Preparation method and application of methylpyrazole-chitosan oligosaccharide derivative

Abstract

Belongs to the technical field of pine wood nematode disease prevention and control, and particularly relates to a preparation method and application of a pyrazole-chitosan oligosaccharide derivative. Chloroacetyl chloride serves as an acylating agent, chitosan oligosaccharide is acetylated, and chloroacetyl chitosan oligosaccharide is generated; then, a pyrazole molecule with nematicidal activity is connected to the Cl site of the chloracetyl chitosan oligosaccharide through ammonolysis reaction, and the novel pyrazole-chitosan oligosaccharide derivative is obtained. The derivative has relatively high contact activity on pine wood nematodes, and LC50 of 24 hours and LC50 of 48 hours are 4.814 mM and 4.061 mM respectively; the chitosan oligosaccharide can remarkably inhibit multiple physiological indexes such as movement, feeding and service life of the pine wood nematodes, and is good in stability, low in molecular weight of the chitosan oligosaccharide, good in water solubility, green and environment-friendly, and the plant toxicity is remarkably reduced. The derivative has a good application prospect in the aspect of controlling the pine wood nematode disease and can be applied to prevention and control of the pine wood nematode disease.

Description

Technical Field:

[0001] The present invention belongs to the technical field of prevention and control of pine wilt disease, and specifically relates to a preparation method and application of a mipryzole-chitosan oligosaccharide derivative. Background Art:

[0002] Pine wilt disease is a highly destructive forest disease caused by Bursaphelenchus xylophilus, which is characterized by fast spreading speed, strong lethality, and difficulty in prevention and control, and is an important international quarantine object. This disease was first discovered in Nanjing, China in 1982 and then spread rapidly, with an obvious trend of northward and westward expansion; up to now, the affected area in China has reached more than 25 million mu, causing huge economic and ecological losses. The main prevention and control means of this disease include physical method, biological method, and chemical method, among which the drug injection method is one of the most efficient prevention and control means. However, the types of chemical drugs currently used are limited, the drug resistance of Bursaphelenchus xylophilus is increasing day by day, and these agents have the characteristics of high toxicity and high residue, and are relatively toxic to pine trees. Therefore, it is urgent to develop nematicidal agents with low toxicity, high efficiency, and environmental protection. Summary of the Invention:

[0003] The purpose of the present invention is to overcome the deficiencies of existing prevention and control drugs such as high toxicity and poor stability, and to provide a new type of green and environmentally friendly chitosan oligosaccharide derivative for killing Bursaphelenchus xylophilus.

[0004] To achieve the above purpose, the present invention provides a mipryzole-chitosan oligosaccharide derivative, which modifies the chitosan oligosaccharide backbone with the nematicidal active molecule mipryzole to synthesize the mipryzole-chitosan oligosaccharide derivative, and its structural formula is:

[0005]

[0006] The present invention also provides a preparation method of the mipryzole-chitosan oligosaccharide derivative, which selects chloroacetyl chloride as an acylating agent to be connected to the N position of chitosan oligosaccharide to acetylate chitosan oligosaccharide to generate chloroacetyl chitosan oligosaccharide; then mipryzole is introduced at the Cl position of chloroacetyl chitosan oligosaccharide through an ammonolysis reaction, and the reaction formula is as follows:

[0007] (1)

[0008] (2)

[0009] Wherein, n = 13; m = 7; o = 6; p = 6.

[0010] The preparation steps of the mipryzole-chitosan oligosaccharide derivative of the present invention are as follows:

[0011] (1) Dissolve chitosan oligosaccharide in a solvent, then dropwise add chloroacetyl chloride, heat under reflux, then cool, filter by suction, wash the filter cake and dry it to obtain chloroacetyl chitosan oligosaccharide; the solvent is prepared by mixing chloroform and pyridine;

[0012] (2) Dissolve chloroacetyl chitosan oligosaccharide in dimethylformamide, add methylpyrazole, and heat for reaction; after cooling, filter by suction, wash the filter cake and dry it to obtain a methylpyrazole-chitosan oligosaccharide derivative.

[0013] In step (1) of the present invention, the heating reflux temperature is 70 °C and the reflux time is 15 - 20 h.

