A tea saponin oxazole derivative, and a preparation method and application thereof

Abstract

The application discloses a tea saponin genin oxazole derivative, a preparation method and application thereof. The tea saponin genin oxazole derivative provided by the application has a molecular structure as shown in formula (I), wherein R1 is H or Cl, and R2 is H or a phenyl group. The tea saponin genin oxazole derivative is synthesized by using tea saponin genin and 4-oxazole carboxylic acid compounds as raw materials. The tea saponin genin oxazole derivative has a good control effect on root-knot nematodes, the anti-root-knot nematode activity of the tea saponin genin oxazole derivative is more than 10 times that of tea saponin, and the tea saponin genin oxazole derivative can inhibit the superoxide dismutase activity of root-knot nematodes, and the mechanism is different from that of abamectin. The derivative can be prepared into an agriculturally acceptable preparation such as a water dispersible granule or a soluble powder, and can efficiently control root-knot nematodes.

Description

Technical Field

[0001] This invention belongs to the field of biopesticide technology, specifically relating to a tea saponin oxazole derivative, its preparation method, and its application. Background Technology

[0002] Root-knot nematode disease has become a bottleneck restricting the rapid development of my country's vegetable planting industry. my country has a large vegetable planting area, but most vegetable-producing areas face the threat of nematode diseases, resulting in huge economic losses every year. Root-knot nematodes can cause vegetable yield reductions of more than 50%, and in some severely affected areas, even total crop failure. Current methods for controlling root-knot nematodes include: resistance breeding with long cycles and low success rates; high-temperature killing requiring enormous human, material, and financial resources, making it difficult to promote; biological control, although harmless to humans and animals and environmentally friendly, has limited practical effectiveness; and chemical control faces problems such as pesticide resistance, environmental pollution, and food residues.

[0003] On the other hand, the scale of my country's camellia oil industry continues to expand, generating a large amount of camellia seed meal while producing camellia oil. However, the resource utilization of camellia seed meal is still in its initial stage, and its active ingredients have not been developed and utilized, resulting in a great waste of resources and restricting the development of the camellia oil industry. Tea saponin is the main active ingredient in camellia seed meal, which has a certain inhibitory effect on root-knot nematodes, but the effect is weak (Chen Lianghong. Study on the nematicidal activity of tea saponin against southern root-knot nematodes [J]. Agricultural Disaster Research, 2018, 8(01): 1-5), and its insecticidal effect needs to be improved. However, no research has been reported on its effect on vegetable root-knot nematodes through structural modification. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the existing technology, this paper aims to provide a tea saponin oxazole derivative, its preparation method and application.

[0005] The first objective of this invention is to provide a tea saponin oxazole derivative having a molecular structure as shown in formula (I), wherein R1 is H or Cl and R2 is H or phenyl; Formula (I).

[0006] Preferably, the tea saponin oxazole derivative is any one or more of the compounds shown in formula (1) to formula (3); Equation (1); Equation (2); Equation (3).

[0007] Preferably, the preparation method of the tea saponin oxazole derivative includes the following steps: S1. Dissolve 10 g of tea saponin and 15-20 g of tert-butyldimethylchlorosilane in 50-150 mL of N,N-dimethylformamide, add 15-20 g of imidazole and 0.2-0.5 g of 4-dimethylaminopyridine, react at 20℃-30℃ for 10-14 h, dilute with 50-450 mL of distilled water, extract with 50-450 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain intermediate 1; In step S1, the amount of tert-butyldimethylchlorosilane used is 1.5 to 2 times the mass of tea saponin; the volume-to-mass ratio of N,N-dimethylformamide to tea saponin is 5 to 15 mL: 1 g; the amount of imidazole used is 1.5 to 2 times the mass of tea saponin; the amount of 4-dimethylaminopyridine used is 0.02 to 0.05 times the mass of tea saponin; and the amounts of distilled water and ethyl acetate are 1 to 3 times the volume of 4-dimethylaminopyridine, respectively. S2. Dissolve 10 g of intermediate 1 with 2-4 g of sodium triacetoxyborohydride in 50-150 mL of dichloromethane, react at 20-30°C for 1-3 h, dilute with 50-450 mL of distilled water, extract with 50-450 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain intermediate 2. In step S2, the amount of sodium triacetoxyborohydride is 0.2 to 0.4 times the mass of intermediate 1; the volume-to-mass ratio of dichloromethane solvent to intermediate 1 is 5 to 15 mL: 1 g; and the amounts of distilled water and ethyl acetate are 1 to 3 times the volume of dichloromethane, respectively. S3. Dissolve 10 g of intermediate 2 and 1-2 g of 4-oxazolyl carboxylic acid compound in 50-150 mL of pyridine, add 1-2 g of 4-dimethylaminopyridine and 1.5-2.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 30℃-50℃ for 24-36 h, dilute with 50-450 mL of distilled water, extract with 50-450 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain intermediate 3; In step S3, the 4-oxazole carboxylic acid compound is 4-oxazole carboxylic acid, 2-chloro-4-oxazole carboxylic acid, or 5-phenyloxazole-4-carboxylic acid, and its amount is 0.1 to 0.2 times the mass of intermediate 2; the volume-to-mass ratio of pyridine to intermediate 2 is 5 to 15 mL: 1 g; the amount of 4-dimethylaminopyridine is 0.1 to 0.2 times the mass of intermediate 2; the amount of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.15 to 0.25 times the mass of intermediate 2; and the amounts of distilled water and ethyl acetate are 1 to 3 times the volume of pyridine, respectively. S4. Dissolve 10 g of intermediate 3 with 1-4 mL of tetrabutylammonium fluoride in 30-50 mL of anhydrous tetrahydrofuran, react at 20-30°C for 1-3 h, dilute with 30-150 mL of distilled water, extract with 30-150 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain the tea saponin oxazole derivative. In step S4, the volume-to-mass ratio of tetrabutylammonium fluoride to intermediate 3 is 0.1-0.4 mL:1 g, and the volume-to-mass ratio of anhydrous tetrahydrofuran to intermediate 3 is 3-5 mL:1 g; the amounts of distilled water and ethyl acetate are 1-3 times the volume of anhydrous tetrahydrofuran.

