A berberine hydrochloride-resveratrol supramolecular self-assembled nanopesticide, its preparation method and application

Abstract

This invention discloses a berberine hydrochloride-resveratrol self-assembled nanopesticide, its preparation method, and its application. The preparation method involves dissolving berberine hydrochloride and resveratrol separately in an organic solvent, mixing them in a molar ratio of 3:1–1:3, adding the mixture to deionized water, and then adding an alkaline solution; the mixture then self-assembles to obtain nanoparticles. Based on the principle of supramolecular construction, this invention utilizes the high compatibility of berberine hydrochloride and resveratrol in charge distribution and spatial geometry. Through multiple intermolecular forces such as electrostatic attraction, π-π stacking, and hydrogen bonding, the precise recognition and directional assembly of the two active molecules in the aqueous phase are achieved. This nanopesticide requires no additional synthetic adjuvants or nanocarriers, achieving confined enrichment of the active ingredients at the nanoscale, resulting in a significant synergistic effect. This nanopesticide significantly enhances the inhibitory activity against various pathogens such as *Botrytis cinerea*, *Fusarium oxysporum*, *Phytophthora capsici*, and *Rhizoctonia solani*, and exhibits excellent environmental biosafety.

Description

Technical Field

[0001] This invention belongs to the field of pesticide formulation technology, specifically relating to a berberine hydrochloride-resveratrol supramolecular self-assembled nanopesticide, its preparation method, and its application. Background Technology

[0002] Plant diseases pose a serious threat to global food security. While chemical fungicides offer significant control, their long-term use has led to increasingly prominent "3R" problems (resistance, residues, and resurgence). Plant-derived fungicides offer advantages such as environmental friendliness and diverse mechanisms of action, but their application is limited by poor solubility, insufficient stability, and unstable field efficacy. Existing nanocarrier technologies can improve their performance, but the introduction of carriers increases production costs, and the toxicological risks and metabolic fate of the carrier materials themselves remain unclear. Therefore, developing a nano-formulation technology that requires no external carrier, has a simple process, and is highly efficient and synergistic is of great significance for promoting the modern application of plant-derived pesticides.

[0003] Currently, there are no reports on the technology related to the self-assembly of berberine hydrochloride and resveratrol. Summary of the Invention

[0004] The purpose of this invention is to address the problems of poor water solubility, low bioavailability, and unclear environmental toxicology of existing pesticide carriers in the prior art by providing a berberine hydrochloride-resveratrol nanopesticide based on supramolecular self-assembly technology. This pesticide utilizes the intermolecular interactions between active ingredients to achieve synergistic self-assembly, exhibiting high pesticide loading, environmental friendliness, and synergistic effects.

[0005] The technical solution adopted in this invention is as follows: This invention provides a method for preparing berberine hydrochloride-resveratrol self-assembled nanopesticides, characterized by comprising the following steps: (1) Dissolve berberine hydrochloride in an organic solvent to obtain an organic solution of berberine hydrochloride; (2) Dissolve resveratrol in an organic solvent to obtain a resveratrol organic solution; (3) The berberine hydrochloride organic solution and the resveratrol organic solution are mixed to obtain a mixed organic phase; the molar ratio of berberine hydrochloride to resveratrol is 3:1–1:3; (4) Under stirring conditions, the mixed organic phase is added to deionized water, an alkaline solution is added, and the reaction is continued by stirring to form a nanoparticle suspension through self-assembly. (5) The nanoparticle suspension is purified to remove solvent and unassembled molecules, and then dried to obtain the nanopesticide powder form; In the method, in steps (1) and (2), the organic solvent is at least one of methanol, ethanol or acetone; the concentration of the berberine hydrochloride organic solution in step (1) is 10–80 mmol / L; the concentration of the resveratrol organic solution in step (2) is 10–80 mmol / L. In the method, in step (4), the alkaline solution is a 0.5 mmol / L sodium hydroxide aqueous solution; the volume ratio of the alkaline solution to the mixed organic phase is 1:20-1:50; In the method, in step (4), the volume ratio of the mixed organic phase to deionized water is 1:(10–200); the stirring speed is 600–1200 rpm; and the stirring reaction time is 2–4 hours.