[0014] In step (2) of the present invention, the heating reaction temperature is 80 °C and the reaction time is 8 - 10 h.

[0015] In the present invention, the mass-volume ratio of chitosan oligosaccharide to the solvent is (1 - 3) g: 10 mL; the volume ratio of chloroform to pyridine in the solvent is 1:1.

[0016] In the present invention, the mass-volume ratio of chitosan oligosaccharide to the total chloroacetyl chloride is 24 g: 202.5 mL.

[0017] In the present invention, the mass ratio of chloroacetyl chitosan oligosaccharide to methylpyrazole is 1.5:1.35; the mass-volume ratio of chloroacetyl chitosan oligosaccharide to dimethylformamide is 1.5 g: 16.5 mL.

[0018] The present invention also provides the application of the methylpyrazole-chitosan oligosaccharide derivative in preventing and controlling pine wilt disease or killing pine wood nematodes. The LC 50 of the methylpyrazole-chitosan oligosaccharide derivative to pine wood nematodes at 24 h and 48 h are 4.814 mM and 4.061 mM respectively.

[0019] Compared with the prior art, the present invention utilizes the excellent properties of chitosan oligosaccharide such as slow release and film formation, and the high nematocidal activity of methylpyrazole to synthesize a new type of green and environmentally friendly chitosan oligosaccharide derivative. This derivative has a relatively high contact killing activity against pine wood nematodes, and the LC 50 at 24 h and 48 h are 4.814 mM and 4.061 mM respectively; it can significantly inhibit multiple physiological indexes of pine wood nematodes such as movement, feeding, and lifespan, and has good stability, low molecular weight of chitosan oligosaccharide, good water solubility, environmental friendliness, and significantly reduced phytotoxicity. This derivative has good application prospects in controlling pine wilt disease and can be applied to the prevention and control of pine wilt disease. Description of the Drawings:

[0020] Figure 1 It is the infrared spectrum (a) and nuclear magnetic resonance hydrogen spectrum (b) of chitosan oligosaccharide and methylpyrazole-chitosan oligosaccharide derivative involved in the present invention. Detailed Embodiments:

[0021] The technical solution of the present invention will be fully described below through embodiments in conjunction with the accompanying drawings.

[0022] Example 1:

[0023] This example relates to a preparation method of a miprazole-chitosan oligosaccharide derivative, and the specific steps are as follows:

[0024] (1) Accurately weigh 24 g of chitosan oligosaccharide and dissolve it in 240 mL of a solvent (the solvent is prepared by mixing chloroform and pyridine in a volume ratio of 1:1), let it stand for 24 h, then dropwise add 135 mL of chloroacetyl chloride, reflux at 70 °C for 10 h, then add 67.5 mL of chloroacetyl chloride, and continue refluxing for 8 h, or dropwise add 202.5 mL of chloroacetyl chloride at one time and reflux at 70 °C for 18 h; after cooling, filter by suction, wash the filter cake twice with absolute ethanol, and dry at 60 °C to obtain chloroacetyl chitosan oligosaccharide;

[0025] (2) Accurately weigh 1.5 g of chloroacetyl chitosan oligosaccharide and dissolve it in 16.5 mL of dimethylformamide (DMF), add 1.35 g of miprazole, and react at 80 °C for 10 h; after cooling, filter by suction, wash the filter cake twice with absolute ethanol, and dry at 60 °C to obtain the miprazole-chitosan oligosaccharide derivative.

[0026] Mix chitosan oligosaccharide and the miprazole-chitosan oligosaccharide derivative with KBr respectively and press them into tablets, and use an infrared absorption spectrometer (Thermo Nicolet iS10) to measure the infrared absorption spectra of the two; use a nuclear magnetic resonance spectrometer (Bruker500) to measure the nuclear magnetic resonance hydrogen spectrum (1H NMR, 500 MHz) of chitosan oligosaccharide and the miprazole-chitosan oligosaccharide derivative with tetramethylsilane (TMS) as the internal standard. The results are as Figure 1 shown. As can be seen from Figure 1 a, the infrared absorption spectrum of the miprazole-chitosan oligosaccharide derivative has obvious signal peaks at 1630 - 1750 cm -1 , which coincides with the stretching vibration absorption peak positions of C=O, C=N, and C=C in the miprazole structure; according to Figure 1 b, the signal peaks at δ7.30 and δ1.94 in the 1 1H NMR of the miprazole-chitosan oligosaccharide derivative coincide with the absorption peak positions of =CH and -CH3 in the miprazole structure respectively. Based on the above analysis, miprazole was successfully grafted onto the chitosan oligosaccharide molecule to obtain the miprazole-chitosan oligosaccharide derivative.