[0008] Preferably, the washing, dehydration and evaporation of the organic layer are performed as follows: the organic layer is washed with 12.5~225 mL of saturated sodium chloride aqueous solution, then dehydrated with 5~10 g of anhydrous sodium sulfate for 8~12 h, and ethyl acetate is removed by evaporation at 50℃~60℃; The amount of saturated sodium chloride aqueous solution used is 0.25 to 0.5 times the volume of ethyl acetate; the amount of anhydrous sodium sulfate used is 0.5 to 1 times the mass of tea saponin or intermediate.

[0009] The principle of the preparation method of the tea saponin oxazole derivative is as follows: the hydroxyl group on the tea saponin is protected by tert-butyldimethylchlorosilane to ensure the correct site for subsequent reactions, thus obtaining intermediate 1; then, the aldehyde group on intermediate 1 is reduced to a hydroxyl group by sodium triacetoxyborohydride, thus obtaining intermediate 2; then, a 4-oxazole carboxylic acid compound reacts with the reduced hydroxyl group to obtain intermediate 3; finally, the tert-butyldimethylchlorosilane on intermediate 3 is removed by tetrabutylammonium fluoride, thus obtaining the tea saponin oxazole derivative.

[0010] Preferably, the tea saponin is a compound obtained by acid-base hydrolysis and extraction of tea oil saponin, and its preparation method includes the following steps: S1. Dissolve tea oil saponin in an 80%–90% (v / v) methanol aqueous solution according to a mass-to-volume ratio of 1 g: 15–25 mL. Adjust the pH of the solution to 1–3 with 2–4 mol / L hydrochloric acid aqueous solution. Reflux the reaction at 70–85 °C for 5–6 h. Add 5–8 times the volume of distilled water to the methanol aqueous solution and let it stand for 12–24 h to obtain a precipitate. S2. According to the mass-volume ratio of precipitate to methanol aqueous solution 1 g: 15~25 mL, dissolve the precipitate obtained in step S1 in a methanol aqueous solution with a volume fraction of 80%~90%, adjust the pH of the solution to 11~13 with a sodium hydroxide aqueous solution with a mass fraction of 5~10%, reflux at 70℃~85℃ for 5~6 h, add 5~8 times the volume of distilled water of methanol aqueous solution, let stand for 12~24 h, and obtain the precipitate; S3. Take the precipitate obtained in step S2 according to the mass-to-volume ratio of precipitate to chloroform 1 g: 5~10 mL and extract it with 100~200 mL of chloroform. After the solvent evaporates, tea saponin is obtained. The structure of the obtained tea saponin is shown in formula (4): Equation (4).

[0011] A second objective of this invention is to provide an agent for controlling root-knot nematodes, which contains the aforementioned tea saponin oxazole derivative.

[0012] Preferably, the preparation for controlling root-knot nematodes comprises: 30 parts by weight of tea saponin oxazole derivative, 5 parts by weight of sodium lignosulfonate, 2 parts by weight of anhydrous sodium sulfate, 1 part by weight of sodium dodecyl sulfate and 62 parts by weight of kaolin.

[0013] Preferably, the preparation for controlling root-knot nematodes comprises: 10 parts by weight of tea saponin oxazole derivative, 5 parts by weight of polyvinylpyrrolidone K30, and 85 parts by weight of anhydrous glucose.

[0014] A third objective of this invention is to provide the application of the aforementioned tea saponin oxazole derivative in the preparation of formulations for controlling root-knot nematodes.

[0015] A fourth objective of this invention is to provide the application of the aforementioned tea saponin derivative or the aforementioned formulation in the control of root-knot nematodes.

[0016] Compared with the prior art, the advantages of the present invention are: (1) The tea saponin oxazole derivative of the present invention has a good control effect on root-knot nematodes. Its anti-root-knot nematode activity is more than 10 times higher than that of tea saponin, which is based on the synergistic effect of tea saponin oxazole derivative on multiple targets.

[0017] (2) The preparation method of the present invention is simple, can be industrialized and prepared, and is easy to promote and apply. Attached Figure Description

[0018] Figure 1 These are the infrared spectra of tea saponin and the tea saponin oxazole derivatives prepared in Examples 1-3.

[0019] Figure 2 This is the 1H NMR spectrum of tea saponin.

[0020] Figure 3 This is the 1H NMR spectrum of the tea saponin derivative prepared in Example 1.

