Application of steviosin or substance containing steviosin in preparation of nematode control product

Abstract

The invention relates to the technical field of biological control, and discloses an application of steviosin or a substance containing the steviosin in preparation of a nematode control product. According to a caenorhabditis elegans toxicity tracking method, stevia rebaudiana mother liquor sugar is separated according to the polarity of the substance; two compounds are separated and purified from mother liquor sugar by using normal phase silica gel, reverse phase silica gel, LH20 gel and liquid chromatography in sequence, the compounds are identified by using mass spectrum and nuclear magnetism, the compounds are determined to be Steretin F and Steretin E, the Steretin F and the Steretin E are proved to have nematicidal toxicity, the Steretin F has the strongest toxicity, LC50 is 1.57, the toxicity effect is stronger than that of thiazoline and weaker than that of abamectin, and the Steretin F and the Steretin E have the nematicidal toxicity. A new direction is provided for application of stevia rebaudiana, and an important theoretical and practical basis is provided for development and large-scale application of natural insecticidal compounds.

Description

Technical Field

[0001] The present invention relates to the application of steviol or a substance containing the steviol in the preparation of a product for preventing and controlling nematodes, and belongs to the technical field of biological control. Background Art

[0002] Plant-parasitic nematodes (PPNs) are major agricultural pathogens. Chemical nematicides, due to their rapid efficacy and known mode of action, remain the mainstream control means. However, their long-term use has led to environmental pollution, toxicity to non-target organisms, and nematode resistance. Therefore, screening nematicidal compounds from natural sources such as plants and microorganisms has become a research hotspot. Natural compounds, with their diverse active ingredients and modes of action, can reduce the risk to non-target organisms, accelerate biodegradation, and delay the emergence of resistance. In particular, plant-derived pesticides have received much attention in terms of insecticidal and antibacterial activities, environmental protection promotion, and resistance prevention.

[0003] So far, 348 plant species in 81 families have been found to contain nematicidal components, among which the Compositae family is particularly prominent. The secondary metabolites with nematicidal activity mainly include alkaloids, terpenoids, phenols, flavonoids, and sulfur-containing compounds. Terpenoids, as the secondary metabolites with the highest structural diversity, are composed of isoprene units and can be further divided into monoterpenes (such as thymol), caryophyllene, diterpenes, and triterpenes, etc., and are also the main components of essential oils. Natural products and their residues can not only enhance agricultural productivity but also regulate plant parasitic organisms.

[0004] Stevia rebaudiana Bertoni is a perennial herbaceous plant of the Asteraceae family and one of the most important economic crops for natural sweeteners in the world. Its chemical composition is complex, mainly including steviol glycosides, flavonoids, diterpenes and their derivatives, phenols and their derivatives, polysaccharides and volatile oils. These compounds show a wide range of biological activities, such as anti-diabetes, antioxidant, anti-hypertension, anti-caries, anti-obesity, anti-virus, anti-bacterial and anti-tumor. At present, steviol glycosides (SGs) extracted from stevia leaves by water extraction have become the most widely used zero-calorie natural high-intensity sweetener in the world. It is estimated that by 2027, the global consumption of SGs powder will reach 10,254.93 tons, with an annual growth rate of 7-8%. Mother liquor sugar (MLS), a byproduct of stevioside extraction, contains about 60% SGs and other bioactive ingredients such as flavonoids and polyphenols in its dry weight, but its sweetness is complex and has a bitter aftertaste, which cannot meet the commercial production standards of SGs. With the growth of SGs demand, large-scale stevia cultivation and the accumulation of MLS, the stevia industry faces severe environmental and sustainability challenges. Currently, in the context of the circular economy, MLS is an underestimated natural raw material resource, and the backlog of MLS has become a core issue in the industry. Exploring the value of MLS may bring more commercial value. How to redevelop and utilize mother liquor sugar has always been a problem that has plagued the stevia extraction industry, especially the separation and utilization of MLS bioactive components (especially those with nematicidal potential) is still limited.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide the use of stevioside or a substance containing stevioside in the preparation of a nematode control product, aiming to separate and identify nematicidal compounds from MLS and evaluate their toxicity, in order to discover potential biopesticide compounds, turn stevia by-products into treasure, and provide a theoretical basis for the development of natural nematicidal compounds.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] In a first aspect, a use of stevioside or a substance containing the stevioside in the preparation of a product for controlling nematodes, wherein the stevioside is selected from Sterebin E, Sterebin F or a mixture of the two.

[0009] Furthermore, the nematode is a plant parasitic nematode or Caenorhabditis elegans.

[0010] Further, the plant parasitic nematodes are selected from root-knot nematodes, cyst nematodes, Bursaphelenchus xylophilus, stem nematodes, root-lesion nematodes, sting nematodes, dagger nematodes and Aphelenchoididae.

[0011] Further, the product is an insecticide.

[0012] Further, the dosage form of the insecticide is selected from sprays, powders, granules or fumigants.

[0013] Further, the substance containing steviol glycoside is Stevia rebaudiana, stevia mother liquor sugar, ethyl acetate extract of stevia mother liquor sugar, and extract of ethyl acetate extract of stevia mother liquor sugar.