[0006] In the method, in step (5), the purification is carried out by dialysis, the molecular weight cutoff of the dialysis bag is 200–2000 Da, the volume ratio of sample solution to dialysis water is 1:(50–200), and the dialysis time is 8–36 h.

[0007] The berberine hydrochloride-resveratrol self-assembled nanopesticides obtained by the above method also fall within the scope of protection of this invention. The hydrated particle size of the nanopesticides is 70–255 nm.

[0008] The method described above, or the application of the berberine hydrochloride-resveratrol self-assembled nanopesticide in the prevention and control of plant diseases, also falls within the scope of protection of this invention. The plant disease mentioned is Botrytis cinerea (…). Botrytis cinerea Fusarium oxysporum (Fusarium oxysporum) Fusarium fujikuroi Rhizoctonia solani ( ) Rhizoctonia solani ) and Phytophthora capsici ( Phytophthora capsici One or more of them.

[0009] The advantages of this invention are: 1. By driving intermolecular interactions through the antisolvent effect, molecular recognition and assembly between active ingredients occur without the need for surfactants, theoretically allowing the effective ingredient content in the system to reach 100%, significantly reducing the negative environmental impact and production costs of pesticide adjuvants; 2. This method transforms poorly soluble resveratrol and easily crystallizing berberine into uniformly dispersed nanoparticles, which not only improves the dispersion stability of the agent but also significantly enhances the antibacterial activity by utilizing the synergistic effect generated after the self-assembly of the two natural products; 3. The preparation process is carried out at room temperature, without the need for complex chemical synthesis steps, meeting the requirements of green pesticide formulations.

[0010] In summary, this invention is based on the principle of supramolecular construction, utilizing the high compatibility of berberine hydrochloride (a cationic isoquinoline alkaloid) and resveratrol (a polyphenolic compound) in charge distribution and spatial geometry. Through multiple intermolecular forces such as electrostatic attraction, π-π stacking, and hydrogen bonding, it achieves precise recognition and directional assembly of dual-active molecules in the aqueous phase. This nanopesticide requires no additional synthetic adjuvants or nanocarriers, achieving confined enrichment of active ingredients at the nanoscale, resulting in a significant synergistic effect. Bioactivity assays confirm that this nanopesticide significantly enhances the inhibitory activity against various pathogens such as Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici, and Rhizoctonia solani compared to corresponding single agents and physical mixtures, and exhibits excellent environmental biosafety. This invention not only improves the bioavailability of plant-derived active ingredients but also provides a new approach for developing highly efficient, low-toxicity, and high-load-bearing green nanopesticides. Attached Figure Description

[0011] Figure 1 This is a scanning electron microscope (SEM) image of berberine hydrochloride-resveratrol nanopesticide sample 1 (alkaline solution: organic phase = 1:20, v:v).

[0012] Figure 2 SEM image of the berberine hydrochloride-resveratrol assembled sample (without alkali solution added).

[0013] Figure 3 SEM image of the berberine hydrochloride-resveratrol assembly sample (alkaline solution: organic phase = 1:10, v:v).

[0014] Figure 4 The hydrated particle size distribution and zeta potential diagram of berberine hydrochloride-resveratrol nanopesticide sample 1 are shown.

[0015] Figure 5 This is a transmission electron microscope (TEM) image of sample 1, a nanopesticide containing berberine hydrochloride and resveratrol.

[0016] Figure 6 The X-ray diffraction (XRD) pattern of berberine hydrochloride-resveratrol nanopesticide sample 1 is shown.

[0017] Figure 7 The image shows the Fourier transform infrared (FT-IR) spectrum of sample 1, which is a berberine hydrochloride-resveratrol nanopesticide. The left image shows the infrared spectrum across the full wavenumber range (400–4000 cm⁻¹), and the right image shows a magnified view of the fingerprint region at 500–1800 cm⁻¹.