[0021] Figure 4This is the 1H NMR spectrum of the tea saponin derivative prepared in Example 2.

[0022] Figure 5 This is the 1H NMR spectrum of the tea saponin derivative prepared in Example 3.

[0023] Figure 6 This is a microscopic image of Southern Root-knot Nematodes that have not been treated with tea saponin-oxazole derivatives.

[0024] Figure 7 This is a microscope image of Southern Root-Knot Nematodes after treatment using Example 1.

[0025] Figure 8 This is a microscope image of Southern Root-Knot Nematodes after treatment using Example 2.

[0026] Figure 9 This is a microscope image of Southern Root-Knot Nematodes after treatment using Example 3.

[0027] Figure 10 This is an electron microscope image of live southern root-knot nematodes that have not been treated with tea saponin oxazole derivatives.

[0028] Figure 11 This is an electron microscope image of *Symplocos spp.* after treatment in Example 1.

[0029] Figure 12 This is an electron microscope image of *Symplocos spp.* after treatment in Example 2.

[0030] Figure 13 This is an electron microscope image of *Strombus heterophylla* treated in Example 3. Detailed Implementation

[0031] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0032] In the following examples, the camellia saponin was produced by Jiangxi Xinzhongye Tea Industry Technology Co., Ltd.; 4-oxazole carboxylic acid (CAS: 23012-13-7) was purchased from Maclean Biochemical Technology Co., Ltd.; 2-chloro-4-oxazole carboxylic acid (CAS: 706789-07-3) was purchased from Beijing Mairuida Technology Co., Ltd.; and 5-phenyloxazole-4-carboxylic acid (CAS: 99924-18-2) was purchased from Anaiji (Shanghai) Pharmaceutical Chemical Co., Ltd.

[0033] The preparation method of tea saponins is as follows: (1) According to the mass-volume ratio of tea oil saponin to methanol aqueous solution 1 g: 15~25 mL, dissolve tea oil saponin in methanol aqueous solution with a volume fraction of 80%~90%, adjust the pH of the solution to 1~3 with 2~4 mol / L hydrochloric acid aqueous solution, reflux at 70℃~85℃ for 5~6 h, add 5~8 times the volume of distilled water of methanol aqueous solution, let stand for 12~24 h, and obtain precipitate; (2) According to the mass-volume ratio of precipitate to methanol aqueous solution 1 g: 15~25 mL, take the precipitate obtained in step (1) and dissolve it in methanol aqueous solution with a volume fraction of 80%~90%. Adjust the pH of the solution to 11~13 with sodium hydroxide aqueous solution with a mass fraction of 5~10%. Reflux the reaction at 70℃~85℃ for 5~6 h. Add 5~8 times the volume of distilled water to the methanol aqueous solution and let it stand for 12~24 h to obtain the precipitate. (3) Take the precipitate obtained in step (2) according to the mass-to-volume ratio of precipitate to chloroform 1 g: 5~10 mL and extract it with 100~200 mL of chloroform. After the solvent evaporates, tea saponin is obtained, the structure of which is shown in formula (4): Equation (4).

[0034] Example 1 (1) Hydroxyl protection: 10 g of tea saponin and 15 g of tert-butyldimethylchlorosilane were dissolved in 50 mL of N,N-dimethylformamide (DMF), and 15 g of imidazole and 0.2 g of 4-dimethylaminopyridine (DMAP) were added. The mixture was allowed to stand at 20 °C for 14 h. After the reaction was completed, 50 mL of distilled water was added to dilute the mixture, and 50 mL of ethyl acetate was used to extract the organic phase. The resulting organic phase was washed with 25 mL of saturated NaCl aqueous solution and dehydrated with 5 g of anhydrous Na2SO4 for 12 h. The ethyl acetate was then evaporated at 50 °C to obtain 22 g of intermediate 1.

[0035] (2) Aldehyde reduction: 10 g of intermediate 1 and 2 g of sodium triacetoxyborohydride were dissolved in 50 mL of dichloromethane and reacted at 20 °C for 3 h. After the reaction was completed, 50 mL of distilled water was added for dilution, and the mixture was extracted with 50 mL of ethyl acetate. The resulting organic phase was washed with 25 mL of saturated NaCl aqueous solution, dehydrated with 5 g of anhydrous Na2SO4 for 12 h, and ethyl acetate was evaporated at 50 °C to obtain 11 g of intermediate 2.

[0036] (3) Structural derivatization: 10 g of intermediate 2 and 1 g of 4-oxazolyl carboxylic acid were dissolved in 50 mL of pyridine, 1 g of DMAP and 1.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) were added, and the reaction was carried out at 30 °C for 36 h. After the reaction was completed, 50 mL of distilled water was added to dilute the mixture, and 50 mL of ethyl acetate was used for extraction. The resulting organic phase was washed with 25 mL of saturated NaCl aqueous solution, dehydrated with 5 g of anhydrous Na2SO4 for 12 h, and ethyl acetate was evaporated at 50 °C to obtain 10.5 g of intermediate 3.

