Preparation method and application of polyhexamethylene biguanide hydrochloride / magnolol co-assembled anti-ultraviolet nano preparation

Abstract

The invention discloses a preparation method and application of a polyhexamethylene biguanide hydrochloride / magnolol co-assembled anti-ultraviolet nano preparation. The preparation method of the polyhexamethylene biguanide hydrochloride / magnolol co-assembled anti-ultraviolet nano pesticide preparation comprises the following steps: dissolving polyhexamethylene biguanide hydrochloride in deionized water to obtain a polyhexamethylene biguanide hydrochloride solution, dissolving magnolol in an alkaline aqueous solution to obtain a magnolol solution, and adding the magnolol solution into the polyhexamethylene biguanide hydrochloride solution to obtain the polyhexamethylene biguanide hydrochloride / magnolol co-assembled anti-ultraviolet nano pesticide preparation. The preparation method comprises the following steps: slowly dropwise adding a magnolol solution into a polyhexamethylene biguanide hydrochloride solution, stirring in a dark place at a certain temperature, centrifuging, discarding supernate, freeze-drying and precipitating to obtain powder, and dissolving the powder into deionized water to obtain the polyhexamethylene biguanide hydrochloride / magnolol co-assembled anti-ultraviolet nano pesticide preparation. The nano preparation is prepared by a co-assembly method in water, and has better ultraviolet resistance. Compared with an existing method, the preparation method is simple in condition, convenient to operate and low in cost.

Description

Technical Field

[0001] The invention relates to the preparation of a polyhexamethylene biguanide hydrochloride / magnolia nobile anti-ultraviolet nano pesticide preparation and application thereof in preventing and controlling fungal plant pathogens, and belongs to the field of pesticide chemical industry. Background Art

[0002] Pathogenic fungi cause a global crop yield reduction of about 20% each year, and continue to cause about 10% losses after harvest. At present, the prevention and control of plant fungal diseases mainly rely on chemical pesticides. However, the large-scale use of chemical pesticides brings serious environmental pollution and aggravates ecological imbalance. In addition, the long-term use of chemical pesticides leads to drug resistance in pathogens, which further increases the difficulty of disease prevention and control. Therefore, it is very necessary to find environmentally friendly natural antibacterial agents to replace chemical pesticides. my country is rich in medicinal plant resources. Screening natural antibacterial active ingredients from them can not only effectively prevent and control diseases, but also avoid environmental pollution and drug resistance caused by chemical pesticides. However, natural pesticides have disadvantages such as low bioavailability and poor photostability in practical applications.

[0003] In order to overcome these shortcomings, nanopesticide preparations have been favored in recent years due to their unique physical and chemical properties. Nanomaterials have high specific surface area and excellent sustained-release and targeting properties. They can be highly effective in antibacterial activities at lower concentrations, prolong the duration of efficacy and reduce the frequency of use. These properties make them show broad application prospects in disease control. However, the preparation process of traditional nanopesticide preparations is relatively complicated and usually requires the use of adjuvants or organic solvents, which not only increases production costs, but also leads to new environmental problems, thus limiting their promotion and application.

[0004] The co-assembly preparation does not require the addition of auxiliary agents during the preparation process, which not only reduces costs and improves biocompatibility, but also reduces harm to the environment. These advantages make it have important application value.

[0005] After a large number of screenings, it was found that magnolol in the extract of Chinese medicine Magnolia officinalis has a strong inhibitory effect on plant pathogens. On this basis, polyhexamethylene biguanide hydrochloride was used as a carrier, and according to the structural characteristics of polyhexamethylene biguanide hydrochloride and magnolol, a polyhexamethylene biguanide hydrochloride / magnolsol co-assembled anti-ultraviolet nanopesticide preparation was prepared in water. Compared with the traditional nanoformulation preparation method, this method requires simple conditions, is easy to operate, has low cost, stable preparation, and has anti-ultraviolet effect. So far, no reports have been found on the preparation of polyhexamethylene biguanide hydrochloride / magnolsol co-assembled nanopesticide preparations and their anti-plant pathogenic fungi activity. Summary of the invention

[0006] In view of the fact that existing chemical pesticides and nanopesticide preparations are harmful to humans and the environment and have short-lived efficacy, the present invention aims to overcome these defects and seek a green, environmentally friendly and highly effective antifungal nanopesticide preparation. The present invention relates to a preparation method of a polyhexamethylene biguanide hydrochloride / magnolia nobile nanopesticide preparation with significant antibacterial activity and its anti-plant pathogenic fungi activity.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nano pesticide preparation, which is nano-scale. The technical route of the polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nano pesticide preparation comprises the following steps:

[0009] (1) The structures of polyhexamethylene biguanide hydrochloride (PHMB) and magnolol (MN) are as follows:

[0010]

[0011] (2) Preparation of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation (PM NFs):

[0012] Dissolve polyhexamethylene biguanide hydrochloride in deionized water to obtain a polyhexamethylene biguanide hydrochloride solution, and dissolve magnolol in an alkaline aqueous solution. Slowly drop magnolol solution into a glass bottle containing polyhexamethylene biguanide hydrochloride solution, stir in the dark at a certain temperature, discard the supernatant after centrifugation, dry and precipitate to obtain powder, and disperse the powder in deionized water to obtain an aqueous solution of the polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation. Disperse 1 mg of the reagent in 10 mL of deionized water to obtain an aqueous solution of 0.1 mg / mL polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation.

[0013] The alkaline aqueous solution has a pH of 9-11;

[0014] The concentration of the polyhexamethylene biguanide hydrochloride solution is 0.1 mg / mL to 10 mg / mL, preferably 0.1 mg / mL to 1 mg / mL;

[0015] The concentration of the magnolol solution is 0.01 mg / mL to 1 mg / mL, preferably 0.1 mg / mL to 1 mg / mL;

[0016] The mass ratio of the polyhexamethylene biguanide hydrochloride solution to the magnolol solution is 1:5 to 100:1, and the preferred mass ratio is 1:1 to 10:1;

[0017] The stirring temperature range is 18-60°C, preferably 20-30°C, the stirring speed is 500-800rpm, and the light-proof stirring time is 0.25-1h;

[0018] The centrifugal speed is 3000-5000 rpm, and the centrifugal time is 15-20 min;

[0019] The drying method is freeze drying.

[0020] (3) Structural characterization of polyhexamethylene biguanide hydrochloride / magnolia officinalis co-assembled nanopesticide preparation

[0021] The ultraviolet absorption peaks of polyhexamethylene biguanide hydrochloride, magnolol, and polyhexamethylene biguanide hydrochloride / magnolsol co-assembled nanopesticide preparations were measured using a UV-visible absorption spectrometer. The test solution was dissolved in deionized water, and the UV-visible absorption spectrometer was preheated for 10 minutes, with a scanning wavelength range of 200nm to 850nm. 1.5mL of deionized water was used as a reference solution. At the same time, transmission electron microscopy, nuclear magnetic resonance, infrared spectroscopy, and thermogravimetric analysis of polyhexamethylene biguanide hydrochloride, magnolol, and polyhexamethylene biguanide hydrochloride / magnolsol co-assembled nanopesticide preparations were carried out.

[0022] (4) Storage stability of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation

[0023] Referring to the requirements of the National Standard for Determination of Pesticide Stability (GB / T 19136-2003), 20 mL of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nano-solution was placed in a clean vial, sealed and placed in a 0°C refrigerator, a 25°C room temperature desiccator and a 54°C constant temperature box for temperature stability experiments. The samples to be tested were taken out after storage for 7d and 14d, respectively, and their particle size distribution and polymer dispersity index (PdI) were determined by dynamic light scattering.

[0024] (5) Determination of the UV resistance of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparations

[0025] 20 mL of the nanoformulation and magnolol solution were respectively placed in a quartz glass tube, which was placed 20 cm away from a 36 W ultraviolet lamp. 1.0 mL of samples were taken at different time intervals, and the concentration of magnolol in the solution was determined by HPLC.

[0026] The polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nano pesticide preparation of the present invention is used for preventing and controlling plant pathogenic fungi, including Fusarium graminearum, Helminthosporium maydis, Alternaria alternata, Eutypella microtheca, Fusarium sambucinum, Fusarium proliferatum, Fusarium oxysporum, Monilinia fructicola and the like.

[0027] Compared with the prior art, the advantages of the present invention are:

[0028] The polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation is co-assembled in water, which has the advantages of simple preparation conditions, convenient operation, low cost, synergistic antibacterial properties, etc.

[0029] In summary, the polyhexamethylene biguanide hydrochloride / magnolia bark phenol co-assembled nano pesticide formulation provided in this application has a mature preparation technology and is easy to operate. The prepared nano formulation has good light stability and good application effect, and has important application potential for plant disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Ultraviolet absorption spectra of the synthesized nanoformulation, magnolol and polyhexamethylene biguanide hydrochloride in Example 1.

[0031] Figure 2 : Transmission electron micrograph of the synthesized nanoformulation in Example 1.

[0032] Figure 3 : The nuclear magnetic resonance spectra of the synthesized nano preparation, magnolol and polyhexamethylene biguanide hydrochloride in Example 1.

[0033] Figure 4 : Infrared spectra of the synthesized nano preparation, magnolol and polyhexamethylene biguanide hydrochloride in Example 1.

