Preparation and application of anisodus acutangula anti- root-knot nematode active alkaloid

Abstract

The application discloses a preparation method and application of anisodus tanguticus active alkaloid against root-knot nematodes, and belongs to the field of biological pesticides.The preparation method comprises the following steps: S1, drying anisodus tanguticus plants in Guangxi: after the anisodus tanguticus plants in Guangxi are prepared and cleaned, the plants are dried in a drying machine with a drying temperature of 50 DEG C until the weight is constant; S2, anisodus tanguticus powder preparation: the dried plants are crushed by using a high-speed crusher, and then sieved by using a 40-mesh sieve; the large particles after sieving are repeatedly crushed and sieved, and the powder after sieving is collected to obtain the anisodus tanguticus powder; and S3, obtaining an extract: the prepared anisodus tanguticus powder is extracted by using 95% ethanol as an extracting agent, and the extraction is carried out by using a Soxhlet extraction method in a micro-boiling state for 2 hours.The preparation method and application of the anisodus tanguticus active alkaloid against root-knot nematodes have the advantages that the antibacterial property and the insecticidal property of the anisodus tanguticus are fully utilized, and the anisodus tanguticus is more suitable for the demand of people for low-toxic and high-efficiency nematocidal agents.

Description

Technical Field

[0001] This invention relates to the field of plant-derived nematicides, and in particular to the preparation and application of an active alkaloid of Lycoris radiata that is effective against root-knot nematodes. Background Technology

[0002] Root-knot nematodes (Meloidogyne spp.) are one of the most important pathogenic organisms that harm crops. They account for about 5% of the total annual losses in agricultural production worldwide caused by various disasters.

[0003] Long-term use of traditional highly toxic nematicides can not only lead to increased nematode resistance, but also affect the environment and human health. Researchers are paying more attention to some low-toxicity and highly effective nematicides.

[0004] Plant-derived secondary metabolites often possess specific biological activities, thus providing abundant resources for the research and development of plant-derived insecticides, which can serve as excellent alternatives to organic synthetic chemical pesticides. Currently, only one plant-derived nematicide (matrine) is registered in China. Therefore, we have been dedicated to developing plant-derived nematicides with superior efficacy compared to matrine. Summary of the Invention

[0005] This invention discloses the preparation and application of an active alkaloid for resisting root-knot nematodes from Lycoris radiata, aiming to solve the technical problem of how to prepare a plant-derived nematicide with better effects than matrine.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The preparation of an active alkaloid of Lycoris radiata against root-knot nematodes includes the following specific steps:

[0008] S1: Drying Guangxi Lycoris plants: Prepare Guangxi Lycoris plants, clean them and set them aside. Dry them in a dryer at a drying temperature of 50℃ until constant weight.

[0009] S2: Preparation of Guangxi Lycoris powder: Take out the dried plant and crush it with a high-speed pulverizer, then sieve it with a 40-mesh sieve. Repeat the crushing and sieving steps for the large particles after sieving, and collect the sieved powder to obtain Guangxi Lycoris powder.

[0010] S3: Obtaining the extract: The prepared Guangxi Lycoris powder was extracted with 95% ethanol as the extractant and extracted by Soxhlet extraction at a gentle boiling state for 2 hours to obtain a mixed extract. The mixed extract was then subjected to rotary evaporation to obtain a reddish-brown paste extract.

[0011] S4: purification: collect the red-brown paste extract in S3, dissolve with 1% hydrochloric acid, filter, extract with equal volume of chloroform, take the upper acid water layer after standing and separating; adjust the pH value to 9-10 with 10% sodium hydroxide solution, extract with chloroform for 3 times, combine the 3 times of chloroform layer, and then get Guangxi lycoris alkaloids by rotary evaporation.