[0037] (4) Deprotection: 10 g of intermediate 3 and 1 mL of tetrabutylammonium fluoride (TBAF) (Maclean, catalog number T819125, specification: 1.0 M dissolved in tetrahydrofuran, water content 5%) were dissolved in 30 mL of anhydrous tetrahydrofuran (THF) solution and reacted at 20 °C for 3 h. After the reaction was completed, 30 mL of distilled water was added for dilution, and the mixture was extracted with 30 mL of ethyl acetate. The resulting organic phase was washed with 15 mL of saturated NaCl, dehydrated with 5 g of anhydrous Na2SO4 for 12 h, and the ethyl acetate was evaporated at 50 °C to obtain 6 g of tea saponin oxazole derivative.

[0038] Structural analysis by infrared spectroscopy, nuclear magnetic resonance, etc., revealed that the above-mentioned tea saponin oxazole derivative has the structural formula shown in formula (1): Equation (1).

[0039] Example 2 (1) Hydroxyl protection: 10 g of tea saponin and 18 g of tert-butyldimethylchlorosilane were dissolved in 100 mL of DMF, and 18 g of imidazole and 0.3 g of DMAP were added. The reaction was carried out at 25 °C for 12 h. After the reaction was completed, 200 mL of distilled water was added to dilute the mixture, and 200 mL of ethyl acetate was used for extraction. The resulting organic phase was washed with 50 mL of saturated NaCl aqueous solution, dehydrated with 8 g of anhydrous Na2SO4 for 10 h, and ethyl acetate was evaporated at 60 °C to obtain 24 g of intermediate 1.

[0040] (2) Aldehyde reduction: 10 g of intermediate 1 and 3 g of sodium triacetoxyborohydride were dissolved in 100 mL of dichloromethane and reacted at 25 °C for 2 h. After the reaction was completed, 200 mL of distilled water was added for dilution, and the mixture was extracted with 200 mL of ethyl acetate. The resulting organic phase was washed with 50 mL of saturated NaCl aqueous solution, dehydrated with 8 g of anhydrous Na2SO4 for 10 h, and ethyl acetate was evaporated at 60 °C to obtain 12 g of intermediate 2.

[0041] (3) Structural derivatization: 10 g of intermediate 2 and 1.5 g of 2-chloro-4-oxazolic acid were dissolved in 100 mL of pyridine, 1.5 g of DMAP and 2 g of EDC.HCl were added and reacted at 40 °C for 24 h. After the reaction was completed, 200 mL of distilled water was added to dilute the mixture, and 200 mL of ethyl acetate was used for extraction. The resulting organic phase was washed with 50 mL of saturated NaCl aqueous solution, dehydrated with 8 g of anhydrous Na2SO4 for 10 h, and ethyl acetate was evaporated at 60 °C to obtain 11 g of intermediate 3.

[0042] (4) Deprotection: 10 g of intermediate 3 and 2 mL of TBAF (1.0 M dissolved in tetrahydrofuran, with a water content of 5%) were dissolved in 40 mL of anhydrous THF solution and reacted at 25 °C for 2 h. After the reaction was completed, 80 mL of distilled water was added for dilution, and the mixture was extracted with 80 mL of ethyl acetate. The resulting organic phase was washed with 20 mL of saturated NaCl, dehydrated with 8 g of anhydrous Na2SO4 for 10 h, and the ethyl acetate was evaporated at 60 °C to obtain 7 g of tea saponin oxazole derivative.

[0043] Structural analysis, including infrared and nuclear magnetic resonance, revealed that the above-mentioned tea saponin oxazole derivative has the structural formula shown in formula (2): Equation (2).

[0044] Example 3 (1) Hydroxyl protection: 10 g of tea saponin and 20 g of tert-butyldimethylchlorosilane were dissolved in 150 mL of DMF, and 20 g of imidazole and 0.5 g of DMAP were added. The reaction was carried out at 30 °C for 10 h. After the reaction was completed, 450 mL of distilled water was added to dilute the mixture, and 450 mL of ethyl acetate was used for extraction. The resulting organic phase was washed with 150 mL of saturated NaCl aqueous solution, dehydrated with 10 g of anhydrous Na2SO4 for 8 h, and ethyl acetate was evaporated at 60 °C to obtain 25 g of intermediate 1.

[0045] (2) Aldehyde reduction: 10 g of intermediate 1 and 4 g of sodium triacetoxyborohydride were dissolved in 150 mL of dichloromethane and reacted at 30 °C for 1 h. After the reaction was completed, 450 mL of distilled water was added for dilution, and the mixture was extracted with 450 mL of ethyl acetate. The resulting organic phase was washed with 150 mL of saturated NaCl aqueous solution, dehydrated with 10 g of anhydrous Na2SO4 for 8 h, and the ethyl acetate was evaporated at 60 °C to obtain 13 g of intermediate 2.

[0046] (3) Structural derivatization: 10 g of intermediate 2 and 2 g of 5-phenyloxazole-4-carboxylic acid were dissolved in 150 mL of pyridine, 2 g of DMAP and 2.5 g of EDC.HCl were added, and the reaction was carried out at 50 °C for 30 h. After the reaction was completed, 450 mL of distilled water was added for dilution, and 450 mL of ethyl acetate was used for extraction. The resulting organic phase was washed with 150 mL of saturated NaCl aqueous solution, dehydrated with 10 g of anhydrous Na2SO4 for 8 h, and ethyl acetate was evaporated at 60 °C to obtain 11 g of intermediate 3.