[0014] Further, the preparation method of the stevia mother liquor sugar includes: water-extracting stevia, filtering, extracting with macroporous resin and drying;

[0015] And / or, the preparation method of the ethyl acetate extract of stevia mother liquor sugar includes: extracting stevia mother liquor sugar with ethyl acetate, removing ethyl acetate after obtaining the ethyl acetate layer, and then redissolving and extracting with a mixed solvent of dichloromethane and methanol;

[0016] And / or, the preparation method of the extract of the ethyl acetate extract of stevia mother liquor sugar includes:

[0017] S1. The ethyl acetate extract of stevia mother liquor sugar is subjected to gradient elution on a normal-phase silica gel column with three ratios of water, chloroform and methanol as mixed eluents. After collecting the eluate and removing the mixed eluents, it is then redissolved with a mixed solvent of dichloromethane and methanol and dried to obtain a first gradient component, a second gradient component and a third gradient component;

[0018] Among them, the volume ratio of chloroform to methanol in the first gradient mixed eluent is 95:5; the volume ratio of chloroform to methanol to water in the second gradient mixed eluent is 80:20:1.5; the volume ratio of chloroform to methanol to water in the third gradient mixed eluent is 50:50:5;

[0019] S2. The second gradient component obtained in step S1 is subjected to gradient elution on a reverse-phase silica gel column with methanol at concentrations of 30%, 50%, 70% and 90% respectively. Each gradient is eluted with 3 column volumes, and the first, second and third column volume components of different concentrations of methanol are collected and dried respectively, including the first column volume elution component of 90% methanol concentration;

[0020] S3. The first column volume elution component of 90% methanol concentration obtained in step S2 is separated by an LH-20 gel column and isocratically eluted with anhydrous methanol to obtain an eluate;

[0021] Among them, when the eluent is sampled, it is collected at regular intervals or in a quantified manner into multiple components. After detection, similar components are combined. Finally, 5 components numbered from 90-1-1 to 90-1-5 are obtained after drying by rotary evaporation under reduced pressure. Among them, the 90-1-3 component is a combined set of components collected at 60-64% of the total amount of the eluent after the eluent is sampled;

[0022] S4. For the 90-1-3 component obtained in step S3, use an Eclipse Plus C 18 column, and elute with methanol gradients of 50%, 70%, 90% and 100% concentrations respectively to obtain the 70% methanol elution component;

[0023] The extract of the ethyl acetate extract of the stevia mother liquor sugar includes the second gradient component obtained in step S1, the first column volume elution component of 90% methanol concentration obtained in step S2, the eluent obtained in step S3, the 90-1-3 component, and the 70% methanol elution component obtained in step S4.

[0024] In a second aspect, a nematode insecticide includes: an active ingredient and a pharmaceutically acceptable additive or excipient, wherein the active ingredient is selected from Sterebin E or Sterebin F or a mixture of the two, or the stevia mother liquor sugar described in the first aspect, or the ethyl acetate extract of the stevia mother liquor sugar, or the extract of the ethyl acetate extract of the stevia mother liquor sugar.

[0025] Furthermore, the concentration of the active ingredient in the insecticide is 0.01 mg / mL or more (for example, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, etc.).

[0026] Furthermore, the excipient is selected from 8% methanol or water or a penetrant or a synergist; and / or, the additive is selected from a compounded insecticidal ingredient.

[0027] For the nematode insecticide of the present invention, there are no special limitations on the additives and excipients, and those already existing in the prior art can be used. For example, the penetrant can be common silicone-based (Silwet L-77, Trisiloxane), alcohol ether-based (AEO series, OP-10), ester-based (methyl oleate), sulfonate-based (sodium dodecylbenzene sulfonate, sodium dodecyl sulfate), etc. The synergist can be piperonyl butoxide (PBO) and silicone-based synergists, and the compounded insecticidal ingredient can be abamectin, fosthiazate, etc.

[0028] Compared with the prior art, the technical solution of the present invention has at least the following technical effects:

[0029] Through an acute toxicity tracking experiment using C. elegans as a model, the present invention separates the stevia mother liquor sugar, screens out the highly active components Sterebin F and Sterebin E, and proves that both Sterebin F and E have nematocidal toxicity, and Sterebin F has the strongest toxicity, with its LC 50 being 1.57, and its toxicity effect is stronger than that of thiazoline and weaker than that of abamectin. This will provide a sustainable alternative solution for synthetic pesticides, reducing environmental and health risks. Moreover, it will also provide a new direction for the application of stevia, providing an important theoretical and practical basis for the development and large-scale application of natural insecticidal compounds. The utilization of by-products such as MLS complies with the principles of circular economy, can improve resource efficiency, and promote soil health and biodiversity. At the industrial level, the development of plant-derived nematocidal preparations will promote green technology innovation, meet the needs of eco-friendly pest management, thereby reducing the dependence on harmful chemicals and building a global sustainable agricultural system. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a flow chart for extracting and separating natural nematocidal compounds from the MLS solution in the present invention;

[0031] Figure 2 is a graph showing the acute toxicity results of different concentrations of MLS solution on Caenorhabditis elegans after 24 hours;

[0032] Figure 3 is a graph showing the content of three extraction components (A) obtained by polar extraction of the MLS solution and the acute toxicity results of (B) on Caenorhabditis elegans at different concentrations after 24 hours. Note: In Figure (B), the results of the water extract on nematodes at different concentrations after 24 hours were statistically analyzed using a one-sided independent samples t-test;

[0033] Figure 4 is a graph showing the content of the normal-phase different gradient elution components of (A) the n-butanol extract and the ethyl acetate extract, and the acute toxicity results of (B) the n-butanol extract and (C) the ethyl acetate extract on Caenorhabditis elegans at different concentrations after 24 hours. Note: In Figure (B), the results of the normal-phase different gradient elution components of the n-butanol extract on nematodes at different concentrations after 24 hours were statistically analyzed using Welch's test;