[0018] Figure 8Comparative images and statistical charts of the efficacy of berberine hydrochloride aqueous solution (BBR AS), resveratrol soluble solution (RES SL), and berberine hydrochloride-resveratrol nanopesticide sample 1 (BR NPs) against greenhouse control of rice sheath blight. Detailed Implementation

[0019] The present invention will be further illustrated below through embodiments. It should be understood that the following embodiments are intended to help understand the method and beneficial effects of the present invention, but do not limit the scope of protection of the present invention. Any improvements or equivalent substitutions made by those skilled in the art under the guidance of the present invention, which do not depart from the spirit of the present invention, should be covered within the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0021] Example 1: Preparation of berberine hydrochloride organic solution Berberine chloride form: purity 98%, molecular weight 371.81, CAS number: 633-65-8, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0022] A certain amount of berberine hydrochloride was weighed and dissolved in a certain amount of methanol to prepare a berberine hydrochloride methanol solution with a concentration of 26.90 mmol / L.

[0023] Example 2 Preparation of resveratrol organic solution Resveratrol: purity ≥99%, molecular weight 228.24, CAS No.: 501-36-0, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0024] A certain amount of resveratrol was weighed and dissolved in a certain amount of methanol to prepare a resveratrol methanol solution with a concentration of 26.90 mmol / L.

[0025] Example 3: Preparation of berberine hydrochloride-resveratrol nanopesticide sample 1 500 μL of berberine hydrochloride methanol solution prepared in Example 1 and 500 μL of resveratrol methanol solution prepared in Example 2 were mixed, with a molar ratio of berberine hydrochloride to resveratrol of 1:1, resulting in a total volume of 1000 μL of mixed organic phase. The mixture was thoroughly mixed using a vortex mixer. The mixed organic phase was then slowly added dropwise to 19 mL of deionized water at a stirring speed of 600 rpm. 50 μL of a 0.5 mmol / L NaOH aqueous solution was added (alkali solution:organic phase = 1:20, v:v). After stirring for 3 h, the resulting suspension was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da, with a volume ratio of suspension to dialysis medium of 1:100. Dialysis was performed for 24 h to remove methanol and free molecules, yielding berberine hydrochloride-resveratrol self-assembled nanopesticide sample 1. Scanning electron microscopy images are shown below. Figure 1 The nanoparticles were found to be spherical, with intact morphology and good dispersion, and no obvious aggregation was observed.

[0026] Comparative Example 1: Control group without added alkaline solution Repeat the steps of Example 3, except that NaOH aqueous solution is not added when the organic phase is added dropwise to the aqueous phase. Scanning electron micrographs are shown below. Figure 2 The results showed that the obtained samples could not form regular nanoparticle structures, mainly exhibiting irregular amorphous film fragments or bulk crystals. This indicates that without an alkaline-induced pretreatment step, effective intermolecular driving forces could not be generated between the active ingredient molecules, making it difficult to trigger the supramolecular self-assembly process.

[0027] Comparative Example 2: Control group with excess alkaline solution added Repeat the steps of Example 3, except that 100 μL of a 0.5 mmol / L NaOH aqueous solution (alkali solution:organic phase = 1:10, v:v) is added. Scanning electron microscopy observation (see...) Figure 3 The results showed that although a particulate structure was formed in the system, severe adhesion, fusion, and agglomeration occurred between the particles, making it impossible to maintain a stable nano-dispersion. The results indicate that there is a critical range for the amount of alkaline inducer added; excessive alkali will disrupt the equilibrium of the assembled system.

[0028] Example 4 Preparation of Berberine Hydrochloride-Resveratrol Nanopesticide Sample 2 250 μL of berberine hydrochloride methanol solution prepared in Example 1 and 750 μL of resveratrol methanol solution prepared in Example 2 were mixed, with a molar ratio of berberine hydrochloride to resveratrol of 1:3. 50 μL of 0.5 mmol / L NaOH aqueous solution was added to the aqueous phase. Other self-assembly and purification steps were the same as in Example 3, resulting in berberine hydrochloride-resveratrol self-assembled nanopesticide sample 2. The hydrated particle size was measured to be 156.44 nm by dynamic light scattering.