[0047] (4) Deprotection: 10 g of intermediate 3 and 4 mL of TBAF (1.0 M dissolved in tetrahydrofuran, with a water content of 5%) were dissolved in 50 mL of anhydrous THF solution and reacted at 30 °C for 1 h. After the reaction was completed, 150 mL of distilled water was added for dilution, and the mixture was extracted with 150 mL of ethyl acetate. The resulting organic phase was washed with 50 mL of saturated NaCl, dehydrated with 10 g of anhydrous Na2SO4 for 8 h, and the ethyl acetate was evaporated at 60 °C to obtain 6.5 g of tea saponin oxazole derivative.

[0048] Structural analysis by infrared spectroscopy, nuclear magnetic resonance, etc., revealed that the above-mentioned tea saponin oxazole derivative has the structural formula shown in formula (3): Equation (3).

[0049] Comparative Example 1 The difference between this comparative example and Example 1 is that the preparation in step (3) is omitted, and intermediate 3 in step (4) is replaced with intermediate 2, while other conditions remain unchanged, to obtain 7.1 g of product, specifically tea saponin with aldehyde group reduced to hydroxyl group, the structure of which is shown in formula (5): Equation (5).

[0050] Comparative Example 2 The difference between this comparative example and Example 1 is that the reaction time in step (3) was changed to 10 h, while other conditions remained the same. The result was 4.6 g of intermediate 3. This indicates that the reaction time was insufficient and the yield was significantly reduced.

[0051] Comparative Example 3 The difference between this comparative example and Example 1 is that the reaction temperature in step (3) was changed to 20°C, while other conditions remained unchanged. The result was 5.2 g of intermediate 3. This indicates that a reaction temperature that is too low is detrimental to the reaction, significantly reducing the yield.

[0052] Comparative Example 4 The difference between this comparative example and Example 1 is that in step (3), 4-oxazole carboxylic acid was replaced with oxazole-2-carboxylic acid (CAS: 672948-03-7, purchased from Hubei Rishengchang New Material Technology Co., Ltd.), while other conditions remained unchanged, and the resulting product was 6.3 g.

[0053] Test Example 1: Structural Characterization of Tea Saponin Axazole Derivatives Fourier transform infrared spectroscopy (Thermo Scientific Nicolet iS10) and nuclear magnetic resonance spectroscopy (Burker Avance III) were used to analyze the infrared and proton spectra of tea saponinogen and the tea saponinogen oxazole derivatives prepared in Examples 1-3.

[0054] Comparison of the infrared spectra of tea saponins ( Figure 1 ), tea saponin oxazole derivative at 1260.36 cm -1 An absorption peak for the asymmetric stretching vibration of the ester bond appeared at 837.38 cm⁻¹. -1 774.25 cm -1 The presence of oxazole ring skeletal vibration indicates that the tea saponin has successfully reacted, yielding the corresponding tea saponin derivative.

[0055] 1H NMR data of tea saponins ( Figure 2 )as follows: 1 H NMR ((400 MHz, DMSO) δ 9.25 (s, 1H),6.88 (s, 1H), 5.49 (t, J = 26.4 Hz, 1H), 5.17 (s, 2H), 4.66 (s, 1H), 4.28 (s,1H), 3.66-3.97 (m, 1H), 3.58 (d, J = 7.9 Hz, 2H), 3.28-3.24 (m, 3H), 1.99 (t,J = 21.2 Hz, 1H), 1.29-1.96 (m, 14H), 1.27 (t, J = 21.3 Hz, 1H), 1.23 (s,3H), 1.02 (t, J = 21.7Hz, 1H), 0.84-0.89 (m, 15H).

[0056] 1H NMR data of the tea saponin derivative prepared in Example 1 ( Figure 3 )as follows: 1H NMR (500MHz, DMSO) δ 7.35 (s, 1H), 7.20 (s, 1H), 6.87 (s, 1H), 5.27 (t, J = 25.5 Hz,1H), 5.17 (s, 2H), 4.65 (s, 1H), 4.29 (s, 1H), 4.04-4.00 (m, 1H), 3.95 (s,2H), 3.66 (d, J = 7.8 Hz, 2H), 3.59 - 3.57 (m, 3H), 2.13 (t, J = 21.4 Hz,1H), 1.35 (m, 14H), 1.23 (t, J = 20.8Hz, 1H), 1.16 (t, J = 21.6Hz, 1H), 0.99(s, 3H), 0.81(m, 15H).

[0057] 1H NMR data of the tea saponin derivative prepared in Example 2 ( Figure 4 )as follows: 1 H NMR (500MHz, DMSO) δ 7.39 (s, 1H), 6.87 (s, 1H), 5.32 (t, J = 25.9 Hz, 1H), 5.27 (s,2H), 4.80 (s, 1H), 4.30 (s, 1H), 4.16 (s, 2H), 3.95 (m, 1H), 3.59 (d, J = 7.9Hz, 2H), 3.16 (m, 3H), 2.13 (t, J = 21.1 Hz, 1H), 1.31 (m, 14H), 1.16 (t, J =20.9Hz, 1H), 1.07 (s, 3H), 0.97 (t, J = 21.3Hz, 1H), 0.85 m, 15H).