[0027] Example 2:

[0028] This example relates to a preparation method of a miprazole-chitosan oligosaccharide derivative, and the specific steps are as follows:

[0029] (1) Weigh accurately 72 g of chitosan oligosaccharide and dissolve it in 240 mL of solvent (the solvent is prepared by mixing chloroform and pyridine in a volume ratio of 1:1). After standing for 24 h, add dropwise 202.5 mL of chloroacetyl chloride, reflux at 70 °C for 15 h, cool and then filter by suction. Wash the filter cake twice with absolute ethanol and dry at 60 °C to obtain chloroacetyl chitosan oligosaccharide;

[0030] (2) Weigh accurately 1.5 g of chloroacetyl chitosan oligosaccharide and dissolve it in 16.5 mL of dimethylformamide (DMF), add 1.35 g of methylpyrazole, and react at 80 °C for 8 h; cool and then filter by suction. Wash the filter cake twice with absolute ethanol and dry at 60 °C to obtain methylpyrazole-chitosan oligosaccharide derivative.

[0031] Example 3:

[0032] This example relates to a preparation method of methylpyrazole-chitosan oligosaccharide derivative, and the specific steps are as follows:

[0033] (1) Weigh accurately 48 g of chitosan oligosaccharide and dissolve it in 240 mL of solvent (the solvent is prepared by mixing chloroform and pyridine in a volume ratio of 1:1). After standing for 24 h, add dropwise 135 mL of chloroacetyl chloride, reflux at 70 °C for 9 h, then add an additional 67.5 mL of chloroacetyl chloride and continue refluxing for 7 h. Cool and then filter by suction. Wash the filter cake twice with absolute ethanol and dry at 60 °C to obtain chloroacetyl chitosan oligosaccharide;

[0034] (2) Weigh accurately 1.5 g of chloroacetyl chitosan oligosaccharide and dissolve it in 16.5 mL of dimethylformamide (DMF), add 1.35 g of methylpyrazole, and react at 80 °C for 9 h; cool and then filter by suction. Wash the filter cake twice with absolute ethanol and dry at 60 °C to obtain methylpyrazole-chitosan oligosaccharide derivative. Example 4:

[0035] This example relates to a preparation method of methylpyrazole-chitosan oligosaccharide derivative, and the specific steps are as follows:

[0036] (1) Weigh accurately 72 g of chitosan oligosaccharide and dissolve it in 240 mL of solvent (the solvent is prepared by mixing chloroform and pyridine in a volume ratio of 1:1). After standing for 24 h, add dropwise 202.5 mL of chloroacetyl chloride, reflux at 70 °C for 20 h, cool and then filter by suction. Wash the filter cake twice with absolute ethanol and dry at 60 °C to obtain chloroacetyl chitosan oligosaccharide;

[0037] (2) Weigh accurately 1.5 g of chloroacetyl chitosan oligosaccharide and dissolve it in 16.5 mL of dimethylformamide (DMF), add 1.35 g of methylpyrazole, and react at 80 °C for 8 h; cool and then filter by suction. Wash the filter cake twice with absolute ethanol and dry at 60 °C to obtain methylpyrazole-chitosan oligosaccharide derivative.