[0029] Example 5 Preparation of berberine hydrochloride-resveratrol nanopesticide sample 3 A 750 μL methanol solution of berberine hydrochloride prepared in Example 1 and a 250 μL methanol solution of resveratrol prepared in Example 2 were mixed, with a molar ratio of berberine hydrochloride to resveratrol of 3:1. 50 μL of a 0.5 mmol / L NaOH aqueous solution was added to the aqueous phase. Subsequent self-assembly and purification steps were the same as in Example 3, yielding berberine hydrochloride-resveratrol self-assembled nanopesticide sample 3. The hydrated particle size was measured to be 197.64 nm by dynamic light scattering.

[0030] Example 6: Characterization of berberine hydrochloride-resveratrol nanopesticide sample 1 6.1 Particle size and Zeta potential 1 mL of the nanopesticide sample 1 prepared in Example 3 was injected into the dynamic light scattering sample cell, and its hydrated particle size was measured to be 132.80 nm (see...). Figure 4 ); Sample 1 of the nano-pesticide prepared in Example 3 was injected into the electrophoretic light scattering sample cell, and its Zeta potential was measured to be 38.2 mV (see Figure 4 The results show that the nanosystem has a small particle size and a high surface charge, which is beneficial for stable dispersion in the aqueous phase.

[0031] 6.2 Morphological observation Five μL of the nano-pesticide sample 1 prepared in Example 3 was spotted onto a copper mesh, air-dried, and its morphology was observed using a transmission electron microscope. The experimental results are as follows: Figure 5 As shown.

[0032] Depend on Figure 1 and Figure 5 The results show that berberine hydrochloride-resveratrol nanopesticide sample 1 is spherical and has a core-shell structure. This specific morphology demonstrates that the components form an ordered complex structure through supramolecular self-assembly, rather than random aggregation.

[0033] 6.3 X-ray diffraction analysis X-ray diffraction (XRD) was used to characterize berberine hydrochloride, resveratrol, an equimolar physical mixture of the two, and nanopesticide sample 1 prepared in Example 3. The results are as follows: Figure 6As shown, berberine hydrochloride and resveratrol technical materials, as well as their physical mixtures, all exhibit a series of sharp and intense characteristic diffraction peaks, indicating that the active ingredients exist in a highly crystalline state. In the diffraction pattern of nanopesticide sample 1, the original characteristic crystalline peaks of berberine hydrochloride and resveratrol have essentially disappeared, replaced by diffuse amorphous peaks. This result indicates that berberine hydrochloride and resveratrol, through intermolecular interactions during self-assembly, transform from their original crystalline state to an amorphous state and assemble into the nanosystem. This transformation further proves that the nanopesticides prepared in this invention form supramolecular self-assembled particles with unique phase structures, rather than simple physical mixtures.

