Preparation method and application of pH response type tannic acid modified pyraclostrobin metal organic framework nanocomposite

Abstract

The invention discloses a preparation method and application of a pH response type tannic acid modified pyraclostrobin metal organic framework nano-composite. The preparation method comprises the following steps: constructing a pyraclostrobin metal organic framework nano-composite by using pyraclostrobin, dimethylimidazole and zinc acetate, and then carrying out interface function modification on the pyraclostrobin-loaded metal organic framework nano-composite by using tannic acid to prepare the tannic acid modified pyraclostrobin metal organic framework nano-composite. The tannic acid modified pyraclostrobin metal organic framework nano-composite endows the pyraclostrobin with the characteristics of sustained and controlled release and pH environment response, meanwhile, Botrytis cinerea thalli are caused to generate excessive active oxygen, and the efficient utilization of the pyraclostrobin in the field of fungal disease prevention and control is enhanced. The metal organic framework nano-composite and the tannic acid have degradability and biocompatibility, and the metal organic framework nano-composite is low in preparation cost, simple in process and suitable for popularization and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a preparation method and application of a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite. Background Art

[0002] Grapes are perennial vines and one of the most widely cultivated fruit trees, primarily grown for fresh consumption and winemaking. However, grape gray mold (Botrytis cinerea) can occur during the flowering, fruit ripening, and storage periods. With the expansion of the grape industry and the increasing planting area, grape diseases caused by gray mold are becoming increasingly serious. This is particularly true during storage, transportation, and marketing, where gray mold can cause widespread mold growth, threatening grape yield and quality.

[0003] Pyraclostrobin (Pyr) is a novel methoxyacrylate QoI fungicide that targets the Cytb gene, inhibiting fungal mitochondrial respiration and hindering ATP production, ultimately leading to apoptosis in pathogenic fungal cells. Pyraclostrobin has a broad spectrum and high efficacy, exhibiting excellent control effects against fungal diseases and is widely used in the prevention and control of crop diseases such as gray mold, downy mildew, sheath blight, and anthracnose. However, pyraclostrobin suffers from shortcomings such as easy photolysis and a short duration of effectiveness during field application, which seriously restrict its efficient use in disease prevention and control. Therefore, it is highly necessary to develop and apply biodegradable, highly efficient pyraclostrobin-loaded nanoparticle drug delivery systems.

[0004] Zeolite imidazolate framework material (ZIF-8) is a hybrid porous nanometal-organic framework (MOF) constructed from zinc ions and the organic ligand 2-methylimidazole. Due to its simple preparation, controllable particle size, large porosity, high drug loading capacity, easy modification, and excellent biocompatibility, metal-organic frameworks are widely used in drug carrier transportation, food preservation and other fields. ZIF-8 has a stable structure under normal physiological conditions. However, under acidic conditions (pH 2.5-6.5), the nitrogen atom of 2-methylimidazole accepts protons and becomes protonated, thereby destroying the coordination between the zinc ion and the imidazole nitrogen atom, causing ZIF-8 to disintegrate, giving ZIF-8 acid-sensitive properties and giving the loaded drug pH environment responsiveness, facilitating intelligent drug delivery and release, thereby improving the drug's biological activity. Summary of the Invention

[0005] The present invention addresses the problems of poor solubility, dispersibility, sedimentation, and stability of pyraclostrobin and provides a method for preparing a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite (Pyr-ZIF-8-TA). The method improves the sustained-release and pH-responsiveness of pyraclostrobin, causes the Botrytis cinerea fungus to produce excessive active oxygen, and thereby enhances the antifungal bioactivity of the pyraclostrobin metal-organic framework nanocomposite.

[0006] Therefore, one of the objects of the present invention is to provide a method for preparing a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite, wherein the pH-responsive metal-organic framework nanocomposite is used to load pyraclostrobin, and then the loaded pyraclostrobin metal-organic framework nanocomposite is modified with tannic acid to obtain a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite.

[0007] The specific preparation method mainly includes the following steps:

[0008] (1) Dissolve zinc acetate and glacial acetic acid in deionized water to prepare solution A;

[0009] (2) dissolving pyraclostrobin and 2-methylimidazole in methanol to prepare solution B;

[0010] (3) adding solution A dropwise to solution B, magnetically stirring for 1 to 12 hours, collecting the precipitate by centrifugation, and washing the precipitate with deionized water to prepare a pyraclostrobin metal-organic framework nanocomposite;

[0011] (4) The pyraclostrobin metal-organic framework nanocomposite was dispersed in 10-100 mL of Tris-HCl buffer, and then 0.005-0.05 g of tannic acid was added. The mixture was magnetically stirred and centrifuged to collect the precipitate. The precipitate was washed with deionized water to obtain a tannic acid-modified pyraclostrobin metal-organic framework nanocomposite.

[0012] In step (1), the concentration of zinc acetate in solution A is 50 to 500 mmol / L, more preferably 150 mmol / L.

[0013] The concentration of the glacial acetic acid in solution A is 0.005 to 0.05% by volume, more preferably 0.02%.