[0034] Figure 5Acute toxicity of the components obtained by reverse-phase elution of the second gradient active components of ethyl acetate extract at different column volumes and methanol concentrations on Caenorhabditis elegans for 24 h at different concentrations. (A) Overall result diagram and (B) partial result diagram. Note: The results of the components obtained at different column volumes and methanol concentrations on nematodes for 24 h at different concentrations were statistically analyzed by one-sided independent sample t-test;

[0035] Figure 6 Acute toxicity result diagram of the components eluted from the first column volume of 90% methanol and the five components obtained by Sephadex LH-20 column chromatography on Caenorhabditis elegans for 24 h at different concentrations. Note: The results of the components obtained by Sephadex LH-20 column chromatography on nematodes for 24 h at different concentrations were statistically analyzed by one-sided independent sample t-test;

[0036] Figure 7 HPLC analysis diagrams of (A) 90-1-3F1 and (B) 90-1-3F2 separated from component 90-1-3 at 230 nm;

[0037] Figure 8 24-hour LC 50 diagram;

[0038] Figure 9 For 90-1-3F1 1 1H-NMR spectrum (600 MHz, CD3OD);

[0039] Figure 10 For 90-1-3F1 13 13C-NMR and DEPT spectra (150 MHz, CD3OD);

[0040] Figure 11 HSQC spectrum of 90-1-3F1;

[0041] Figure 12 HMBC spectrum of 90-1-3F1;

[0042] Figure 13 For 90-1-3F1 1 1H- 1 1H COSY spectrum;

[0043] Figure 14 ESI-MS spectrum of 90-1-3F1;

[0044] Figure 15 For 90-1-3F2 1 1H-NMR spectrum (600 MHz, CD3OD);

[0045] Figure 16 For 90-1-3F2 13 C-NMR and DEPT spectra (150 MHz, CD3OD);

[0046] Figure 17 HSQC spectrum for 90-1-3F2;

[0047] Figure 18 HMBC spectrum for 90-1-3F2;

[0048] Figure 19 For 90-1-3F2 1 H- 1 H COSY spectrum;

[0049] Figure 20 ESI-MS spectrum for 90-1-3F2;

[0050] Figure 21 Graphs of the body lengths of nematodes at 1, 2, 3, 5, and 7 days after treatment with Sterebin E (A) and Sterebin F (B), and graphs of the head swing frequencies of nematodes within 30 seconds at 1, 2, 3, 5, and 7 days after treatment with Sterebin E (C) and Sterebin F (D);

[0051] Note: In the above figures, unless otherwise specified in the statistical analysis, "one-way ANOVA and SNK multiple comparisons" are used. Detailed implementation manners

[0052] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The process parameters without specific conditions noted in the following embodiments are usually in accordance with conventional conditions.

[0053] In the ranges disclosed in the present invention, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in the present invention.

[0054] The present invention is the first attempt to isolate and identify specific monomeric compounds from the by-products of stevioside extraction. By recycling resources, MLS can be used as a raw material resource for manufacturing botanical insecticides, for the development of new safe insecticidal products, which can expand the product chain of the stevia industry and help farmers maximize the economic value of crops, and is of great significance.

[0055] Caenorhabditis elegans is a eukaryote that moves in soil and feeds on bacteria. It is easily isolated from decaying plant matter, as the decaying plant matter provides an abundant source of bacterial food for the nematodes. The nematodes have two sexes, male and hermaphrodite, with hermaphrodites accounting for about 99.8%. The adult nematodes are about 1 mm in length and have a relatively short lifespan of only 2-3 weeks. The development from embryo to sexually reproductive adult undergoes four larval stages, L1-L4, which takes about 3 days. Caenorhabditis elegans has the characteristics of rapid development, easy cultivation, and easy operation. And compared with PPNs, Caenorhabditis elegans is widely used for nematicide screening because it shares the same toxicological mechanisms with PPNs (such as interfering with the nervous system, disrupting the integrity of cell membranes, and inducing oxidative stress, specifically see references [1] Gilleard, J S. "The use of Caenorhabditis elegans in parasitic nematode research." Parasitology 128 Suppl 1. S1 (2004): S49; [2] Salinas, Gustavo, and Risi, Gastón. "Caenorhabditis elegans nature and nurture gift to nematode parasitologists." Parasitology (2017): 1-9; [3] Costa, Joana C., C. J. Lilley, and P. E. Urwin. "Caenorhabditis elegans as a model for plant-parasitic nematodes." Nematology 9.1 (2007): 3-16.). Its easy culturing property and genetic manipulability still make it an ideal model for high-throughput screening.

[0056] The present invention aims to isolate and identify nematicidal compounds from MLS, evaluate their toxicity and establish an extraction and purification process. Through acute toxicity tracking experiments using C. elegans as a model, highly active ingredients are screened to provide a sustainable alternative to synthetic pesticides and reduce environmental and health risks. The utilization of by-products such as MLS conforms to the principles of circular economy, can improve resource efficiency, and promote soil health and biodiversity. At the industrial level, the development of plant-derived nematicidal preparations will promote green technology innovation, meet the needs of eco-friendly pest management, thereby reducing the dependence on harmful chemicals and building a global sustainable agriculture system.