[0034] 6.4 Infrared Spectroscopy Analysis Fourier transform infrared spectroscopy (FT-IR) was used to characterize berberine hydrochloride, resveratrol, an equimolar physical mixture of the two, and nanopesticide sample 1 prepared in Example 3. The results are as follows: Figure 7 As shown, berberine hydrochloride at 2849 cm⁻¹ -1 and 1633cm -1 The peaks at the positions of the methoxy C–H stretching vibration and the quaternary ammonium group C=N are respectively displayed. + Characteristic peaks, 1503 and 1566 cm⁻¹ -1 This is an absorption related to the C=C skeleton vibration of the aromatic ring. Resveratrol at 3182 cm⁻¹ -1 The peaks show the OH stretching vibration of the phenolic hydroxyl group at 1633, 1604, 1582, and 1509 cm⁻¹. -1 These peaks are correlated with vinyl C=C and aromatic C=C vibrations, respectively. Compared to the physical mixture, the OH absorption peak in nanopesticide sample 1 red-shifts to 3065 cm⁻¹. -1 The intensity decreased significantly, indicating that hydrogen bonding occurred between the two components. 1633cm -1 The multiple sets of absorption signals in the vicinity are shifted as a whole and at 1600cm -1 A broad peak forms at this point, reflecting C=N + Electrostatic interactions with phenolic hydroxyl groups, and changes in the electronic environment caused by aromatic π–π stacking. Furthermore, resveratrol at 828 cm⁻¹... -1 The out-of-plane bending vibration peak of C–H plane shifted to 836 cm⁻¹ -1 The changes suggest a conformational adjustment of the aromatic ring. These changes were not observed in the physical mixture, indicating that nanopesticide sample 1 was formed through self-assembly driven by multiple intermolecular forces, including hydrogen bonding, electrostatic interactions, and π-π interactions.

[0035] Example 7: Determination of the synergistic effect of berberine hydrochloride-resveratrol self-assembled nanoparticles on various plant pathogens. The mycelial growth rate method was used to determine the inhibitory activity of berberine hydrochloride-resveratrol self-assembled nano-pesticide (BR NPs) on the mycelial growth of 4 plant pathogens.

[0036] Test pathogens: Botrytis cinerea ( Botrytis cinerea ), Fusarium fujikuroi ( Fusarium fujikuroi ), Rhizoctonia solani ( Rhizoctonia solani ), and Phytophthora capsici ( Phytophthora capsici ).

[0037] Solid medium: PDA was used for fungi, and V8 agar was used for oomycetes (both were sterilized at 121 °C).

[0038] Preparation of poisoned medium: Berberine hydrochloride single agent, resveratrol single agent, and equimolar ratio mixture of berberine hydrochloride and resveratrol (physical mixture) were dissolved in methanol respectively, and then added to the medium according to the volume ratio of 1:100; BR NPs were diluted with sterile water, vortexed and mixed evenly, and then added to the medium. Poisoned plates with concentrations of 12.5, 25, 50, 100, 200, and 400 mg / L (calculated by total active ingredient) were prepared respectively, and the medium added with equal volume of methanol or sterile water was used as the blank control.

[0039] Inoculation: 5-mm mycelial discs were taken from the edge of each pathogen colony and placed in the center of the poisoned plate, and cultured in the dark at 25±1 °C.

[0040] Data investigation and analysis: When the colony diameter of the blank control was close to 2 / 3 of the culture dish diameter, the cross method was used to measure the colony diameter of each treatment. Log-Probit regression analysis was performed on the logarithm of the mass concentration of different药剂处理 and the corresponding inhibitory rate probability value using SPSS to establish a toxicity regression equation and calculate the effective median inhibitory concentration (EC 50 ).

[0041] Calculation of combined toxicity: According to the Agricultural Industry Standard NY / T 1156.6-2006 of the People's Republic of China, "Guidelines for Indoor Bioassay of Pesticides - Fungicides - Part 6: Determination of Combined Action of Mixtures", the co-toxicity coefficient (CTC) was calculated by the Sun Yunpei method and the synergistic coefficient (SR) was calculated by the Wadley method respectively.

[0042] Evaluation criteria for synergism: When CTC≥120 or SR≥1.5, it showed a synergistic effect; when CTC≤80 or SR≤0.5, it showed an antagonistic effect; when 80<CTC<120 or 0.5<SR<1.5, it showed an additive effect. The specific test results are shown in Table 1.

[0043] Table 1 Synergistic evaluation of berberine hydrochloride and resveratrol self-assembled nano-pesticide on 4 plant pathogens

[0044] Note: The molar ratios in the table should be converted to mass ratios and then substituted into the formulas for calculation.