[0012] By using Guangxi lycoris to prepare lycorine, Guangxi lycoris is distributed in Guangxi, China, and the whole plant contains 8 kinds of alkaloids, among which lycorine, galanthamine and other compounds not only can be used as raw materials for pharmaceuticals, but also have certain antibacterial properties and toxicity to some moths. By using lycoris plant as raw material, the powder obtained after crushing and sieving is prepared into a red-brown paste extract by Soxhlet extraction and rotary evaporation. The Soxhlet extraction method has the advantages of saving solvent, high completion degree and high utilization rate of raw materials, which can save the preparation cost. Finally, lycorine is obtained by purifying the extract, which can fully utilize the antibacterial and insecticidal properties of Guangxi lycoris, and has excellent control effect, which is more suitable for people's demand for low toxicity and high efficiency of nematicide.

[0013] The application of a lycoris anti- root knot nematode active alkaloid, comprising the following specific steps:

[0014] S01: alkaloid qualitative detection: different kinds of reagents are mixed with the Guangxi lycoris alkaloids obtained in S4, and qualitative and quantitative detection are carried out respectively;

[0015] S02: toxicity determination: indoor toxicity determination is carried out by using lycorine;

[0016] S03: pot experiment: relative control effect data is obtained by pot experiment using lycorine;

[0017] In S01, the reagents used in the alkaloid qualitative detection are bismuth potassium iodide reagent, iodine-potassium iodide reagent, mercury potassium iodide reagent and silicotungstic acid reagent, and the standard lycorine is used as the control for quantitative detection, and the content is detected by ultraviolet spectrophotometer;

[0018] In S02, the specific determination method of toxicity determination is to dissolve lycorine with methanol and then dilute with distilled water to prepare a gradient concentration solution, with 3 times of repetition, 1% ethanol as control, and the insecticidal effect is determined at 24h, 48h and 72h;

[0019] In S03, the pot experiment comprises the following specific steps:

[0020] S031: treatment group setting: a total of 6 treatment groups are set, each treatment group is set as 1 pot, and each pot is set as 3 plants, and 4 repetitions are set;

[0021] S032: Irrigate the hairy node melon seedlings: select the uniform growth, four true leaves of hairy node melon seedlings are transplanted to the flowerpots containing 3000g nematode disease soil, and each concentration of lycoris base solution, abamectin agent, sophoramine agent and water is irrigated during transplanting;

[0022] S033: Obtain the control effect information: after 30 days, the control effect information of lycoris base is obtained, wherein the control effect (%)=(control disease index-treatment disease index) / control disease index*100;

[0023] In the S031, the effective content of the 6 treatment groups set in the treatment setting is respectively lycoris base 0.5mg / plant, 1mg / plant, 2mg / plant, sophoramine 1mg / plant and water control.

[0024] The control effect of lycoris base is tested by using qualitative detection, quantitative detection, toxicity detection and potting test, especially by setting multiple treatment groups to simulate the control effect comparison of lycoris base with other agents in the same use environment, and the comparison values obtained in multiple tests describe lycoris base in many aspects, so that the control effect of lycoris base can be accurately obtained, which has certain representativeness.

[0025] As can be seen from the above, a preparation of lycoris anti- root knot nematode active alkaloid includes the following specific steps:

[0026] S1: Dry Guangxi lycoris plants: prepare the washed Guangxi lycoris plants, set the drying temperature to 50℃ in the dryer, and dry to constant weight;

[0027] S2: Guangxi lycoris powder preparation: take out the dried plants, crush them with a high-speed crusher, and then sieve them with a 40-mesh sieve. The large particles after sieving are repeatedly crushed and sieved, and the sieved powder is collected to obtain Guangxi lycoris powder;

[0028] S3: Obtain the extract: prepare the Guangxi lycoris powder, use 95% ethanol as the extracting agent, and extract it by Soxhlet extraction method under micro-boiling state for 2 hours to obtain a mixed extract. The mixed extract is then rotary evaporated to obtain a red-brown paste extract;

[0029] S4: Purification: collect the red-brown paste extract in S3, dissolve it with 1% hydrochloric acid, filter it, extract it with an equal volume of chloroform, and separate the layers after standing. Adjust the pH value to 9-10 with 10% sodium hydroxide solution, and extract it with chloroform for 3 times. Combine the 3 chloroform layers, and then rotary evaporate to obtain Guangxi lycoris alkaloid. The preparation and application of the lycoris anti-root knot nematode active alkaloid provided by the present application have the technical effects of fully utilizing the antibacterial and insecticidal properties of Guangxi lycoris, excellent control effect, and being more suitable for people's demand for low-toxicity and high-efficiency nematicides. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 A flow chart of preparation of the Lycoris squillaceum active alkaloid against root-knot nematode is proposed for the present application.