[0058] 1H NMR data of the tea saponin derivative prepared in Example 3 ( Figure 5 )as follows: 1 H NMR (500MHz, DMSO) δ 8.60 (d, J = 8.4 Hz, 2H), 8.41 (s, 1H), 7.48 (t, J = 9.6 Hz, 3H), 6.86 (s, 1H), 5.31 (s, 2H), 5.16 (t,J = 25.8 Hz, 1H), 4.69 (s, 1H), 4.20 (s,1H), 4.02 (s, 2H), 3.73 - 3.68 (m, 1H), 3.58 (d, J = 7.3 Hz, 2H), 3.19 - 3.15(m, 3H), 2.02 (t, J = 20.4 Hz, 1H), 1.80 - 1.23 (s, 18H), 1.10 (t, J = 21.5 Hz,1H), 0.91 (m, 15H).

[0059] A comparison of the proton NMR spectra of tea saponin and the examples shows that the aldehyde characteristic peak at δ 9.25 of the tea saponin oxazole derivative disappeared; in Example 1, an ester peak appeared at δ 3.95, and a characteristic peak of hydrogen on the oxazole ring appeared at δ 7.35-δ 7.20; in Example 2, an ester peak appeared at δ 4.16, and a characteristic peak of hydrogen on the oxazole ring appeared at δ 7.39; in Example 3, an ester peak appeared at δ 4.02, a characteristic peak of hydrogen on the oxazole ring appeared at δ 8.41, and characteristic peaks of hydrogen on the benzene ring appeared at δ 8.60 and δ 87.48. This indicates that the examples successfully prepared the tea saponin oxazole derivative.

[0060] Elemental analysis was performed on tea saponin and the tea saponin oxazole derivatives prepared in Examples 1-3, and the results are shown in Table 1. Based on the mass fraction of each element, the atomic ratio in tea saponin was calculated to be C:H:O=30:48:6, the atomic ratio in the tea saponin oxazole derivative prepared in Example 1 was C:H:O:N=31:51:8:1, the atomic ratio in the tea saponin oxazole derivative prepared in Example 2 was C:H:O:N:Cl=34:50:8:1:1, and the atomic ratio in the tea saponin oxazole derivative prepared in Example 3 was C:H:O:N=40:55:8:1, all of which are consistent with the theoretical values.

[0061] Table 1. Elemental content of tea saponins and tea saponin oxazole derivatives Test Example 2: Anti-root-knot nematode activity of the tea saponinogen oxazole derivatives and comparative products prepared in Examples 1-3. The tea saponin oxazole derivatives prepared in Examples 1-3 and the products prepared in Comparative Examples 1-4 were dissolved in 5% DMSO to prepare solutions with a concentration of 20.48 mg / mL. These solutions were then diluted sequentially at a stock solution:solvent (5% DMSO) volume ratio of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, and 1:1024 to create 11 concentration gradient solutions. 150 μL of each concentration gradient solution was then mixed with 100 μL of *Symplocos spp.* (a type of nematode). Meloidogyne incognita A suspension of 2000 second-instar larvae was mixed thoroughly, with 5% DMSO as a negative control and avermectin as a positive control. The larvae were incubated at 27°C for 72 hours, and the drug concentration at which 90% of the southern root-knot nematodes died was determined. Microscopic observation was used; stiffness and immobility indicated death, while undulating movements indicated survival.

[0062] The results are shown in Table 2. The tea saponin oxazole derivatives prepared in Examples 1-3 showed good killing effect on second-instar larvae of *Strombus heterophylla*. Compared with tea saponin, the tea saponin oxazole derivatives prepared in Examples 1-3 showed a significantly lower killing concentration against root-knot nematodes and an activity increase of more than 10 times. The insecticidal activity of the products of Comparative Examples 1, 2, and 3 was weak, indicating that the synthesis reaction in step (3) was indispensable. Although only the reaction temperature and reaction time were changed in Comparative Examples 2 and 3, the product structure remained unchanged, but the yield was significantly reduced, and there were more unreacted substances in the product, resulting in a decrease in its activity. This indicates that the reaction conditions of Examples 1-3 were optimized. The insecticidal activity of the product of Comparative Example 4 was weak, indicating that not all compounds containing oxazole structures can achieve good control of root-knot nematodes when prepared with tea saponin. This may be because the different carboxyl positions of the derivatives created steric hindrance, affecting its interaction with the nematode target.

[0063] Live and dead southern root-knot nematodes treated with the drug were observed under a microscope and a scanning electron microscope, respectively. The results showed that the live southern root-knot nematodes that were not treated with tea saponin oxazole derivatives had intact structures. Figure 6 , Figure 10 ), after treatment with the tea saponin oxazole derivatives obtained in Examples 1-3, the root-knot nematodes shrank and ruptured. Figures 7-9 , Figures 11-13 This indicates that the tea saponin-oxazole derivatives obtained in Examples 1-3 have good anti-root-knot nematode effects. The nematodes treated with the comparative derivatives showed no significant changes.

