Preparation method and application of functional nano-pesticide based on random combinatorial library of polyhexamethylene biguanide hydrochloride analogue and phenolic hydroxyl group-containing natural small molecules

Abstract

The invention discloses a preparation method and application of a functional nano-pesticide based on a random combinatorial library of polyhexamethylene biguanide hydrochloride analogues and phenolic hydroxyl group-containing natural small molecules. A PHMB analogue containing a mono-guanidyl or biguanidyl structure and a natural small molecule compound with 1-3 phenolic hydroxyl structures are selected, nano-particles with good dispersion coefficients can be formed in a sodium hydroxide aqueous solution with the pH value of 9-11 without additional carriers or stabilizers, and the limitation that traditional nano-pesticides depend on macromolecular carriers is broken through. The obtained nano-particles can be enriched on the surface of a plant pathogenic fungus cell membrane by utilizing the electrostatic adsorption effect of guanidyl, and the delivery effect of natural small molecules is enhanced, so that the antifungal activity of the nano-particles is improved. An antibacterial experiment result shows that part of the composition has a synergistic antibacterial effect on fusarium graminearum, and the diameter of an inhibition zone is superior to that of a single component. The method is high in construction efficiency, has good expansibility, can form a nano pesticide preparation library, and is suitable for plant disease prevention and control and environment-friendly material development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nano-pesticides, and particularly relates to a construction method of a co-assembled nano-pesticide based on a guanidine-containing compound and a natural small molecule containing a phenolic hydroxyl group and application thereof. The nanoparticles do not require additional carriers and stabilizers, have a synergistic antibacterial effect, and are suitable for green prevention and control of plant diseases and a structure-function data modeling platform. BACKGROUND

[0002] Fungal diseases seriously restrict global agricultural production during the growth period and after harvest. At present, crop disease control still highly depends on traditional chemical pesticides. However, long-term and large-scale use of chemical pesticides and their non-degradable characteristics often lead to a series of problems such as increased pathogen resistance, accumulation of pesticide residues, and ecological imbalance, which not only pollute the environment but also challenge the quality of agricultural products and food safety.

[0003] With the rise of green agriculture, the development of safe, environmentally friendly and efficient new pesticide formulations has become a research hotspot. Among them, nano-pesticides based on natural products and functional polymers have been proven to have good application prospects due to their small particle size, strong loading capacity, good targeting and controlled release characteristics. Although the research and development of nano-pesticides is rapid, there are still many problems: (1) the structure of the material is single, which is difficult to meet the multifunctional demand; (2) the solubility and light stability of natural small molecule active ingredients are limited although they have biological activity; (3) there is an excess of carriers, and there is still a lack of functional nano-pesticide formulations that can synergistically enhance antibacterial activity. To avoid the shortcomings of existing pesticide formulations such as single structure, excess carriers and environmental unfriendliness, constructing a nano-pesticide library with diverse structure, synergistic function and high-throughput screening has become one of the key strategies. Guanidine-containing compounds (such as polyhexamethylene biguanide PHMB) are widely used in the field of antibacterial and antiseptic due to their strong cationic nature and good biocompatibility. It is known that they can interact with negatively charged cell membranes to form a physical barrier. At the same time, the phenolic hydroxyl structure in natural products often has biological activities such as antioxidant, antibacterial and fungicidal, but lacks an efficient delivery mechanism. Therefore, based on the PHMB analogs with broad-spectrum antibacterial activity, this application combines the phenolic hydroxyl-containing natural small molecules with a wide source and high biological activity to construct a functional nano-pesticide library through co-assembly strategy. This strategy can fully utilize the diversity of analog structure modification, quickly form stable nano-pesticides, and screen new nano-pesticide formulations with synergistic antibacterial effect, providing an innovative solution for efficient screening of pesticide carriers and green pesticide development. SUMMARY

[0004] To solve the potential threat of traditional pesticides and some nano pesticides to the ecosystem, and the problems of short-lasting efficacy and insufficient targeting, the present application aims to overcome the above-mentioned defects, and provides a green, safe and synergistic anti-fungal nano pesticide construction strategy. By mixing a guanidine-containing compound and a natural small molecule containing phenolic hydroxyl in a sodium hydroxide aqueous solution, a nano particle with uniform particle size, stable structure and synergistic antibacterial effect is formed. Not only the anti-fungal activity and utilization rate of natural products are significantly improved, but also the high-throughput combination of functional nano pesticide library is provided, which provides an ideal data basis for the development of machine learning models based on structure-function relationship, and provides an efficient solution for green plant disease control.