[0031] Figure 2 A flow chart of application of the Lycoris squillaceum active alkaloid against root-knot nematode is proposed for the present application.

[0032] Figure 3 A potting test flow chart of application of the Lycoris squillaceum active alkaloid against root-knot nematode is proposed for the present application.

[0033] Figure 4 A quantitative detection result chart of alkaloid qualitative detection test of application of the Lycoris squillaceum active alkaloid against root-knot nematode is proposed for the present application.

[0034] Figure 5 A toxicity curve chart of toxicity determination test of application of the Lycoris squillaceum active alkaloid against root-knot nematode is proposed for the present application. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments.

[0036] The preparation and application of the Lycoris squillaceum active alkaloid against root-knot nematode disclosed in the present application are mainly applied to the scene of preparation and application of lycorine.

[0037] Reference Figure 1 The preparation of the Lycoris squillaceum active alkaloid against root-knot nematode comprises the following specific steps:

[0038] S1: drying Lycoris squillaceum plants: prepare Lycoris squillaceum plants after cleaning for standby, set the drying temperature to 50℃ in the drying machine and dry to constant weight;

[0039] S2: Lycoris squillaceum powder preparation: take out the dried plants and use a high-speed pulverizer to crush, and then use a 40-mesh sieve to sieve; the large particles after sieving are repeatedly crushed and sieved, and the sieved powder is collected to obtain Lycoris squillaceum powder;

[0040] S3: obtaining the extract: the prepared Lycoris squillaceum powder is extracted with 95% ethanol as the extracting agent, and is extracted by the Soxhlet extraction method under a micro-boiling state for 2 hours to obtain a mixed extract; the mixed extract is then subjected to rotary evaporation to obtain a red-brown paste-shaped extract;

[0041] S4: purification: collect the reddish-brown paste extract in S3, dissolve with 1% hydrochloric acid, filter, extract with an equal volume of chloroform, separate the upper acid water layer after standing, adjust the pH value to 9-10 with 10% sodium hydroxide solution, extract with chloroform for 3 times, combine the 3 times of chloroform layer, and then evaporate to obtain Guangxi lycoris alkaloids. Guangxi lycoris is distributed in Guangxi, China, and the whole plant contains 8 kinds of alkaloids. Among them, lycorine, galanthamine and other alkaloids not only can be used as raw materials for pharmaceuticals, but also have certain antibacterial properties and toxicity to some moths. By using lycoris plants as raw materials, the powder obtained after crushing and sieving is extracted by Soxhlet extraction method and rotary evaporation to prepare reddish-brown paste extract. The Soxhlet extraction method has the advantages of saving solvent, high degree of completion and high utilization rate of raw materials, which can save the preparation cost. Finally, lycorine obtained by purifying the extract can fully utilize the antibacterial and insecticidal properties of Guangxi lycoris, and has excellent control effect, which is more suitable for people's demand for low toxicity and high efficiency of nematicides.

[0042] Reference Figure 2 The application of lycoris alkaloids with nematode resistance activity, comprising the following specific steps:

[0043] S01: alkaloid qualitative detection: different kinds of reagents are used to mix with the Guangxi lycoris alkaloids obtained in S4, and qualitative and quantitative detection are carried out respectively;

[0044] S02: toxicity determination: indoor toxicity determination is carried out by using lycorine;

[0045] S03: pot experiment: relative control effect data is obtained by pot experiment using lycorine.

[0046] Reference Figure 2 In a preferred embodiment, in S01, the reagents used in alkaloid qualitative detection are bismuth potassium iodide reagent, iodine-potassium iodide reagent, mercury potassium iodide reagent and silicotungstic acid reagent, and the content is detected by ultraviolet spectrophotometer with lycorine standard as control.