[0064] Table 2. Killing effect of tea saponin-oxazole derivatives on root-knot nematodes. Test Example 3: Inhibitory effect of tea saponin-oxazole derivatives on superoxide dismutase in root-knot nematodes The tea saponin oxazole derivatives prepared in Examples 1-3 and the products of Comparative Examples 1-4 were dissolved in 5% DMSO to prepare a solution of 0.64 mg / mL. The solution was diluted with solvent (5% DMSO) at volume ratios of 1:1, 1:2, and 1:4 to form three gradients. 150 μL of each concentration gradient solution was mixed with 100 μL of a suspension of about 2000 second-instar root-knot nematode larvae. The solvent (5% DMSO) was used as a negative control. The mixture was incubated at 27°C for 72 h. After 72 h, the nematodes were washed three times with sterile water, then centrifuged at 5000 rpm for 5 min to remove the supernatant. 200 μL of lysis buffer (prepared with 10 μL Triton, 10 μL benzoyl fluoride (PMSF), and 980 μL 1× PBS at pH 7.4) was added, and the mixture was lysed at 0℃ for 40 min. The homogenate was then sonicated at 40% power for 3 min. The superoxide dismutase (SOD) activity was measured and calculated using a superoxide dismutase (SOD) assay kit (purchased from Nanjing Jiancheng Biotechnology Institute, catalog number A001-1-1). The negative control activity was taken as 100%, and the inhibition rate of each drug on SOD activity was calculated.

[0065] The results are shown in Table 3. The results indicate that the oxazole derivative of tea saponin has a significant inhibitory effect on the SOD enzyme activity of second-instar larvae of root-knot nematodes. Compared with tea saponin, the oxazole derivatives of tea saponin obtained in Examples 1-3 significantly improved the inhibition rate of SOD enzyme activity in root-knot nematodes. The products of Comparative Examples 1, 2, and 3 all showed weak inhibitory effects on enzyme activity, indicating that the synthesis reaction in step (3) is indispensable for improving the ability of the oxazole derivative of tea saponin to inhibit the SOD enzyme activity of second-instar larvae of root-knot nematodes, and the reaction conditions have been optimized to ensure that the reactants react fully and synthesize the oxazole derivative of tea saponin. The product of Comparative Example 4 showed a weak inhibitory effect on enzyme activity, but it did not achieve the effect of the oxazole derivatives of tea saponin obtained in Examples 1-3, indicating that not all compounds containing an oxazole structure can react with tea saponin to generate oxazole derivatives of tea saponin that can inhibit the SOD enzyme activity of root-knot nematodes. The weak inhibitory effect of avermectin on SOD indicates that the tea saponin derivative of the present invention has a different mechanism of action than avermectin.

[0066] Table 3. Inhibition rate (%) of tea saponin-oxazole derivatives on SOD enzyme activity in root-knot nematodes Example 4: Application of formulations prepared from tea saponin derivatives oxazole 1. Application of water-dispersible granules prepared from tea saponin-oxazole derivatives Take 30 g of the tea saponin oxazole derivative prepared in Example 1, and mix it with 5 g of dispersant sodium lignosulfonate, 2 g of disintegrant anhydrous sodium sulfate, 1 g of wetting agent sodium dodecyl sulfate, and 62 g of excipient kaolin. Add water and knead in a mixer, then granulate by extrusion, dry, and pass through a 40-60 mesh sieve to obtain the tea saponin oxazole derivative water-dispersible granules.

[0067] Application method: Dilute the tea saponin oxazole derivative water-dispersible granules 500 times with water and apply them to the root zone soil of the crop by root drenching or furrow application. Apply by root drenching on the day of tomato transplanting, using 500 mL of the solution per plant, ensuring full penetration into the main root layer. Apply a second root drenching with the tea saponin oxazole derivative water-dispersible granules 15 days after the first application. Investigate the incidence of root-knot nematodes in tomatoes 30 days after the second application.

[0068] 2. Application of soluble powders prepared from tea saponin derivatives and oxazole derivatives 10 g of the tea saponin oxazole derivative prepared in Example 2, 5 g of the synergistic dispersant polyvinylpyrrolidone K30, and 85 g of the carrier anhydrous glucose were pulverized and passed through a 100-mesh sieve. The above materials were mixed in a three-dimensional mixer for 30 min until homogeneous to obtain a soluble powder of the tea saponin oxazole derivative.

[0069] Application method: (1) Application: Before transplanting tomato plants, mix the above-mentioned soluble powder of tea saponin oxazole derivative with an appropriate amount of dry fine soil at a dosage of 1 kg per mu, and apply it evenly to the soil surface, and then plow it into the soil. Investigate the incidence of root-knot nematodes 30 days after application.

[0070] (2) Hole application: When transplanting tomato plants, apply the above-mentioned soluble powder of tea saponin oxazole derivative at a dosage of 1.0 g per hole directly into the planting hole, mix it slightly with the soil in the hole, and then plant the seedling. Water thoroughly immediately after application to promote the diffusion of the agent in the root zone. Investigate the incidence of root-knot nematodes 30 days after application.

[0071] The results showed that the incidence of root-knot nematodes in tomato plants without the use of formulations containing tea saponin-oxazole derivatives was 50%–70%. After treatment with water-dispersible formulations of tea saponin-oxazole derivatives, the incidence of root-knot nematodes in tomato plants decreased to 8.7%, with a control effect of 92.2%. After treatment with soluble powder formulations of tea saponin-oxazole derivatives, the incidence of root-knot nematodes in tomato plants decreased to 5.1%, with a control effect of 86.7%, and no significant root knot formation was observed in either formulation. Both formulations significantly reduced the damage caused by southern root-knot nematodes, indicating that the formulations prepared from tea saponin-oxazole derivatives can effectively control root-knot nematodes in vegetables.