[0057] In the present invention, MLS is first separated according to the polarity of substances, and then the components in MLS are separated and purified in turn by normal-phase silica gel, reverse-phase silica gel, LH-20 gel, and liquid chromatography. Taking Caenorhabditis elegans as a model organism, the activities of different separated components are tracked, and the acute toxicity effects of different separated components on nematodes are evaluated and explored. By exploring the active ingredients of the by-products of Stevia rebaudiana leaf extraction and their acute toxicity on nematodes, potential leading compounds of biological insecticides are expected to be discovered, turning the waste of Stevia rebaudiana by-products into treasures, and providing a theoretical basis for the development of natural insecticidal compounds. The specific materials and methods are as follows:

[0058] 1. Materials and Methods

[0059] 1.1 Materials and Reagents

[0060] Mother liquor sugar (MLS) was provided by Shandong Haigen Biotechnology Co., Ltd. (Shandong, China). It is a by-product of recovering rebaudioside A (RA) and stevioside (STV) after water extraction, filtration, macroporous resin extraction and drying of "Puxing No. 6" Stevia rebaudiana. Caenorhabditis elegans and Escherichia coli OP 50 strains were derived from the Laboratory of Microbial Secondary Metabolism (College of Life Sciences, Yangtze University, Jingzhou, China). Analytical grade reagents such as ethyl acetate (batch number: 20221205), n-butanol (batch number: 20230810), methanol (batch number: 20221011), chloroform (batch number: 20230505), dichloromethane (batch number: 20240902) and petroleum ether (batch number: 20220118) were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Silica gel for chromatography (normal-phase filler, 200-300 mesh, Qingdao Ocean Chemical Industry, batch number: 20230508), C 18 silica gel (reverse-phase filler, 40-60μm, Qingdao Bangkai High-Tech Materials, batch number: PCH2024060432) and Sephadex LH-20 gel (Amersham Biosciences, UK, batch number: JS250176) were used for column chromatography separation.

[0061] 1.2 Culturing and Synchronization of Caenorhabditis elegans

[0062] The nematodes were routinely cultured on nematode growth medium (NGM) plates containing Escherichia coli OP 50 at 20 °C. Referring to the method of Calahorro et al. (Calahorro, F., Holden-Dye, L., O’Connor, V., 2021. Impact of drug solvents on C. elegans pharyngeal pumping. Toxicol. Rep. 8, 1240 - 1247.), the nematode eggs were synchronized to obtain L1 larvae and L4-stage nematodes.

[0063] 1.3 Determination of Nematode Lethality (24-Hour Exposure Experiment)

[0064] The synchronized L4-stage nematodes were selected for the experiment. 200 μL of the test substance solution and approximately 30 nematodes were added to each well of a 96-well plate, and an equal volume of solvent solution was used as a negative control. After incubating for 24 hours at 20 °C in the dark, the status of the nematodes was observed and the number of dead individuals was recorded. The criterion for nematode death was the cessation of pharyngeal pumping and a rigid, motionless body.

[0065] Lethality (%) = (Number of dead nematodes / Total number of test nematodes) × 100

[0066] 1.4 Evaluation of Body Length and Head Wiggling Frequency

[0067] Using a stereomicroscope (Olympus CX31, Japan), key indicators of nematode growth and development, namely body length and head swing frequency, were recorded. Approximately 30 L1-stage nematodes were transferred to wells containing 200 μL of Sterebin E and F solutions at different concentrations (0, 0.1, 0.3, 1 mg / mL) and incubated at 20 °C. Nematodes were collected on the 1st, 2nd, 3rd, 5th, and 7th days after treatment, washed 2 - 3 times, and then transferred to blank NGM plates. Their body lengths and the number of head swings within 30 seconds were recorded (Liu, S.L., Wu, Q.Q., Zhong, Y.R., He, Z.Z., Wang, Z., Li, R., Wang, M.H., 2023. Fosthiazate exposure induces oxidative stress, nerve damage, and reproductive disorders in nontarget nematodes. Environ. Sci. Pollut. Res. 30(5), 12522 - 12531.). Head swing was defined as a change in the bending direction of the midbody.

[0068] 1.5 Separation of bioactive components of the mother liquor sugar

[0069] Accurately weigh 500 g of MLS powder and extract it with 2500 mL of water at room temperature. Based on the component with the highest activity against nematodes, the bioactive components of the MLS solution were separated, and the specific process is as Figure 1 shown.

[0070] 1.5.1 Extraction of bioactive components of MLS with polar solvents

[0071] The MLS solution was extracted step by step with solvents of decreasing polarity (water, ethyl acetate, n-butanol). First, extract with 30 mL of water-saturated ethyl acetate (ethyl acetate: water = 5:1). After separating the ethyl acetate layer, the aqueous phase was extracted three times, and the ethyl acetate layers were combined and collected. Subsequently, the aqueous phase was extracted with 30 mL of water-saturated n-butanol (n-butanol: water = 5:1). After separating the n-butanol layer, it was repeated three times, and the n-butanol layers were combined and collected, and the aqueous phase was retained. Each layer was evaporated to dryness by rotary evaporation. The solid was dissolved in 3 mL of a mixed solvent (dichloromethane: methanol = 1:1) and extracted three times. After combining the solutions, they were naturally volatilized and dried. Finally, the extracts of the ethyl acetate layer, n-butanol layer, and aqueous layer were dissolved in sterile water to prepare 0, 10, 30, 50 mg / mL solutions for acute toxicity evaluation.