[0047] Reference Figure 2 In a preferred embodiment, in S02, the specific determination method of toxicity determination is to dissolve lycorine with methanol and then dilute with distilled water to prepare a gradient concentration solution, with 3 times of repetition, 1% ethanol as control, and the insecticidal effect is determined at 24h, 48h and 72h.

[0048] Reference Figure 3 In a preferred embodiment, in S03, the pot experiment comprises the following specific steps:

[0049] S031: treatment group setting: 6 treatment groups are set, and each treatment group is set to 1 pot, and 3 plants per pot, 4 repeats are set;

[0050] S032: irrigation of luffa seedlings: select luffa seedlings with uniform growth, four true leaves, transplant to pots containing 3000g nematode disease soil, and irrigate with each concentration of lycoris base solution, abamectin, sophoramine and water at the time of transplanting;

[0051] S033: obtain the prevention effect information: after 30 days, test and obtain the prevention effect information of lycoris base, wherein the prevention effect (%) = (control disease index - treatment disease index) / control disease index * 100.

[0052] Reference Figure 3 In a preferred embodiment, S031, the effective content of the 6 treatment groups set in the treatment setting is lycoris base 0.5mg / plant, 1mg / plant, 2mg / plant, sophoramine 1mg / plant and water control. The prevention effect of lycoris base is tested by using qualitative detection, quantitative detection, toxicity detection and potting test, and especially by setting multiple treatment groups to simulate the prevention effect comparison of lycoris base with other pesticides in the same use environment. The comparison values obtained in multiple tests describe lycoris base in many aspects, so that the prevention effect of lycoris base can be accurately obtained, which has certain representativeness.

[0053] Example:

[0054] 1. First, mix Guangxi lycoris alkaloids with bismuth potassium iodide reagent, iodine-potassium iodide reagent, mercury potassium iodide reagent and silicotungstic acid reagent, and perform qualitative detection. The detection results are shown in the following table:

[0055] Table 1: qualitative detection results

[0056]

[0057] 2. Then, quantitative detection is performed. The detection results are shown in Figure 4 as a control calibration. The content of Guangxi lycoris alkaloids is calculated to be 3.82%;

[0058] 3. Indoor toxicity determination, lycoris base is dissolved with methanol and then diluted with distilled water to prepare a gradient concentration solution. 1% methanol is used as a control. The insecticidal effect is determined at 24h, 48h and 72h, wherein LC50 = 116.01μg / mL, and the toxicity curve is shown in Figure 5 ;

[0059] 4. A pot experiment was conducted, and 6 treatment groups were set up, respectively, Lycoris base 0.5 mg / plant, 1 mg / plant, 2 mg / plant, matrine 1 mg / plant and water control, and each treatment group was set as 1 pot, 3 plants per pot, 4 replicates; Select uniform growth, four true leaves of the hairy node melon seedlings were transplanted into pots containing 3000g nematode disease soil, and each concentration of Lycoris base solution, abamectin, matrine and water were irrigated at the time of transplanting, and the inspection was carried out after 30 days;

[0060] 5. The test results show that the control effect of Lycoris base is higher than that of the commonly used plant source nematocide matrine, and the pot experiment results are shown in the following table.

[0061] Table 2: Pot experiment results

[0062]

[0063] Working principle: Guangxi Lycoris is distributed in Guangxi, China, and the whole plant contains 8 kinds of alkaloids, among which Lycoris base, galanthamine and other compounds can not only be used as pharmaceutical raw materials, but also have certain antibacterial properties and toxicity to some moths. By using Lycoris plants as raw materials, the powder obtained after crushing and sieving is prepared into a red-brown paste by Soxhlet extraction and rotary evaporation. The Soxhlet extraction method has the advantages of saving solvent, high degree of completion and high utilization rate of raw materials, which can save the preparation cost. Finally, the Lycoris base obtained by purifying the extract can fully utilize the antibacterial and insecticidal properties of Guangxi Lycoris, and has excellent control effect, which is more suitable for people's demand for low toxicity and high efficiency of nematocide. In addition, the control effect of Lycoris base is tested by qualitative detection, quantitative detection, toxicity detection and pot experiment, especially by setting multiple treatment groups to simulate the control effect comparison of Lycoris base with other pesticides under the same use environment, and the comparison values obtained in multiple tests describe Lycoris base in many aspects, so that the control effect of Lycoris base can be accurately obtained, which has certain representativeness.