[0038] Example 5:

[0039] This example involves the in vitro pine wood nematode killing activity and stability experiment of the 1-methylpyrazole-chitosan oligosaccharide derivative prepared in Example 1. The specific steps are as follows:

[0040] I. In vitro pine wood nematode killing activity experiment of 1-methylpyrazole-chitosan oligosaccharide derivative

[0041] 1. Prepare a 100 μL pine wood nematode treatment system in a 96-well plate so that the pine wood nematode concentration is about 1 nematode / μL; separately set aqueous solutions of chitosan oligosaccharide, 1-methylpyrazole, and 1-methylpyrazole-chitosan oligosaccharide derivative with a series of gradient concentrations (2.7 mM, 3.6 mM, 3.9 mM) to treat the pine wood nematodes. Use the pine wood nematodes treated with sterile water as the control group. Set three biological replicates for each group and culture them in the dark at 25 °C. Count the corrected mortality rate after 24 h. The formula for calculating the corrected mortality rate is as follows:

[0042]

[0043] The results are shown in Table 1. The experimental results show that the LC 50 values of chitosan oligosaccharide and 1-methylpyrazole after 24 h are 15.502 mM and 9.054 mM respectively, and the LC 50 value of the 1-methylpyrazole-chitosan oligosaccharide derivative after 24 h is 4.814 mM. Compared with chitosan oligosaccharide and 1-methylpyrazole, the nematode killing activity of the 1-methylpyrazole-chitosan oligosaccharide derivative is greatly improved.

[0044] Table 1 Corrected mortality rate of pine wood nematodes after 24 h

[0045]

[0046] Table 2 Corrected mortality rate of pine wood nematodes after 48 h

[0047]

[0048] 2. Prepare a 100 μL pine wood nematode treatment system in a 96-well plate so that the nematode concentration is about 1 nematode / μL; separately set aqueous solutions of chitosan oligosaccharide, 1-methylpyrazole, and 1-methylpyrazole-chitosan oligosaccharide derivative with a series of gradient concentrations (0.6 mM, 1.2 mM, 1.8 mM, 2.4 mM) to treat the pine wood nematodes. Use the nematodes treated with sterile water as the blank control. Set three biological replicates for each group. After dark treatment for 24 h, count the number of times the nematode heads swing within 1 min under a stereomicroscope to characterize their motility. The results are shown in Table 2. The experimental results show that compared with the control group, the chitosan oligosaccharide treatment group, and the 1-methylpyrazole treatment group, the motility of the pine wood nematodes in the 1-methylpyrazole-chitosan oligosaccharide derivative treatment group is significantly reduced, and the inhibition efficiency has a linear relationship with its concentration. At a concentration of 2.4 mM, the number of swings in the 1-methylpyrazole-chitosan oligosaccharide derivative treatment group decreased by 43% compared with the 1-methylpyrazole treatment group (Table 2).

[0049] Table 3 Number of head swings of Bursaphelenchus xylophilus within 1 minute

[0050]

[0051] 3. Use an aqueous solution of 2.4 mM methylpyrazole-chitosan oligosaccharide derivative to dark-treat Bursaphelenchus xylophilus in a 1.5 mL centrifuge tube for 6 h; centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the nematodes with 20 μL of sterile water, and then inoculate them onto a PDA medium plate covered with Botrytis cinerea mycelia, and incubate in the dark at 25°C. Take pictures to record the feeding situation of Bursaphelenchus xylophilus, and use ImageJ software to measure the area of the feeding circle to evaluate the feeding ability of Bursaphelenchus xylophilus. Using the same method, use an aqueous solution of 2.4 mM chitosan oligosaccharide, an aqueous solution of 2.4 mM methylpyrazole, and sterile water to treat the nematodes as controls, and set three biological replicates for each group. The experimental results show that the feeding ability of chitosan oligosaccharide on Bursaphelenchus xylophilus is comparable to that of the sterile water treatment group, and the feeding area of the nematodes is 2706 mm 2 , indicating that chitosan oligosaccharide has little effect on the feeding ability of nematodes; the feeding areas of the nematodes in the methylpyrazole-chitosan oligosaccharide derivative treatment group and the methylpyrazole treatment group are 959 mm 2 and 344 mm 2 , respectively, indicating that the feeding ability of the nematodes is strongly inhibited.