[0045] Experimental results showed that, within a molar ratio range of 1:3 to 3:1, the self-assembled nanoparticles prepared by the process described in this invention (Examples 3, 4, and 5) exhibited significant inhibitory activity against all four tested pathogens, and their control efficacy was significantly superior to that of the physical mixture group and the single-agent component. In the determination of *Botrytis cinerea*, *Fusarium oxysporum*, and *Phytophthora capsici*, the co-toxicity coefficient (CTC) of different proportions of BR NPs was significantly greater than 120, and the synergistic effect coefficient (SR) was greater than 1.5, showing a synergistic effect. In particular, a significant "antagonistic-to-synergistic" phenomenon was observed: in the experiments with *Fusarium oxysporum* and *Phytophthora capsici*, the physical mixture showed obvious antagonistic effects due to the differences in chemical or biological properties between the components (CTCs were 61.10 and 68.68); while the nanostructures formed by supramolecular self-assembly not only overcame the antagonism between the components, but also successfully transformed it into a high degree of synergistic effect. Among them, the nano-pesticide sample 1 (Example 3) achieved the highest CTC of 297.37 against *Fusarium oxysporum*. Furthermore, for Rhizoctonia solani, the co-toxicity coefficient (CTC) of nano-pesticide samples at different molar ratios all exceeded the threshold of 120, indicating that they have a synergistic effect.

[0046] In summary, the self-assembled nanopesticides described in this invention can construct stable delivery systems within a 1:3 to 3:1 range through intermolecular hydrogen bonds, electrostatic interactions, and π-π interactions. This unique phase structure not only significantly enhances the efficacy of natural products but also demonstrates outstanding technical effectiveness in overcoming component antagonism, proving the enormous application potential of this nanosystem.

[0047] Example 8: Determination of the control effect of berberine hydrochloride-resveratrol nanopesticide on rice sheath blight Under greenhouse conditions, the control efficacy of berberine hydrochloride-resveratrol nanopesticides (BR NPs) against rice sheath blight was evaluated using an artificial inoculation method, with commercially available berberine aqueous solution (BBR AS) and resveratrol soluble concentrate (RES SL) used as controls. Rice plants of uniform growth were selected and sprayed with solutions at concentrations of 200 mg / L and 400 mg / L, ensuring uniform coverage of the plant surface. The control group was sprayed with water. Twenty-four hours after application, Rhizoctonia solani mycelia were inoculated onto the leaf sheaths of the stems using a toothpick inoculation method. The diameter of lesions was recorded on days 3, 7, and 14 after application to evaluate the control efficacy of each treatment. Each treatment consisted of 20 rice plants, with three replicates. The results showed that, 3-14 days after application, the berberine hydrochloride-resveratrol nanopesticide was superior to two commercially available pesticides in controlling rice sheath blight. At a concentration of 400 mg / L, the control efficacy of the berberine hydrochloride-resveratrol nanopesticide against rice sheath blight was 70.32-82.75%. Figure 8 Furthermore, observations at a concentration of 400 mg / L revealed that the tested rice plants grew normally without any phytotoxicity symptoms, indicating that the formulation has good biological safety.

[0048] Example 9: Zebrafish Toxicity Determination The acute toxicity of nano-pesticides to zebrafish (Danio rerio) was determined using a static test method, and their environmental safety was assessed. The experiment was conducted according to the "Guidelines for Environmental Safety Evaluation of Chemical Pesticides" (GB / T 31270.12-2014). Healthy zebrafish, 2.0-3.0 cm in length, were used as the test organisms. The ambient temperature was maintained at (25±1) ℃, and the photoperiod was 12h / 12h (light / dark). Five treatment groups were set up: nano-pesticide sample 1 (prepared in Example 3), nano-pesticide sample 2 (prepared in Example 4), nano-pesticide sample 3 (prepared in Example 5), berberine hydrochloride technical control group, and resveratrol technical control group. A series of mass concentration gradients of 10, 18, 32, 56, and 80 mg / L (based on active ingredient) were prepared using a 1% Tween-80 aqueous solution as a co-solvent. A 1% Tween-80 aqueous solution was also used as a blank control. Zebrafish were randomly assigned to glass containers containing 2 L of the drug solution, with 10 fish per treatment group and three biological replicates. No feeding or oxygenation was provided during the experiment. Symptoms of poisoning and the number of deaths in the zebrafish were continuously observed and recorded within 96 hours of the start of the experiment. Log-Probit regression analysis was performed using SPSS statistical software to calculate the 96-hour median lethal concentration (LC50). The experimental results are shown in Table 2.