[0064] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any skilled person in the art can make equivalent replacement or change according to the technical solution and inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. The application of an active alkaloid from *Lycoris radiata* in the fight against root-knot nematodes, characterized in that, The preparation method of the Lycoris radiata anti-root-knot nematode active alkaloid includes the following specific steps: S1: Drying Guangxi Lycoris plants: Prepare Guangxi Lycoris plants, clean them and set them aside. Dry them in a dryer at a drying temperature of 50℃ until constant weight. S2: Preparation of Guangxi Lycoris powder: Take out the dried plant and crush it with a high-speed pulverizer, then sieve it with a 40-mesh sieve. Repeat the crushing and sieving steps for the large particles after sieving, and collect the sieved powder to obtain Guangxi Lycoris powder. S3: Obtaining the extract: The prepared Guangxi Lycoris powder was extracted with 95% ethanol as the extractant and extracted by Soxhlet extraction at a gentle boiling state for 2 hours to obtain a mixed extract. The mixed extract was then subjected to rotary evaporation to obtain a reddish-brown paste extract. S4: Purification: Collect the reddish-brown paste-like extract from S3, dissolve it in 1% hydrochloric acid, filter it, extract it with an equal volume of chloroform, let it stand to separate the layers, and take the upper acidic aqueous layer. The pH was adjusted to 9-10 with 10% sodium hydroxide solution, and then extracted three times with chloroform. The three chloroform layers were combined and then rotary evaporated to obtain the Guangxi Lycoris alkaloids. The dosage of the Guangxi Lycoris alkaloids is 1 mg / plant or 2 mg / plant.

2. The application according to claim 1, characterized in that, The specific steps include the following: S01: Qualitative detection of alkaloids: Different types of reagents were used to mix the Guangxi Lycoris alkaloids obtained in S4, and qualitative and quantitative detections were performed respectively. S02: Toxicity determination: Indoor toxicity determination was conducted using Lycoris radiata alkaloids from Guangxi. S03: Pot experiment: Data on the relative control effect of lycoris radiata alkaloids from Guangxi were obtained through a pot experiment.

3. The application according to claim 2, characterized in that, In S01, the reagents used for qualitative detection of alkaloids are potassium bismuth iodide reagent, potassium iodide-mercuric iodide reagent, and silicotungstic acid reagent, respectively. For quantitative detection, lycorine standard is used as a control, and the content is detected by ultraviolet spectrophotometer.

4. The application according to claim 2, characterized in that, In S02, the specific method for determining toxicity is to dissolve the Guangxi Lycoris alkaloids in methanol and then dilute them with distilled water to prepare solutions with gradient concentrations. The solution is repeated three times, with 1% ethanol as a control, and the insecticidal effect is determined at 24h, 48h, and 72h.

5. The application according to claim 2, characterized in that, In step S03, the pot experiment includes the following specific steps: S031: Treatment group settings: There are a total of 6 treatment groups, and each treatment group is set to 1 pot, with 3 plants per pot and 4 repetitions. S032: Irrigating hairy gourd seedlings: Select hairy gourd seedlings with uniform growth and four true leaves and transplant them into flowerpots containing 3000g of nematode-infected soil. At the time of transplanting, irrigate with lycorine solution, abamectin, matrine solution and water of different concentrations respectively. S033: Obtain efficacy information: Test after 30 days to obtain the control efficacy information of Lycoris radiata alkaloids in Guangxi, where the control efficacy (%) = (control disease index - treatment disease index) / control disease index * 100.

6. The application according to claim 5, characterized in that, In S031, the effective contents of the six treatment groups set in the treatment settings are 0.5 mg / plant, 1 mg / plant, and 2 mg / plant of Guangxi Lycoris radiata alkaloids, 1 mg / plant of matrine, and water control, respectively.

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