[0005] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] 1. The present application provides a nano pesticide preparation formed by co-assembly of a guanidine-containing compound and a natural small molecule containing phenolic hydroxyl, characterized in that:

[0007] A compound A containing at least one guanidine functional group is selected, the guanidine group can be a mono-guanidine group or a di-guanidine group structure, and the compound A includes but is not limited to polyhexamethylene mono-guanidine, polyamino propyl di-guanidine, guanidine hexanediamine polymer hydrochloride, etc. A natural small molecule compound B containing 1-3 phenolic hydroxyl structures is selected, and the compound B includes but is not limited to honokiol, hesperetin, curcumin, alizarin, etc. Compound A and compound B are mixed in a mass ratio of 1:5-20:1 in a sodium hydroxide aqueous solution with pH 9-11, the stirring temperature range is 18-60℃, and the stirring time in the dark is 0.25-3h, and the nano particles can be co-assembled. The nano particles can exist stably in water without adding any exogenous stabilizer.

[0008] 2. The method supports the combination construction of different guanidine-containing compounds and phenolic hydroxyl-containing natural small molecules, has the potential of high-throughput construction of functional nano pesticide preparation library, and has verified the universality and adaptability through 70 kinds of combination assembly experiments. The obtained data can be used as a training sample for machine learning model, and provides basic data support for intelligent nano pesticide combination screening.

[0009] 3. The nano particles are suitable for preventing and controlling various plant pathogenic fungi (such as fusarium graminearum), and various combinations show synergistic antibacterial effect, and the synergistic effect can be quantitatively evaluated by the diameter of the inhibition zone.

[0010] The present application overcomes the problems of high toxicity, easy resistance of existing chemical pesticides, and single structure and difficulty in regulation of some nano-pesticides by constructing a co-assembled nano-pesticide library based on PHMB analogues and natural small molecules containing phenolic hydroxyl groups. The method is simple to operate, does not require additional carriers and stabilizers, is green and environmentally friendly, and the constructed library has diverse structures and rich functions, and can efficiently screen new nano-pesticide materials with synergistic antifungal effect. In addition, the combination strategy has high data potential, providing a reliable foundation for subsequent construction of structure-function relationship database and intelligent design of pesticide materials based on machine learning, and has advanced technology and broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 Particle size distribution of the polyaminopropyl biguanide-hesperidin co-assembled nano-pesticide described in Example 1

[0012] Figure 2 UV spectrum and nano-pesticide physical map of the polyaminopropyl biguanide-hesperidin co-assembled nano-pesticide described in Example 1

[0013] Figure 3 Antifungal activity of polyaminopropyl biguanide-curcumin nanoparticles and polyhexamethylene guanidine hydrochloride-myricetin nanoparticles against Fusarium graminearum

[0014] Figure 4 Flowchart of constructing 70 nano-pesticide structure-function relationship database described in Example 8 DETAILED DESCRIPTION

[0015] The present application will be further described below through specific examples and drawings. It should be noted that the following described examples are only preferred partial embodiments of the present application, for exemplarily illustrating the principles and technical solutions of the present application, and are not intended to exhaust or limit the present application to the specific details described. Based on the disclosure of the present application, those skilled in the art can conceive other possible embodiments or make various modifications, replacements and variations without inventive labor, which should be considered to fall within the protection scope of the present application defined by the appended claims.

[0016] Example 1: Preparation of polyhexamethylene biguanide hydrochloride analogue (polyaminopropyl biguanide) / phenolic hydroxyl-containing natural small molecule B2 (hesperidin) co-assembled nano-pesticide preparation

[0017] 5 mg of PHMB analog A4 (polyaminopropyl biguanide) was dissolved in 5 mL of deionized water to obtain a 1 mg / mL polyaminopropyl biguanide solution. 25 mg of a phenolic hydroxyl-containing natural small molecule B2 (hesperidin) was dissolved in 5 mL of pH 9 sodium hydroxide aqueous solution to obtain a 5 mg / mL hesperidin solution. 1 mL of hesperidin solution was added dropwise to a glass bottle containing 1 mL of polyaminopropyl biguanide solution. The mixture was stirred at 18°C ​​in the dark for 0.25 h, then centrifuged, the supernatant was discarded, and the precipitate was dried to obtain a polyaminopropyl biguanide-hesperidin solid powder, abbreviated as A4-B2, with the structure shown below. 1 mg of the polyaminopropyl biguanide-hesperidin solid powder was dispersed in 5 mL of deionized water to obtain an aqueous solution of the 0.2 mg / mL polyaminopropyl biguanide-hesperidin co-assembled nano-pesticide formulation.