[0034] Figure 5 : Thermogravimetric diagrams of the synthesized nanoformulation, magnolol and polyhexamethylene biguanide hydrochloride in Example 1.

[0035] Figure 6 : Dynamic light scattering particle size distribution diagram of the stability test of the synthetic nanoformulation in Example 1.

[0036] Figure 7 : Fitting diagram of the anti-ultraviolet stability and degradation rate of the synthesized nanoformulation and magnolol in Example 1.

[0037] Figure 8: In Example 2, the particle size distribution diagram of the nanopesticide preparation.

[0038] Fig. 9 : In Example 3, the particle size distribution diagram of the nanopesticide preparation. DETAILED DESCRIPTION

[0039] The present invention is further described below by specific embodiments and drawings. The following describes only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by other technicians in the field without creative work should fall within the scope of protection of the claims of this application.

[0040] Example 1: Preparation of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation

[0041] Dissolve 20 mg of polyhexamethylene biguanide hydrochloride in 5 mL of deionized water to obtain a 4 mg / mL polyhexamethylene biguanide hydrochloride solution, and dissolve 10 mg of magnolol in 10 mL of a pH=10 alkaline aqueous solution to obtain a 1 mg / mL magnolol solution. Slowly drop 100 μL of magnolol solution into a glass bottle containing 1 mL of polyhexamethylene biguanide hydrochloride solution, stir at room temperature in the dark, centrifuge after stirring for 1 hour, discard the supernatant, and dry the precipitate to obtain a powder. Disperse 1 mg of the powder in 10 mL of deionized water to obtain the 0.1 mg / mL aqueous solution of the polyhexamethylene biguanide hydrochloride / magnolia co-assembled nanopesticide preparation.

[0042] The aqueous solution of the polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation was detected by ultraviolet spectroscopy. The results are shown in the attached figure. Figure 1 The nanoparticles were observed using a transmission electron microscope, and the results are shown in the attached Figure 2 As shown; the obtained nanoformulation was analyzed by nuclear magnetic resonance, and the results are attached Figure 3 As shown; the obtained nanoformulation was analyzed by infrared spectroscopy, and the results are shown in the attached Figure 4 The obtained nanoformulation was analyzed by thermogravimetric analysis, and the results are shown in the attached Figure 5 shown.

[0043] The above structural characterization of the nanopesticide preparation proves the successful synthesis of the nanoformulation.

[0044] 20 mL of 4 mg / mL polyhexamethylene biguanide hydrochloride / magnolia bark phenol co-assembled nano solution was placed in three clean vials, and placed in a 0°C refrigerator, a 25°C room temperature dryer, and a 54°C constant temperature box for temperature stability experiments. The samples were taken out after 7 days and 14 days of storage, and their particle size distribution and PdI were determined by dynamic light scattering. Figure 6This indicates that the nanoformulation is stable and meets the requirements of (GB / T 19136-2003).

[0045] Take 20mL of 4mg / mL polyhexamethylene biguanide hydrochloride / magnolia phenol co-assembled nanopesticide preparation and 0.1mg / mL magnolia phenol solution respectively, and place them 20cm away from a 36W ultraviolet lamp. Take 1.0mL samples at intervals of 0h, 3h, 6h, 9h, 12h, 15h, 24h, and 36h, and determine the concentration of magnolia phenol in the solution by HPLC, and fit it by the rate equation. Figure 7 This shows that the anti-ultraviolet ability of the nanopreparation is significantly higher than that of magnolol solution.

[0046] Example 2: Preparation of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation

[0047] Dissolve 40 mg of polyhexamethylene biguanide hydrochloride in 5 mL of deionized water to obtain an 8 mg / mL polyhexamethylene biguanide hydrochloride solution, and dissolve 10 mg of magnolol in 10 mL of a pH=9 alkaline aqueous solution to obtain a 1 mg / mL magnolol solution. Slowly drop 1 mL of magnolol solution into a glass bottle containing 1 mL of polyhexamethylene biguanide hydrochloride solution, stir in the dark at 60°C, centrifuge and discard the supernatant after stirring for 30 minutes, and dry the precipitate to obtain a powder. Disperse 2 mg of the powder in 10 mL of deionized water to obtain the 0.2 mg / mL aqueous solution of the polyhexamethylene biguanide hydrochloride / magnolia co-assembled nanopesticide preparation. Figure 8 The particle size distribution of the nanoformulation is shown.