[0072] 1.5.2 Normal-phase chromatography separation

[0073] Referring to the method of Grauso et al. (Grauso, L., Cesarano, G., Zotti, M., Ranesi, M., Sun, W., Bonanomi, G., Lanzotti, V., 2020. Exploring Dittrichia viscosa (L.) Greuter phytochemical diversity to explain its antimicrobial, nematicidal and insecticidal activity. Phytochem. Rev. 19, 659 - 689.), chromatographic separation was carried out using methanol as the solvent. A silica gel column (300 mm × 4.6 mm, 5 μm) was wet - packed. After thorough rinsing, samples of the ethyl acetate layer and n - butanol layer were added respectively, that is, a methanol solution of the ethyl acetate extract at 5 mg / mL and a methanol solution of the n - butanol extract at 5 mg / mL were added. Gradient elution was carried out at a flow rate of 3 BV / h: the first gradient was chloroform:methanol (V / V) = 95:5 (4 - 5 column volumes), the second gradient was chloroform:methanol:water (V / V / V) = 80:20:1.5 (5 column volumes), and the third gradient was chloroform:methanol:water (V / V / V) = 50:50:5. The elution fractions of each gradient were collected respectively. After rotary evaporation and weighing, they were dissolved in 5 mL of a mixed solvent (dichloromethane:methanol = 1:1) and dried to obtain three gradient fractions of the ethyl acetate layer and three gradient fractions of the n - butanol layer. Solutions of 0, 1, 2.14, 4.68, 10 mg / mL were prepared with water as the solvent for the n - butanol layer fractions, and solutions of 0, 1, 1.26, 1.58, 2.0 mg / mL were prepared with water as the solvent for the ethyl acetate layer fractions for toxicity evaluation.

[0074] 1.5.3 Reverse - phase chromatographic separation

[0075] The second - gradient fraction of the ethyl acetate layer was dissolved in methanol to form a sample solution at 20 mg / mL. 250 mL of the sample solution was transferred to a C 18 silica gel column (250 mm × 4.5 mm, 5 μm) for reverse - phase liquid chromatographic separation. Gradient elution was carried out with 30%, 50%, 70%, 90% methanol (flow rate 3 BV / h). Each gradient was eluted for 3 column volumes and the fractions were collected respectively. After evaporation and drying, they were dissolved in methanol to form solutions of different concentrations, and the nematicidal toxicity was evaluated at concentrations of 0, 1, 3, 5 mg / mL.

[0076] 1.5.4 Sephadex LH - 20 column chromatography separation

[0077] The eluate fraction of the first column volume of 90% methanol (90-1, 20 mg / mL, 25 mL) was loaded onto a Sephadex LH-20 column (400 mm × 20 mm, 5 μm), eluted with anhydrous methanol (flow rate 1 mL / min), with a total elution volume of approximately 80 mL. One tube was collected every 1 - 2 mL at the sample outlet, and a total of 50 tubes were collected. The components of each tube were detected by TLC. After combining similar components, they were dried by rotary evaporation under reduced pressure at 40°C to obtain five components, namely 90-1-1 to 90-1-5. Among them, the 90-1-1 component is the combined fraction collected from the 1st to 26th tubes after the eluate was sampled, the 90-1-2 component is the combined fraction collected from the 27th to 29th tubes after the eluate was sampled, the 90-1-3 component is the combined fraction collected from the 30th to 32nd tubes (i.e., approximately at 60 - 64% of the total elution volume after the eluate was sampled), the 90-1-4 component is the combined fraction collected from the 33rd to 37th tubes after the eluate was sampled, and the 90-1-5 component is the combined fraction collected from the 38th to 50th tubes after the eluate was sampled.

[0078] 1.5.5 High Performance Liquid Chromatography (HPLC) Separation

[0079] The Agilent 1260 Infinity II system (USA) was used. Referring to the method of Cevasco-Contreras et al. (Cevasco-Contreras, M.D.P., Borgo, J., Celentano, A.M., Elso, O.G., Bach, H., Catalán, C.A.N., Bivona, A.E., Vaca, H.R., Rosenzvit, M.C., Sülsen, V.P., 2024. Extracts and terpenoids from Stevia species as potential anthelmintics for neglected tropical diseases caused by cestode parasites. Molecules 29(18), 4430.), separation was carried out on an Eclipse Plus C 18 column (250 mm × 4.6 mm, 5 μm). The 90-1-3 component was dissolved in methanol to form a 1 mg / mL 90-1-3 methanol solution (60 mL), and then eluted with a gradient of 50%, 70%, 90%, 100% methanol (flow rate 1.0 mL / min), and two characteristic peaks were observed.

[0080] 1.6 Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and Nuclear Magnetic Resonance (NMR) Analysis

[0081] UHPLC-MS was completed by Qingdao Standard Testing Co., Ltd., using a Thermo Scientific Dionex Ultimate 3000 system (USA) and a Thermo Scientific Acclaim C 18 column (2.1×100 mm, 2.2 μm). The dried sample (50 mg) of 90-1-3 was dissolved in 100 μL of methanol and injected (10 μL). The peak components were identified by retention time, mass-to-charge ratio (m / z), and fragmentation pattern. The NMR data were collected on a Bruker 600 MHz AVANCE NEO spectrometer (Germany) at the State Key Laboratory of Microbial Technology, Shandong University (Qingdao, China). The sample of 90-1-3 was dissolved in CD3OD solvent (25 °C) for testing.

[0082] 1.7 Preparative HPLC separation of Sterebin F

[0083] Using an UItiMate 3000 system (USA) and a SinoChrom ODS-BP column (250 mm×10 mm, 5 μm), 90-1-3 was dissolved in methanol and loaded. The loading concentration was 20 mg / ml. Sterebin E and Sterebin F were separated with 75% methanol as the mobile phase (flow rate 4.0 mL / min). The collected fractions were evaporated to dryness for subsequent analysis and activity testing.

[0084] 1.8 Statistical analysis

[0085] The experimental data were sorted and graphed using Microsoft Excel 2019. All experiments were independently repeated three times, and the results were expressed as mean ± standard deviation (SD). When the data met the homogeneity of variance, one-way analysis of variance (ANOVA) and SNK multiple comparisons were used; when the variance was heterogeneous, Welch's test was used; for non-normal data, an independent samples t-test was used. The significance level was set at 0.05, and the results of multiple comparisons were represented by letter marks.