[0018] The uniformity and stability of this nanopesticide were determined using dynamic light scattering, and the results are shown in the attached figure. Figure 1 and appendix Figure 2 As shown, this demonstrates that polyaminopropyl biguanide and hesperidin can co-assemble in water to form nano-pesticide formulations.

[0019]

[0020] Example 2: Preparation of nanopesticide formulations co-assembled with polyhexamethylene biguanide hydrochloride analog (N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide) / phenolic hydroxyl-containing natural small molecule B1 (myricetin)

[0021] 1 mg of PHMB analog A1 (N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide) was dissolved in 5 mL of deionized water to obtain a 0.2 mg / mL N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide solution; 1 mg of a phenolic hydroxyl-containing natural small molecule B1 (myricetin) was dissolved in 1 mL of pH 10 sodium hydroxide aqueous solution to obtain a 1 mg / mL myricetin solution. 2 mL of myricetin solution was added dropwise to a glass bottle containing 1 mL of N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide solution. The mixture was stirred at 25 °C in the dark for 0.5 h, then centrifuged, the supernatant was discarded, and the precipitate was dried to obtain N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide-myricetin powder, abbreviated as A1-B1, with the structure shown below. 1 mg of N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide-myricetin powder was dispersed in 1 mL of deionized water to obtain an aqueous solution of the 1 mg / mL N-(6-aminohexyl)-N'-(4-chlorophenyl)biguanide-myricetin co-assembled nanopesticide formulation. The uniformity and stability of the nanopesticide were determined by dynamic light scattering, and the results are shown in Table 1 (A1-B1).

[0022]

[0023] Example 3: Preparation of polyhexamethylene biguanide hydrochloride analogue (N-(4-chlorophenyl)-N'-[6-[[(cyanoamino)iminomethyl]amino]hexyl]biguanide) / phenolic hydroxyl-containing natural small molecule B4(honokiol) co-assembled nano-pesticide preparation

[0024] Example 3 differs from Example 1 in that the PHMB analogue A2 (N-(4-chlorophenyl)-N'-[6-[[(cyanoamino)iminomethyl]amino]hexyl]biguanide) and the phenolic hydroxyl-containing natural small molecule B4 (honokiol), honokiol was dissolved in sodium hydroxide aqueous solution with pH = 11, according to the mass ratio of N-(4-chlorophenyl)-N'-[6-[[(cyanoamino)iminomethyl]amino]hexyl]biguanide to honokiol 1:20, stirring at 45°C in the dark, after stirring for 1 h, centrifugation was performed to discard the supernatant, and the precipitate was dried to obtain N-(4-chlorophenyl)-N'-[6-[[(cyanoamino)iminomethyl]amino]hexyl]biguanide-honokiol powder, referred to as A2-B4, the structure of which is shown below. The uniformity and stability of the nano-pesticide were determined by a dynamic light scattering instrument, and the results are shown in Table 1 (A2-B4).

[0025]

[0026] Example 4: Preparation of polyhexamethylene biguanide hydrochloride analogue (polyhexamethylene guanidine hydrochloride) / phenolic hydroxyl-containing natural small molecule B5 (magnolol) co-assembled nano-pesticide preparation

[0027] Example 4 differs from Example 1 in that the PHMB analogue A3 (polyhexamethylene guanidine hydrochloride) and the phenolic hydroxyl-containing natural small molecule B5 (magnolol), magnolol was dissolved in sodium hydroxide aqueous solution with pH = 9, according to the mass ratio of polyhexamethylene guanidine hydrochloride to magnolol 10:1, stirring at 60°C in the dark, after stirring for 1 h, centrifugation was performed to discard the supernatant, and the precipitate was dried to obtain polyhexamethylene guanidine hydrochloride-magnolol powder, referred to as A3-B5, the structure of which is shown below. The uniformity and stability of the nano-pesticide were determined by a dynamic light scattering instrument, and the results are shown in Table 1 (A3-B5).