[0048] Example 3: Preparation of polyhexamethylene biguanide hydrochloride / magnolia nobile co-assembled nanopesticide preparation

[0049] Dissolve 1 mg of polyhexamethylene biguanide hydrochloride in 1 mL of deionized water to obtain a 1 mg / mL polyhexamethylene biguanide hydrochloride solution, and dissolve 1 mg of magnolol in 1 mL of a pH=11 alkaline aqueous solution to obtain a 1 mg / mL magnolol solution. Slowly drop 10 μL of magnolol solution into a glass bottle containing 1 mL of polyhexamethylene biguanide hydrochloride solution, stir in the dark at 18°C, centrifuge and discard the supernatant after stirring for 45 minutes, and dry the precipitate to obtain a powder. Disperse 1 mg of the powder in 10 mL of deionized water to obtain the 0.1 mg / mL aqueous solution of the polyhexamethylene biguanide hydrochloride / magnolia co-assembled nanopesticide preparation. Fig. 9 The particle size distribution of the nanoformulation is shown.

[0050] Example 4: In vitro inhibitory activity of the nanopesticide preparation obtained by the present invention on plant pathogenic fungi

[0051] The mycelium growth rate method was used to determine the inhibitory activity of the nano preparation of the present invention against Fusarium graminearum, Helminthosporium maydis, Alternaria alternata, Eutypella microtheca, Fusarium sambucinum, Fusarium proliferatum, Fusarium oxysporum, and Monilinia fructicola.

[0052] Experimental method: The nanoformulation (PM NFs), magnolol (MN), and polyhexamethylene biguanide hydrochloride (PHMB) in Example 1 of the present invention were dissolved in DMSO solution, and the drugs were diluted with 0.1% Tween aqueous solution to a final concentration of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.5625 μg / mL. The above-mentioned concentrations of drugs were prepared into drug-containing plates containing 0.5% DMSO solution. A bacterial cake with a diameter of 5 mm was taken from the edge of the hyphae cultured on the PDA plate for three days and placed in the center of the solidified drug-containing plate with the hyphae facing down. The inoculated plate was transferred to a constant temperature incubator and cultured at 28°C. When the hyphae of the blank control grew all over the plate, the hyphae growth diameter on each plate was measured, the inhibition rate of hyphae growth by each treatment group was calculated, and the EC of the drug was calculated by SPSS Statistics 27 software. 50 value.

[0053] Table 1 EC values ​​of in vitro antibacterial activity of the nanoformulations of the present invention against various plant pathogenic fungi 50 value

[0054]

[0055] According to the co-toxicity coefficient analysis, the addition of PHMB does not reduce the antibacterial activity of magnolol (MN), and the antibacterial activity of the nanopesticide preparation is higher than that of magnolol alone. The high antifungal activity of the nanopesticide preparation PM NFs of the present invention lays a foundation for its further practical application.

[0056] The protection scope of the present invention includes but is not limited to the above embodiments. The protection scope of the present invention shall be based on the claims. Any replacement, deformation, and improvement of the present technology that can be easily thought of by those skilled in the art shall fall within the protection scope of the present invention.

Claims

1. A co-assembled anti-ultraviolet nanopesticide preparation containing polyhexamethylene biguanide hydrochloride and magnolol, characterized in that: Its structural formula is as follows:

2. A method for preparing a co-assembled anti-ultraviolet nanopesticide preparation containing polyhexamethylene biguanide hydrochloride and magnolol as claimed in claim 1, characterized in that: (1) weighing polyhexamethylene biguanide hydrochloride and dissolving it in deionized water to obtain a polyhexamethylene biguanide hydrochloride solution; (2) weighing magnolol and dissolving it in an alkaline aqueous solution to obtain a magnolol solution; (3) adding the magnolol solution dropwise to the polyhexamethylene biguanide hydrochloride solution while stirring, Stirring in the dark, discarding the supernatant after centrifugation, freeze-drying the precipitate to obtain powder, and dispersing the powder in deionized water to obtain the co-assembled anti-ultraviolet nano-pesticide preparation of polyhexamethylene biguanide hydrochloride and magnolol; The concentration of the polyhexamethylene biguanide hydrochloride solution is 0.1 mg / mL to 10 mg / mL, the concentration of the magnolol solution is 0.01 mg / mL to 1 mg / mL, and in a pH = 9-11 alkaline aqueous solution, the mass ratio of polyhexamethylene biguanide hydrochloride to magnolol is 1:5 to 100:1, the stirring temperature ranges from 18 to 60° C., and the light-proof stirring time is 0.25 to 1 hour.

3. The nanopesticide preparation according to claim 1, characterized in that: It is used against the following phytopathogenic fungi: Fusarium graminearum, Helminthosporium maydis, Alternaria alternata, Eutypellamicrotheca, Fusarium sambucinum, Fusarium proliferatum, Fusarium oxysporum or Monilinia fructicola.

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