[0072] Based on the above examples and comparative examples, the provided tea saponin oxazole derivative has the molecular structure shown in formula (I), wherein R1 is H or Cl, and R2 is H or phenyl. It exhibits good control effects against root-knot nematodes.

[0073] Formula (I).

Claims

1. A tea saponinogen oxazole derivative, characterized in that, It has a molecular structure as shown in formula (I), wherein R1 is H or Cl, and R2 is H or phenyl; Formula (I).

2. The tea saponin oxazole derivative according to claim 1, characterized in that, The tea saponin oxazole derivative is any one or more of the compounds shown in formula (1) to formula (3); Equation (1); Equation (2); Equation (3).

3. The tea saponin oxazole derivative according to claim 1, characterized in that, Its preparation method includes the following steps: S1. Dissolve 10 g of tea saponin and 15-20 g of tert-butyldimethylchlorosilane in 50-150 mL of N,N-dimethylformamide, add 15-20 g of imidazole and 0.2-0.5 g of 4-dimethylaminopyridine, react at 20℃-30℃ for 10-14 h, dilute with 50-450 mL of distilled water, extract with 50-450 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain intermediate 1; S2. Dissolve 10 g of intermediate 1 with 2-4 g of sodium triacetoxyborohydride in 50-150 mL of dichloromethane, react at 20-30°C for 1-3 h, dilute with 50-450 mL of distilled water, extract with 50-450 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain intermediate 2. S3. Dissolve 10 g of intermediate 2 and 1-2 g of 4-oxazolyl carboxylic acid compound in 50-150 mL of pyridine, add 1-2 g of 4-dimethylaminopyridine and 1.5-2.5 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, react at 30℃-50℃ for 24-36 h, dilute with 50-450 mL of distilled water, extract with 50-450 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain intermediate 3; S4. Dissolve 10 g of intermediate 3 with 1-4 mL of tetrabutylammonium fluoride in 30-50 mL of anhydrous tetrahydrofuran, react at 20-30°C for 1-3 h, dilute with 30-150 mL of distilled water, extract with 30-150 mL of ethyl acetate, wash, dehydrate and evaporate the organic layer to obtain the tea saponin oxazole derivative.

4. The tea saponin-oxazole derivative according to claim 3, characterized in that, The washing, dehydration, and evaporation of the organic layer are as follows: the organic layer is washed with 12.5~225 mL of saturated sodium chloride aqueous solution, then dehydrated with 5~10 g of anhydrous sodium sulfate for 8~12 h, and ethyl acetate is removed by evaporation at 50℃~60℃.

5. The tea saponin-oxazole derivative according to claim 3, characterized in that, The tea saponin is a compound obtained by acid-base hydrolysis and extraction of tea oil saponin, and its preparation method includes the following steps: S1. Dissolve tea oil saponin in an 80%–90% (v / v) methanol aqueous solution according to a mass-to-volume ratio of 1 g: 15–25 mL. Adjust the pH of the solution to 1–3 with 2–4 mol / L hydrochloric acid aqueous solution. Reflux the reaction at 70–85 °C for 5–6 h. Add 5–8 times the volume of distilled water to the methanol aqueous solution and let it stand for 12–24 h to obtain a precipitate. S2. According to the mass-volume ratio of precipitate to methanol aqueous solution 1 g: 15~25 mL, dissolve the precipitate obtained in step S1 in a methanol aqueous solution with a volume fraction of 80%~90%, adjust the pH of the solution to 11~13 with a sodium hydroxide aqueous solution with a mass fraction of 5~10%, reflux at 70℃~85℃ for 5~6 h, add 5~8 times the volume of distilled water of methanol aqueous solution, let stand for 12~24 h, and obtain the precipitate; S3. Take the precipitate obtained in step S2 according to the mass-volume ratio of precipitate to chloroform 1 g: 5~10 mL and extract it with 100~200 mL of chloroform. After the solvent evaporates, tea saponin is obtained. The structure of the tea saponin is shown in formula (4). Equation (4).

6. A preparation for controlling root-knot nematodes, characterized in that, It contains the tea saponin oxazole derivative as described in any one of claims 1 to 5.

7. The formulation for controlling root-knot nematodes according to claim 6, characterized in that, Its composition is: 30 parts by mass of tea saponin oxazole derivative, 5 parts by mass of sodium lignosulfonate, 2 parts by mass of anhydrous sodium sulfate, 1 part by mass of sodium dodecyl sulfate and 62 parts by mass of kaolin.

8. The formulation for controlling root-knot nematodes according to claim 6, characterized in that, The preparation for controlling root-knot nematodes comprises: 10 parts by weight of tea saponin oxazole derivative, 5 parts by weight of polyvinylpyrrolidone K30, and 85 parts by weight of anhydrous glucose.

9. The use of the tea saponin derivative according to any one of claims 1 to 5 in the preparation of formulations for controlling root-knot nematodes.

10. The use of the tea saponin derivative of any one of claims 1 to 5 or the preparation of any one of claims 6 to 7 in the control of root-knot nematodes.

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