[0028]

[0029] Example 5: Preparation of polyhexamethylene biguanide hydrochloride analogue (1,1'-hexamethylene bis[5-(4-chlorophenyl)biguanide) / phenolic hydroxyl-containing natural small molecule B6 (phloretin) co-assembled nano-pesticide preparation

[0030] Example 5 is different from Example 1 in that PHMB analogue A5 (1,1'-hexamethylenebis[5-(4-chlorophenyl)biguanide) and phenolic hydroxyl-containing natural small molecule B6 (phloretin), phloretin was dissolved in aqueous sodium hydroxide solution at pH = 10, according to the mass ratio of 1,1'-hexamethylenebis[5-(4-chlorophenyl)biguanide to phloretin 20:1, stirring at 18°C in the dark, stirring for 3h, centrifugation to discard the supernatant, drying the precipitate to obtain 1,1'-hexamethylenebis[5-(4-chlorophenyl)biguanide-phloretin powder, referred to as: A5-B6, the structure is shown below. The uniformity and stability of the nano-pesticide were determined by dynamic light scattering instrument, and the results are shown in Table 1 (A5-B6).

[0031]

[0032] Example 6: Preparation of polyhexamethylene biguanide hydrochloride analogue (N,N"-bis(2-ethylhexyl)-3,12-diaza-2,4,11,13-tetraazatetradecanediamine dihydrochloride) / phenolic hydroxyl-containing natural small molecule B8 (alizarin) co-assembled nano-pesticide preparation

[0033] Example 6 is different from Example 1 in that PHMB analogue A6 (N,N"-bis(2- ethylhexyl)-3,12-diaza-2,4,11,13-tetraazatetradecanediamine dihydrochloride) and phenolic hydroxyl-containing natural small molecule B8 (alizarin), alizarin was dissolved in aqueous sodium hydroxide solution at pH = 11, according to the mass ratio of N,N"-bis(2-ethylhexyl)-3,12-diaza-2,4,11,13-tetraazatetradecanediamine dihydrochloride to alizarin 5:1, stirring at 45°C in the dark, stirring for 0.25h, centrifugation to discard the supernatant, drying the precipitate to obtain N,N"-bis(2-ethylhexyl)-3,12-diaza-2,4,11,13-tetraazatetradecanediamine dihydrochloride-alizarin powder, referred to as: A6-B8, the structure is shown below. The uniformity and stability of the nano-pesticide were determined by dynamic light scattering instrument, and the results are shown in Table 1 (A6-B8).

[0034]

[0035] Example 7: Preparation of polyhexamethylene biguanide hydrochloride analogue (1,1'-hexylbis[5-(p-chlorophenyl)biguanide] diacetate) / phenolic hydroxyl-containing natural small molecule B 10 (curcumin) co-assembled nano-pesticide preparation

[0036] Example 7 is different from Example 1 in that PHMB analogue A7 (1,1'-hexylbis[5-(p- chlorophenyl)biguanide] diacetate) and phenolic hydroxyl-containing natural small molecule B 10(curcumin), curcumin was dissolved in sodium hydroxide aqueous solution with pH = 11, and 1,1'-hexyl bis[5-(p-chlorophenyl)biguanide] diacetate and curcumin were mixed according to the mass ratio of 1:5, stirred at 60°C in the dark, after stirring for 3h, centrifuged to discard the supernatant, dried the precipitate to obtain 1,1'-hexyl bis[5-(p-chlorophenyl)biguanide] diacetate-curcumin powder, referred to as: A7-B 10 , whose structure is shown below. The uniformity and stability of the nano-pesticide were determined by dynamic light scattering instrument, and the results are shown in Table 1 (A7-B 10 ).

[0037]

[0038] Example 8: High-throughput co-assembly library construction of 70 combinations

[0039] To systematically evaluate the adaptability and universality of the present application, the following compounds were selected for combination tests:

[0040] 1. First, from the PHMB analogues (referred to as: A1-A7) and the natural small molecules containing phenolic hydroxyl groups (referred to as: B1-B 10 ), two-by-two pairing was prepared to prepare co-assembled nano-pesticide formulations (A x -B y ), a total of 70 different combinations of nanoparticles were constructed, and the structures are shown below;

[0041]

[0042]

[0043]

[0044]

[0045] 2. The physicochemical properties of the obtained A x -B y nano-pesticides were characterized, including but not limited to the following: (1) particle size; (2) polydispersity index (PDI); (3) Zeta potential; (4) appearance description. The particle size distribution and PDI of the 70 nano-pesticide formulations obtained are shown in Table 1.