[0086] 2. Extraction of bioactive components and evaluation of nematicidal activity

[0087] 2.1 Acute toxicity assessment of mother liquor sugar against nematodes

[0088] To evaluate the nematicidal toxicity of MLS, the mother liquor sugar aqueous solution was used as the initial test material (the negative control was pure water). According to the method in 1.3, a 24-hour acute toxicity experiment was carried out using Caenorhabditis elegans. The results are as Figure 2 shown. MLS showed moderate toxicity, and it was speculated that its toxicity originated from the compounds derived from Stevia leaves.

[0089] 2.2 Polar separation and acute toxicity assessment of mother liquor sugar

[0090] The extraction efficiency of bioactive components is significantly affected by the polarity of the solvent, and the polarity difference plays a key regulatory role in the recovery of chemical components. In this invention, according to different polarities, solvents with decreasing polarity (water, ethyl acetate, n-butanol) were used to stepwise extract the MLS solution. After evaporation, three different polar components were obtained: ethyl acetate extract, n-butanol extract and water extract, and their mass ratios were 3%, 72% and 25% in turn, as Figure 3 shown in

[0091] Figure A. Figure 3 Furthermore, the toxicity of different polar components was analyzed. The results of the average lethality rate after treating nematodes with different polar components for 24 h are shown in

[0092] 2.3 Gradient elution of normal-phase silica gel and acute toxicity assessment

[0093] Based on the toxicity assessment results in 2.2, the gradient elution products of ethyl acetate extract and n-butanol extract were separated by normal-phase chromatography, and different concentrations were prepared for a 24-h acute toxicity test to clarify the toxicity of each component. Among them, the active components of the ethyl acetate extract were the first gradient component, the second gradient component and the third gradient component in the elution order, with proportions of 16%, 43% and 41% respectively, while the n-butanol extract showed a distribution characteristic of 4%, 87% and 9%, specifically as Figure 4 shown in

[0094] Figure A. Figure 4 The toxicity detection results of each gradient separation component of n-butanol extract and ethyl acetate extract are shown in

[0095] 2.4 Gradient elution of reverse-phase silica gel and acute toxicity assessment

[0096] To further isolate the toxic compounds, reverse-phase chromatography was performed on the second gradient fraction of the ethyl acetate extract, and gradient elution was carried out successively with methanol at concentrations of 30%, 50%, 70%, and 90%. Each gradient was eluted with 3 column volumes, and the fractions were collected separately and evaporated to dryness. Evaluation at concentrations of 0, 1, 3, and 5 mg / mL revealed that the fractions eluted with 30% methanol (the second and third column volumes), 50% methanol, and 70% methanol had very low yields and low toxicity, while the remaining fractions had significant toxicity ( Figure 5 A). Among them, the fractions eluted with 90% methanol in the first and second column volumes had higher toxicity, while the fractions eluted with 30% methanol in the first column volume and 90% methanol in the third column volume had lower toxicity ( Figure 5 B), indicating that the toxic substances were mainly enriched in the fractions eluted with 90% methanol in the first two columns.

[0097] 2.5LH-20 Gel Separation and Acute Toxicity Evaluation

[0098] Based on the toxicity evaluation results in 2.4, the fraction eluted with 90% methanol in the first column volume (labeled 90-1) was selected for purification. After separation by Sephadex LH-20 column chromatography, five fractions were obtained (labeled 90-1-1, 90-1-2, 90-1-3, 90-1-4, and 90-1-5).

[0099] The results of the nematode toxicity tests for 90-1, 90-1-1, 90-1-2, 90-1-3, 90-1-4, and 90-1-5 are as Figure 6 shown. Among the five fractions separated by Sephadex LH-20 column chromatography, the toxicity of the 90-1-3 fraction was significantly higher than that of the other fractions, and its toxicity was similar to that of the parent fraction 90-1, indicating that the toxic components were effectively enriched in this fraction during the Sephadex LH-20 separation process.

[0100] 2.6 High Performance Liquid Chromatography (HPLC) Separation and Acute Toxicity Evaluation

[0101] 90-1-3 was separated by high performance liquid chromatography (HPLC) to obtain two monomeric compounds (temporarily named 90-1-3F1 and 90-1-3F2), with retention times of 37-38 minutes ( Figure 7 A) and 35-36 minutes ( Figure 7 B), respectively. The purities of 90-1-3F1 and 90-1-3F2 separated by preparative HPLC reached 95% and 91%, respectively.

[0102] The 24-hour acute toxicity experiment with fosthiazate and avermectin as positive controls showed that both 90-1-3F1 and 90-1-3F2 had nematicidal activity, among which 90-1-3F1 had higher activity. Its 24-hour median lethal concentration (LC 50 ) was 1.57, significantly lower than that of fosthiazate but higher than that of avermectin( Figure 8 ). This result indicates that 90-1-3F1 and 90-1-3F2 have the potential to be used as new nematicidal candidate compounds.

