccharide has potential immunoregulation and tumor inhibition effects, can increase resistance, and the reduction of the content of the exopolysaccharide can influence the stress resistance of the foxtail millet fungus. The oxalic acid content in the non-medicament-treated foxtail millet fungus is 13 mug/mL, EC ₃₀ 、EC ₅₀ 、EC ₇₀ After the treatment, the ratio of the concentration of the compound was reduced by 1.7 times, 5.6 times and 2.9 times, respectively, to 7.4. Mu.g/mL, 5.6. Mu.g/mL and 4.5. Mu.g/mL. The oxalic acid content affects the pathogenicity of germs, and the reduction of the oxalic acid content can reduce the pathogenicity of the foxtail millet germs.

EC of 7-demethoxytylophorine against foxtail millet white hair germ ₇₀ Is 13.1776 mug/mL which is higher than other concentrations, which shows that the sensitivity of 7-demethoxytylophorine to the foxtail millet white hair germ is higher than that of the treatment with other concentrations. 0.1 mg/mL 7-demethoxytylophorine-treated millet seed whiteAfter pathogenic bacteria develop, the intracellular electrolyte of the pathogenic bacteria is oozed, the permeability of cell membranes is increased, the stress resistance is reduced, and the pathogenicity is reduced, thereby laying a theoretical foundation for further defining the action mechanism of 7-demethoxytylophorine on the foxtail millet bacteria.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (6)

1. The application of alkaloid compounds with bactericidal activity separated from the common achyranthes herb is characterized in that: the alkaloid compound is 7-demethoxytylophorine, and can be used for preventing and treating powdery mildew of millet, potato black nevus, corn leaf spot, tomato gray mold and apple rot.

2. Use of alkaloid compounds according to claim 1, characterized in that: can be used for preparing microemulsion, soluble agent and aqueous solution for preventing and treating millet white disease, potato black nevus, corn northern leaf blight, tomato gray mold and apple rot; wherein:

the microemulsion is prepared from the following raw materials in percentage by weight:

7-demethoxytylophorine: 0.25 to 0.5%, organic solvent: 25-35%, emulsifier: 5 to 35 percent of water, and the balance of water, wherein the sum of the weight percentages of the raw materials is 100 percent;

the soluble solution is prepared from the following raw materials in percentage by weight:

7-demethoxytylophorine: 0.25 to 0.5%, surfactant: 5 to 35 percent of organic solvent, and the balance of organic solvent, wherein the sum of the weight percentages of the raw materials is 100 percent;

the water agent is prepared from the following raw materials in percentage by weight:

7-demethoxytylophorine: 0.25 to 0.5%, organic solvent: 20-35%, surfactant: 5-35%, anti-freezing agent: 4 to 10 percent of water, and the balance of water, wherein the sum of the weight percentages of the raw materials is 100 percent.

3. Use of alkaloid compounds according to claim 1, characterized in that: the organic solvent is one or a mixture of more of ethyl acetate, cyclohexanone, methyl oleate, ethanol, methanol, acetone and N, N-dimethylformamide.

4. Use of alkaloid compounds according to claim 1, characterized in that: the emulsifier is one or a mixture of a plurality of alkyl benzene sulfonate, alkylphenol polyoxyethylene ether, polystyrol polyoxyethylene ether, phenethyl phenol polyoxyethylene ether polyoxypropylene ether, span series, tween and agricultural anion-phosphate.

5. Use of alkaloid compounds according to claim 1, characterized in that: the surfactant is one or more of alkyl benzene sulfonate, alkylphenol polyoxyethylene ether, phenethyl phenol polyoxyethylene ether polyoxypropylene ether, castor oil and ethylene oxide adduct, and aralkyl phenol polyoxyethylene ether formaldehyde condensate.

6. Use of alkaloid compounds according to claim 1, characterized in that: the antifreezing agent is ethylene glycol or glycerol.

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