[0049] Table 2. Acute toxicity (96h) of berberine hydrochloride, resveratrol, and self-assembled nanopesticides to zebrafish. Berberine hydrochloride / ＞80 Resveratrol Y = 7.295X - 7.058 51．82 Nanopesticide Sample 1 / ＞80 Nanopesticide Sample 2 / ＞80 Nanopesticide Sample 2 / ＞80 Test results show that the LC50 of the test agent on zebrafish 50 All concentrations were greater than 10 mg / L, classifying them as low toxicity. Notably, the technical grade resveratrol had a 100% lethality at a concentration of 80 mg / L (LC50). 50 = 51.82 mg / L); the nano-pesticide samples 1, 2 and 3 prepared in this invention all showed a mortality rate of less than 50% in zebrafish at the highest test concentration of 80 mg / L (among which the mortality rate of sample 1 at this concentration was only 40%), and their LC... 50 All concentrations were increased to over 80 mg / L. The results confirm that the self-assembled nanodelivery system described in this invention not only enhances biological activity through synergistic effects but also possesses excellent environmental and ecological safety, achieving a dual technical effect of "enhancing efficacy and reducing toxicity," and has broad prospects for agricultural applications.

Claims

1. A method for preparing a self-assembled nanopesticide of berberine hydrochloride-resveratrol, characterized in that, Includes the following steps: (1) Dissolve berberine hydrochloride in an organic solvent to obtain an organic solution of berberine hydrochloride; (2) Dissolve resveratrol in an organic solvent to obtain a resveratrol organic solution; (3) The berberine hydrochloride organic solution and the resveratrol organic solution are mixed to obtain a mixed organic phase; the molar ratio of berberine hydrochloride to resveratrol is 3:1–1:3; (4) Under stirring conditions, the mixed organic phase is added to deionized water and an alkaline solution is added. The reaction is continued by stirring, and then the mixture is purified by dialysis to obtain an aqueous suspension of nanoparticles. The aqueous suspension of nanoparticles is further freeze-dried to obtain a powder, which is the self-assembled nanoparticle pesticide of berberine hydrochloride-resveratrol. In step (4), the alkaline solution is a 0.5 mmol / L sodium hydroxide aqueous solution. The volume ratio of the alkaline solution to the mixed organic phase is 1:20-1:

50.

2. The preparation method according to claim 1, characterized in that, In steps (1) and (2), the organic solvent is at least one of methanol, ethanol or acetone; the concentration of the berberine hydrochloride organic solution in step (1) is 10–80 mmol / L; and the concentration of the resveratrol organic solution in step (2) is 10–80 mmol / L.

3. The preparation method according to claim 1, characterized in that, In step (4), the volume ratio of the mixed organic phase to deionized water is 1:(10–200); the stirring speed is 600–1200 rpm, and the stirring reaction time is 2–4 hours.

4. The preparation method according to claim 1, characterized in that, In step (4), the purification is carried out by dialysis, the molecular weight cutoff of the dialysis bag is 200–2000 Da, the volume ratio of sample solution to dialysis water is 1:(50–200), and the dialysis time is 8–36 h.

5. The berberine hydrochloride-resveratrol self-assembled nanopesticide obtained by the method according to any one of claims 1-4.

6. The nano-pesticide according to claim 5, characterized in that, The hydrated particle size of the nanopesticide is 70–255 nm.

7. The application of the method according to any one of claims 1-4 or the berberine hydrochloride-resveratrol self-assembled nanopesticide according to claim 5 or 6 in the prevention and control of plant diseases.

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