[0103] 3 Structure Identification

[0104] 3.1 Compound Identification Based on Physicochemical Properties and NMR Data

[0105] 90-1-3F1 is a white powder, and it shows a brownish-yellow color when developed with 10% H2SO4-EtOH. Based on its UHPLC-MS data, its molecular formula was deduced to be C 20 H 34 O4 (m / z 303.21 [M - 2H2O + 1]+), with an unsaturation degree of 4. The 1 H-NMR spectrum (600 MHz, CD3OD), 13 C-NMR and DEPT spectra (150 MHz, CD3OD), HSQC spectrum, HMBC spectrum, 1 H- 1 H COSY spectrum and ESI-MS spectrum are as shown in Figures 9 - 14 . Among them, the 1 H-NMR (600 MHz, CD3OD) and 13 C-NMR (150 MHz, CD3OD) spectrum data are shown in Table 1:

[0106] Table 1 1 H NMR (600 MHz) and 13 C NMR (150 MHz) Spectral Data Tables of 90-1-3F1 and 90-1-3F2 in CD3OD

[0107]

[0108] Based on the above, it was determined that 90-1-3F1 is Sterebin F, and its structural formula is as follows:

[0109]

[0110] 90-1-3F2 is a white powder and shows a brownish yellow color when developed with 10% H2SO4-EtOH. According to its UHPLC-MS data, it shows the same mass ion peak as 90-1-3F1 at m / z 303.21 [M-2H2O+1]+, indicating that its molecular formula is C 20 H 34 O4. Specifically, its 1 1H-NMR spectrum (600 MHz, CD3OD), 13 13C-NMR and DEPT spectra (150 MHz, CD3OD), HSQC spectrum, HMBC spectrum, 1 1H- 1 1H COSY spectrum and ESI-MS spectrum are as Figures 15 - 20 shown, in which, 1 1H-NMR (600 MHz, CD3OD) and 13 13C-NMR (150 MHz, CD3OD) spectral data are shown in Table 1. Therefore, it is determined that 90-1-3F2 is Sterebin E, and its structural formula is as follows:

[0111]

[0112] 3.2 Nematicidal activity differences and structure-activity relationships between Sterebin E and Sterebin F

[0113] Sterebin F isolated from MLS and Sterebin E are isomers of each other, both containing a conjugated diene system and belonging to diterpenoid compounds. These two diterpenoid compounds, Sterebin E and Sterebin F, both exhibit nematicidal activity. To further evaluate their effects, the two compounds were prepared into solutions with concentrations of 0, 0.1, 0.3, and 1 mg / mL in 8% methanol and added to the incubation of L1-stage larvae. The body lengths of nematodes and the number of head swings within 30 seconds were measured on the 1st, 2nd, 3rd, 5th, and 7th days after treatment with different concentrations of Sterebin E and F, respectively. The results are as Figure 21 shown.

[0114] Specifically, as can be seen from Figure 21 A and 21B, the body length of nematodes increased significantly with the prolongation of treatment time, but showed a significant inhibition with the increase of concentration. When the treatment concentration was 0.3 - 1.0 mg / mL, the body length of nematodes on the 7th day did not exceed 600 microns; when the treatment concentration was 1.0 mg / mL, the body length of the Sterebin F treatment group was shorter than that of the Sterebin E group. After treatment with Sterebin E, the body length of nematodes on the 7th day did not exceed 585 microns, while after treatment with Sterebin F, the body length of nematodes on the 7th day did not exceed 555 microns. As can be seen from Figure 20As shown in Figure C and Figure 20D, as the exposure time increased, the head swing frequency of nematodes gradually decreased. Compared with the control group (CK), both treatments with Sterebin E and F resulted in a significant decrease in the head swing frequency. When the treatment concentration was 0.1 - 1.0 mg / mL, the number of head swings of nematodes within 30 seconds on the 7th day did not exceed 55 times; and the inhibitory effect was more significant in the high-concentration treatment group (1 mg / mL). After the action of Sterebin E, the number of head swings of nematodes within 30 seconds on the 7th day did not exceed 45 times; while after the action of Sterebin F, the number of head swings of nematodes within 30 seconds on the 7th day did not exceed 40 times, indicating that both had an obvious inhibitory effect on the head swing of nematodes. It is worth noting that the inhibitory degree of Sterebin F on the head swing was significantly higher than that of Sterebin E, suggesting that Sterebin F was more toxic to nematodes. In summary, Sterebin F showed higher nematicidal activity.

[0115] The difference in the activity of Sterebin E and F may be due to the difference in their double bond configurations. The chemical shift values δH 6.13 (1H, d, J = 15.3 HZ), 5.70 (1H, dd, J = 15.3, 9.9 HZ), 5.56 (1H, dd, J = 6.8 HZ), 4.20 (1H, dd, J = 6.8, 2.5 HZ) and 1.81 (3H, s) of H-11, H-12, H-14 and H-15 in the C-9 side chain of Sterebin E showed obvious shifts compared with the signals of Sterebin F; the shifts of the C-16 methyl and C-12 olefin carbon signals of Sterebin F and Sterebin E (Δδ -8.1 and +7.8 ppm respectively) also confirmed the change in the C-13 configuration of the two, and the configurations of the two double bonds were determined to be 11E and 13E respectively; corresponding to the acute toxicity results of the two on L4-stage Caenorhabditis elegans, the cis-olefin structure of Sterebin F showed significant advantages in nematicidal activity, and its median lethal concentration (LC 50 ) was 1.57, about one-eighth of Sterebin E (LC 50 = 15.5). This result indicates that chiral characteristics may play a key role in the regulation of nematicidal activity, but its mechanism of action still needs to be further explored.