[0046] 3. From the 70 nano-pesticide formulations (A x -B y) were randomly selected, and the mycelial growth inhibition method was used to explore the in vitro inhibition activity of the two nano-pesticide formulations on Fusarium graminearum. At the same time, the minimum inhibitory concentration (MIC) of the 70 nano-pesticide formulations obtained was measured. Experimental method: The nano-preparation (polyaminopropyl biguanide-curcumin nanoparticles, polyhexamethylene guanidine hydrochloride-myricetin nanoparticles) of the application, curcumin and myricetin, polyaminopropyl biguanide and polyhexamethylene guanidine hydrochloride were dissolved with a DMSO solution, and the drugs were diluted with a 0.05% Tween aqueous solution to a final concentration of 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, 1.5625 μg / mL. The above-mentioned drug solution of each concentration was added to the pre-sterilized PDA medium, and the final DMSO concentration was controlled to be 0.5%, and then poured into sterile culture dishes, and after solidification, the corresponding concentration of drug treatment PDA plate culture medium was obtained. The mycelial block with a diameter of 5 mm was aseptically cut from the edge of the Fusarium graminearum colony cultured on the potato starch medium for 3 days, and the mycelium was inoculated downward at the center of the solidified drug-containing medium plate. All the inoculated culture dishes were incubated in a 28°C constant temperature incubator in the dark. When the mycelium of the blank control group grew to cover the whole plate, the diameter of the mycelium of each treatment group was measured, and the inhibition rate relative to the control group was calculated. As shown in FIG. 1, the nano-pesticide formulation has a synergistic antibacterial effect. In addition, the MIC of the 70 nano-pesticide formulations obtained is less than 50 μg / mL. Figure 3

[0047] 4. According to the above determination results, the information of each combination A x -B y was standardized and input into the database table item. The fields include: number, structure component, molecular characteristic descriptor, particle size, PDI, zeta potential, inhibition rate, etc.

[0048] 5. The above data is input into the database in the format of Excel, CSV or SQL, and can be imported into the machine learning modeling platform (such as Python+scikit-learn). The database can be used to build a structure-function prediction model to realize the performance prediction and reverse screening of untested combinations, as shown in FIG. 3. Figure 4

[0049] ​​The above data provides a data basis for subsequent construction of structure-function database and training of machine learning model (such as prediction of particle size / Zeta / function group on antibacterial effect). In addition, the platform is not only suitable for the co-assembly system of PHMB analogues and natural small molecules, but also can be extended to other types of high polymer-small molecule, protein-small molecule, metal-organic ligand and other co-assembly pesticide materials; the structure description and function label system can also be extended to multi-dimensional indexes such as ultraviolet resistance, controlled release performance and plant targeting according to actual needs, thereby supporting more extensive screening and intelligent development of green pesticide materials.

[0050] The present application is not limited to the specific embodiments listed in the specification, and any equivalent adjustment, technical transformation or functional extension made on the basis of the technical concept shall fall within the protection scope of the present application, and the right claim shall prevail.

[0051] Table 1. Particle size distribution and stability of 70 kinds of co-assembly nano-pesticide preparations

[0052]

[0053]

[0054]

Claims

1. A method for constructing nanopesticides based on guanidine-containing compounds and phenolic hydroxyl-containing natural small molecules, characterized in that, Includes the following steps: (1) Select compound A containing at least one guanidine functional group, wherein the guanidine group is a monoguanidine or biguanidine structure, and its general structural formula is as follows; (2) Select natural small molecule compound B containing 1 to 3 phenolic hydroxyl structures; (3) Compound A and compound B are mixed in a sodium hydroxide aqueous solution with a mass ratio of 1:5 to 20:1 at pH 9 to 11. The stirring temperature is 18 to 60°C and the stirring time is 0.25 to 3 hours in the dark. They can be co-assembled to form nanoparticles.

2. The method according to claim 1, characterized in that, Seventy types of nanoparticles were synthesized, and their structural formulas are simply referred to as: A 1-7 -B 1-10 The details are as follows:

3. The method according to claim 1, wherein the compound A is selected from: polyhexamethylene monoguanidine hydrochloride, polyaminopropyl biguanide, guanidine hexamethylenediamine polymer hydrochloride, N,N”-bis(2-ethylhexyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediimine dihydrochloride, 1,1'-hexamethylenebis[5-(4-chlorophenyl)biguanide, or an acceptable salt thereof.

4. The method according to claim 1, wherein the compound B is selected from: magnolol, hesperidin, curcumin, myricetin, quercetin, magnolol, paeonol, phloretin, alizarin, or thymol.

5. The method according to claim 2, wherein the nanoparticles are used to control Fusarium graminearum.

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