[0014] In step (2), the molar ratio of pyraclostrobin to 2-methylimidazole is 1:(1-20), more preferably 1:10.

[0015] In step (3), the time for adding solution A dropwise to solution B is 1-20 min, more preferably 5 min.

[0016] In steps (3)-(4), the speed of the magnetic stirring is 300-600 rpm.

[0017] In steps (3)-(4), the centrifugation is carried out at 8000-11000 rpm for 15-60 min.

[0018] In step (4), the volume of the Tris-HCl buffer is 10 to 100 mL, more preferably 20 mL; the mass of tannic acid is 0.005 to 0.05 g, more preferably 0.010 g.

[0019] On the other hand, the present invention also provides a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite prepared according to the above preparation method.

[0020] Preferably, the pH response range of the tannic acid-modified pyraclostrobin metal-organic framework nanocomposite is from acidic to neutral.

[0021] On the other hand, the present invention also provides the use of the pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite in the preparation of antifungal drugs.

[0022] Preferably, the model fungus strain includes Botrytis cinerea.

[0023] On the other hand, the present invention also provides an antifungal drug comprising an effective amount of a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite as an active ingredient.

[0024] The present invention discloses a preparation method and application of a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite. The present invention uses a pH-responsive metal-organic framework nanocomposite to load pyraclostrobin, and then uses tannic acid to modify the pH-responsive metal-organic framework nanocomposite loaded with pyraclostrobin to obtain a tannic acid-modified pyraclostrobin metal-organic framework nanocomposite. The pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite prepared by the present invention causes the grape gray mold Botrytis cinerea fungus to produce excessive active oxygen, thereby improving the antifungal biological activity of the pyraclostrobin metal-organic framework nanocomposite, thereby enhancing the efficient utilization of pyraclostrobin in the field of fungal disease prevention and control, extending the effective period of pyraclostrobin and presenting pH environment responsive characteristics. The metal-organic framework nanocomposite and tannic acid involved in the present invention are degradable and biocompatible, and the preparation process is simple and low-cost, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1These are electron microscopic characterization images of pyraclostrobin metal-organic framework nanocomposites, (A) transmission electron microscopic image of Pyr-ZIF-8; (B) transmission electron microscopic particle size statistical distribution image of Pyr-ZIF-8; (C) transmission electron microscopic image of Pyr-ZIF-8-TA; (D) transmission electron microscopic particle size statistical distribution image of Pyr-ZIF-8-TA.

[0026] Figure 2 It is the zeta potential of pyraclostrobin metal-organic framework nanocomplex.

[0027] Figure 3 This is an analysis of the release performance of pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomplexes.

[0028] Figure 4 Based on DCFH-DA fungal reactive oxygen species fluorescence microscopy images, (A) CK, (B) Pyr-ZIF-8, (C) Pry-ZIF-8-TA; (D) fungal reactive oxygen species fluorescence spectrum; (E) fungal reactive oxygen species fluorescence value. DETAILED DESCRIPTION

[0029] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0030] Example 1: Preparation of pyraclostrobin metal-organic framework nanocomposite

[0031] (1) Dissolve 3 mmol of zinc acetate and 4 μL of glacial acetic acid in 20 mL of deionized water and stir magnetically at 300 rpm at 25°C for 10 min to prepare solution A.

[0032] (2) 1 mmol of pyraclostrobin and 10 mmol of 2-methylimidazole were dissolved in 10 mL of methanol and stirred at 25°C and 300 rpm for 10 min to prepare solution B.

[0033] (3) Solution A was added dropwise to solution B within 5 min, and magnetic stirring was performed at 25°C and 500 rpm for 8 h to prepare a pyraclostrobin metal-organic framework nanocomposite suspension;

[0034] (4) The pyraclostrobin metal organic framework nanocomposite suspension was centrifuged at 25°C and 10,000 rpm for 30 minutes, and the precipitate was collected and washed three times with deionized water to prepare the pyraclostrobin metal organic framework nanocomposite (Pyr-ZIF-8), which was freeze-dried for later use. The morphology of Pyr-ZIF-8 is shown in FIG. Figure 1 As shown in Figure A. From the figure, we know that the average dry particle size of Pyr-ZIF-8 is 22.59nm ( Figure 1 B), and its Zeta potential is 30.38 mV ( Figure 2 ).

[0035] Example 2: Preparation of tannic acid-modified pyraclostrobin metal-organic framework nanocomplex

[0036] (1) Dissolve 3 mmol of zinc acetate and 4 μL of glacial acetic acid in 20 mL of deionized water and stir magnetically at 300 rpm at 25°C for 10 min to prepare solution A.

[0037] (2) 1 mmol of pyraclostrobin and 10 mmol of 2-methylimidazole were dissolved in 10 mL of methanol and stirred at 25°C and 300 rpm for 10 min to prepare solution B.