[0116] Fosthiazate, a typical commercialized organophosphorus nematicide, mainly exerts its effect by inhibiting acetylcholinesterase. In this invention, it is used as a reference to evaluate the application potential of Sterebin E / F. Research shows that the toxicity of natural terpenoids to nematodes, bacteria, and microbial systems is affected by the type and position of functional groups in their molecular structures. The nematicidal activity of terpenoids is closely related to the presence of oxygen-containing functional groups (such as aldehyde groups, ketone groups, or alcohol groups), and the double bond system can enhance their reactivity with nematodes by promoting biological processes related to electron transfer. Both Sterebin F and E contain aromatic rings, hydroxyl groups, and olefin side chains, and these structures may disrupt their integrity by binding to proteins and cell membranes, interfere with the functions of metabolic enzymes in nematode cells, affect energy generation, or disrupt the antioxidant system, thereby triggering oxidative stress and causing cell damage. The olefin double bond chain may interfere with nerve signal transmission by interacting with neurotransmitters or receptors. The activity difference between Sterebin F and E mainly stems from stereochemical differences, and the cis-olefin structure of F may be more conducive to the active process. Its specific biological activity mechanism still needs to be further analyzed.

[0117] Although the two compounds of this invention, Sterebin F and Sterebin E, were identified in 1988, there has been no further research on their activities and so on. Based on the method of activity tracking of Caenorhabditis elegans, this invention obtained highly active sterebin E and sterebin F from the mother liquor sugar of Stevia rebaudiana. As far as we know, this is the first report that these two substances have good nematicidal activity, and it was found that the activity of the cis-olefin structure at the C-13 position is higher than that of the trans structure. The existing mother liquor sugar of Stevia rebaudiana is a commercially available raw material and is relatively cheap. Therefore, the separation and extraction of sterebin E and F from it can be considered as an active ingredient of a suitable botanical pesticide.

[0118] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of steviol glycoside or a substance containing the steviol glycoside in the preparation of a product for preventing and treating nematodes, characterized in that, The steviol glycoside is selected from Sterebin E, Sterebin F or a mixture of the two.

2. The application according to claim 1, wherein: The nematodes are plant parasitic nematodes or Caenorhabditis elegans.

3. The application according to claim 2, characterized in that: The plant parasitic nematodes are selected from root-knot nematodes, cyst nematodes, Bursaphelenchus xylophilus, stem nematodes, root rot nematodes, sting nematodes, dagger nematodes and Aphelenchoididae nematodes.

4. The application according to claim 1, wherein: The product is an insecticide.

5. The application according to claim 4, wherein: The dosage form of the insecticide is selected from sprays, powders, granules or fumigants.

6. The application according to claim 1, characterized in that: The substance containing the steviol glycoside is stevia, stevia mother liquor sugar, ethyl acetate extract of stevia mother liquor sugar, extract of ethyl acetate extract of stevia mother liquor sugar.

7. The application according to claim 6, wherein: The preparation method of the stevia mother liquor sugar includes: subjecting stevia to water extraction, filtration, macroporous resin extraction and drying; and / or, the preparation method of the ethyl acetate extract of stevia mother liquor sugar includes: extracting stevia mother liquor sugar with ethyl acetate, removing ethyl acetate after obtaining the ethyl acetate layer, and then redissolving and extracting with a mixed solvent of dichloromethane and methanol; and / or, the preparation method of the extract of the ethyl acetate extract of stevia mother liquor sugar includes: S1. The ethyl acetate extract of stevia mother liquor sugar is subjected to gradient elution through a normal-phase silica gel column with a mixed eluent of water, chloroform and methanol in three ratios, and the eluent is collected and the mixed eluent is removed, and then redissolved and dried with a mixed solvent of dichloromethane and methanol to obtain a first gradient component, a second gradient component and a third gradient component; Among them, the volume ratio of chloroform to methanol in the first gradient mixed eluent is 95:5; the volume ratio of chloroform to methanol to water in the second gradient mixed eluent is 80:20:1.5; the volume ratio of chloroform to methanol to water in the third gradient mixed eluent is 50:50:5; S2. The second gradient component obtained in step S1 is subjected to gradient elution through a reverse-phase silica gel column with methanol at concentrations of 30%, 50%, 70% and 90% respectively, and each gradient is eluted for 3 column volumes, and the first, second and third column volume components with different methanol concentrations are collected and dried respectively, including the first column volume elution component with 90% methanol concentration; S3. The first column volume elution component with 90% methanol concentration obtained in step S2 is separated by an LH-20 gel column and isocratically eluted with anhydrous methanol to obtain an eluate; Among them, the eluate is collected at regular intervals or quantitatively as multiple components during sampling, and the similar components are combined after detection, and finally 5 components from 90-1-1 to 90-1-5 are obtained after drying by reduced pressure rotary evaporation, and the 90-1-3 component is a component collection collected at 60-64% of the total eluate volume after the eluate is sampled; S4. For the 90-1-3 component obtained in step S3, it is eluted with a methanol gradient of 50%, 70%, 90% and 100% respectively through an Eclipse Plus C 18 column to obtain the eluted fraction with 70% methanol concentration; The extract of the ethyl acetate extract of stevia mother liquor sugar includes the second gradient component obtained in step S1, the first column volume elution component with 90% methanol concentration obtained in step S2, the eluate obtained in step S3, the 90-1-3 component, and the elution component with 70% methanol concentration obtained in step S4.

8. A nematode insecticide, characterized in that, Including: An active ingredient and a pharmaceutically acceptable additive or excipient, wherein the active ingredient is selected from Sterebin E or Sterebin F or a mixture of the two, or the stevia mother liquor sugar described in claim 6, or an ethyl acetate extract of the stevia mother liquor sugar, or an extract of the ethyl acetate extract of the stevia mother liquor sugar.

9. The nematode insecticide according to claim 8, wherein The concentration of the active ingredient in the pesticide is 0.01 mg / mL or more.

10. The nematode insecticide according to claim 8, wherein, The excipient is selected from 8% methanol or water or a penetrant or a synergist; And / or, the additive is selected from compound insecticidal components.

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