[0052] 4. Prepare a 100 μL Bursaphelenchus xylophilus treatment system in a 96-well plate so that the nematode concentration is about 1 nematode / μL and the concentration of methylpyrazole-chitosan oligosaccharide derivative is 2.4 mM; using the same method, use an aqueous solution of 2.4 mM chitosan oligosaccharide, an aqueous solution of 2.4 mM methylpyrazole, and sterile water to treat the nematodes as controls, and set three biological parallels for each group. Count the remaining number of nematodes every 24 h, and record the number of days when the number drops to less than 25% as the lifespan of the nematodes. The experimental results show that the lifespans of the nematodes in the sterile water treatment group and the chitosan oligosaccharide treatment group are both 12 days; while the lifespans of the nematodes in the methylpyrazole treatment group and the methylpyrazole-chitosan oligosaccharide derivative treatment group are significantly shortened. The lifespan of the methylpyrazole treatment group is 9 days, and the lifespan of the methylpyrazole-chitosan oligosaccharide derivative treatment group is 8 days.

[0053] II. Stability experiment of methylpyrazole-chitosan oligosaccharide derivative: Prepare treatment systems of methylpyrazole-chitosan oligosaccharide derivative with concentrations of 3 mM, 5 mM, and 7 mM respectively and divide them into two equal parts. Immediately measure the nematicidal ability of one part, and measure the nematicidal ability of the other part after placing it in a normal temperature and light environment for 7 d. Calculate the corrected mortality rate at 48 h respectively, and calculate the retention rate of the nematicidal ability of the derivative after placing it for 7 d to judge its stability.

[0054]

[0055] The experimental results show that after 7 days of normal temperature and light treatment, the nematicidal activities of different concentrations of the m-tolylpyrazole-chitosan oligosaccharide derivatives are retained at a relatively high level. The retention rates of the nematicidal activities of the derivatives at 3 mM, 5 mM, and 7 mM are 84.03%, 88.22%, and 84.73% respectively, indicating that the m-tolylpyrazole-chitosan oligosaccharide derivatives have good stability.

[0056] Example 6:

[0057] This example involves the control experiment of the m-tolylpyrazole-chitosan oligosaccharide derivative prepared in Example 1 against Bursaphelenchus xylophilus inoculated on black pine seedlings. Specifically:

[0058] Prepare an aqueous solution of the m-tolylpyrazole-chitosan oligosaccharide derivative with a concentration of 2.4 mM in a 1.5 ml centrifuge tube. Dark-treat the Bursaphelenchus xylophilus for 2 h, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the nematodes with 20 μL of the above-mentioned aqueous solution of the m-tolylpyrazole-chitosan oligosaccharide derivative; use a disposable sterile syringe to make an incision at the apical bud of a 6 cm high black pine seedling, inject 20 μL of the nematode suspension into each pine seedling, cover the incision with sterile water-wetted sterilized absorbent cotton to keep it moist, and regularly take pictures to record the wilting situation of the black pine seedlings. Using the same method, use 2.4 mM chitosan aqueous solution, 2.4 mM m-tolylpyrazole aqueous solution, and sterile water to treat Bursaphelenchus xylophilus as controls. Each group has three biological replicates. 10 days after inoculating the nematodes, the pine seedlings in the chitosan treatment group showed severe wilting, the pine seedlings in the m-tolylpyrazole treatment group showed slight wilting, and the pine seedlings in the m-tolylpyrazole-chitosan oligosaccharide derivative treatment group grew well.

[0059] Example 7:

[0060] This example involves the control experiment of the m-tolylpyrazole-chitosan oligosaccharide derivative prepared in Example 1 against Bursaphelenchus xylophilus inoculated on two-year-old black pine seedlings. Specifically:

[0061] Select two-year-old black pine seedlings, use an electric drill to drill a hole obliquely downward at an angle of 45° about 15 cm above the ground on the main trunk of the black pine seedlings, inoculate about 10,000 nematodes into the hole, stuff the hole with sterile water-wetted sterilized absorbent cotton to keep it moist, and seal it with a sealing film. Culture at 25 °C with 16 h of light and 8 h of darkness. After 24 h, remove the sealing film and inject 2 ml of an aqueous solution of the m-tolylpyrazole-chitosan oligosaccharide derivative with a concentration of 2.4 mM into the hole, and seal it with a sealing film again. Regularly take pictures to record the wilting situation of the two-year-old pine seedlings. Each group has three biological replicates, with the application of chitosan, m-tolylpyrazole, and distilled water treatment as controls. 30 days after inoculating the nematodes, the pine seedlings in the distilled water treatment group, chitosan treatment group, and m-tolylpyrazole treatment group all showed wilting, and the pine seedlings in the m-tolylpyrazole-chitosan oligosaccharide derivative treatment group grew relatively well. It shows that the m-tolylpyrazole-chitosan oligosaccharide derivative can effectively kill Bursaphelenchus xylophilus.

[0062] Example 8:

[0063] This example relates to the phytotoxicity experiment of the mepyrazole-chitosan oligosaccharide derivative prepared in Example 1.

[0064] Select black pine seedlings (about 30 days old) that have grown to about 4 cm and have a similar growth state. Use a disposable sterile syringe to make an incision at the apical bud of the black pine seedlings, and inject 10 μL of the aqueous solution of the mepyrazole-chitosan oligosaccharide derivative onto it. Cover the incision with sterile water-moistened sterilized absorbent cotton to keep it moist. At the same time, inject 1 mL of the mepyrazole-chitosan oligosaccharide derivative into the roots of the pine seedlings to simulate the process of injecting the drug into the trunk and enrichment. Add the drug to the absorbent cotton every 48 h and repeat root injection. Observe the growth of the black pine seedlings after 20 days. Using the same method, verify the toxicity of mepyrazole to black pine seedlings. The experimental results show that the indexes such as the length of the radicle and the moisture content of the plant in the mepyrazole-chitosan oligosaccharide derivative treatment group are higher than those in the mepyrazole treatment group, indicating that the toxicity of the mepyrazole-chitosan oligosaccharide derivative is significantly reduced compared with that of mepyrazole.

Claims

1. A fomepizole-chitosan oligosaccharide derivative, characterized in that, It is obtained by modifying the chitosan oligosaccharide backbone with the nematicidal active molecule methylpyrazole, and its structural formula is: Among them, m = 7; p = 6.

2. A method for preparing a fomepizole-chitosan oligosaccharide derivative, characterized in that, First, chloroacetyl chloride is selected as the acylating agent to be connected to the N-position of chitosan oligosaccharide to acetylate chitosan oligosaccharide, generating chloroacetyl chitosan oligosaccharide; then methylpyrazole is introduced through an ammonolysis reaction at the Cl position of chloroacetyl chitosan oligosaccharide.

3. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 2, characterized in that, The preparation steps are as follows: (1) Dissolve chitosan oligosaccharide in a solvent, then dropwise add chloroacetyl chloride, heat under reflux, then cool, filter by suction, and wash and dry the filter cake to obtain chloroacetyl chitosan oligosaccharide; (2) Dissolve chloroacetyl chitosan oligosaccharide in dimethylformamide, add methylpyrazole, and heat for reaction; after cooling, filter by suction, and wash and dry the filter cake to obtain the methylpyrazole-chitosan oligosaccharide derivative.

4. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 3, characterized in that, The solvent is prepared by mixing chloroform and pyridine.

5. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 3, characterized in that, In step (1), the heating reflux temperature is 70 °C and the reflux time is 15 - 20 h.

6. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 3, characterized in that, In step (2), the heating reaction temperature is 80 °C and the reaction time is 8 - 10 h.

7. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 3, wherein The mass-volume ratio of the chitosan oligosaccharide to the solvent is (1 - 3) g:10 mL.

8. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 3, characterized in that, The mass-volume ratio of the chitosan oligosaccharide to the total chloroacetyl chloride is 24 g:202.5 mL.

9. The preparation method of the mirtazapine-chitosan oligosaccharide derivative according to claim 3, characterized in that, The mass ratio of the chloroacetyl chitosan oligosaccharide to methylpyrazole is 1.5:1.35; the mass-volume ratio of the chloroacetyl chitosan oligosaccharide to dimethylformamide is 1.5 g:16.5 mL.

10. Use of the pyrazole-chitosan oligosaccharide derivative according to claim 1 in preventing and controlling pine wilt disease or killing pine wood nematodes, characterized in that, The LC of the pyrazole-chitosan oligosaccharide derivative against Bursaphelenchus xylophilus within 24 h 50 was 4.814 mM.

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