[0038] (3) Solution A was added dropwise to solution B within 5 min, and magnetic stirring was performed at 25°C and 500 rpm for 8 h to prepare a pyraclostrobin metal-organic framework nanocomposite suspension;

[0039] (4) The pyraclostrobin metal organic framework nanocomposite suspension was centrifuged at 25° C. and 10,000 rpm for 30 minutes, the precipitate was collected, and the precipitate was washed three times with deionized water to prepare the pyraclostrobin metal organic framework nanocomposite (Pyr-ZIF-8);

[0040] (5) The pyraclostrobin metal organic framework nanocomposite was dispersed in 20 mL of Tris-HCl (pH = 8.5) buffer, 0.010 g of tannic acid was weighed and added to the above pyraclostrobin metal organic framework nanocomposite aqueous suspension, magnetically stirred at 25 ° C, 500 rpm for 8 h, and then centrifuged at 25 ° C, 10000 rpm for 30 min, the precipitate was collected, and the precipitate was washed with deionized water three times to prepare the tannic acid modified pyraclostrobin metal organic framework nanocomposite (Pyr-ZIF-8-TA), which was freeze-dried for later use. The morphology of Pyr-ZIF-8-TA is as follows: Figure 1 As shown in C. From the figure, we know that the average dry particle size of Pyr-ZIF-8-TA is 26.14nm ( Figure 1 D), and its Zeta potential is -10.05mV ( Figure 2 ).

[0041] The performance evaluation experiment of the pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite of the present invention will be described below.

[0042] 1: Analysis of sustained release performance under different pH environments

[0043] In this study, the dialysis method was used to simulate the drug release of pyraclostrobin and pyraclostrobin metal-organic framework nanocomplexes in vitro. That is, a 20% volume fraction of ethanol aqueous solution was used as the in vitro simulated release medium. The pH of the release medium was adjusted to 7.4, 5.5 and 4.5 using 0.1M HCl and 0.1M NaOH, respectively. 2 mg of the active ingredient of tebuconazole, Pyr original drug, Pyr-ZIF-8 and Pyr-ZIF-8-TA were accurately weighed and placed in dialysis bags (Mw 5000) respectively. The two ends of the dialysis bag were sealed, and the release medium was 20 mL of a sealed system. Then, the drug release was simulated at 28°C and a speed of 180 r / min. 1 mL of release medium was taken at 1h, 2h, 4h, 8h, 12h, 16h, 24h, 36h, 48h, 72h, 96h and 120h, and then 1 mL of fresh corresponding release medium was added to keep the total volume unchanged. When the release medium pH was 7.4 ( Figure 3 A), in the first hour, the percentages of pyraclostrobin released by Pyr original drug, Pyr-ZIF-8 and Pyr-ZIF-8-TA were 24.36%, 8.47% and 5.32%, respectively; after 24 hours of simulated release, the cumulative release amounts of pyraclostrobin from Pyr original drug, Pyr-ZIF-8 and Pyr-ZIF-8-TA were 97.29%, 49.38% and 37.69%, respectively. When the pH of the release medium was 7.4, 5.5 and 4.5, respectively ( Figure 3 B). After 24 hours of simulated release, the percentages of pyraclostrobin released from Pyr-ZIF-8-TA were 37.69% (pH 7.4), 59.37% (pH 5.5), and 61.64% (pH 4.5), respectively. These results indicate that the pyraclostrobin metal-organic framework nanocomposite drug delivery system can effectively reduce the release rate of pyraclostrobin. The tannic acid-modified pyraclostrobin metal-organic framework nanocomposite drug delivery system further reduces the release rate of pyraclostrobin and prolongs its duration of action. The tannic acid-modified pyraclostrobin metal-organic framework nanocomposite exhibits pH-responsive properties, i.e., acidic conditions facilitate the release of pyraclostrobin from the metal-organic framework carrier.

[0044] 2: Antifungal activity analysis

[0045] In this experiment, the antibacterial activity of Pyr original drug, Pyr-ZIF-8 and Pyr-ZIF-8-TA against Botrytis cinerea was determined by using potato dextrose agar medium (HKM / Huankai, 021052) and measuring the mycelial growth diameter using the cross-cross method. Potato dextrose agar medium without Pyr was used as a blank control group. Pyr original drug, Pyr-ZIF-8, and Pyr-ZIF-8-TA active ingredient (pyraclostrobin) were diluted to prepare potato dextrose agar medium with final concentrations of 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL. A 10 mm diameter sterile punch was used to punch holes at the edge of the Botrytis cinerea colony to obtain a 10 mm Botrytis cinerea cake. The cake was then inoculated in the center of potato dextrose agar medium containing pyraclostrobin and the blank control group. The culture was carried out at 28°C for 4 days. The colony growth diameter of each group was measured and the antibacterial activity was evaluated. SPSS software was used for "Probit" analysis to calculate the virulence regression equation, inhibition median concentration, correlation coefficient, and relative virulence. The results are shown in Table 1. The results showed that the EC of Pyr original drug, Pyr-ZIF-8 and Pyr-ZIF-8-TA to Botrytis cinerea 50 The values ​​were 250.37 ng / mL, 103.69 ng / mL and 74.76 ng / mL respectively. Taking pyraclostrobin original drug as the standard agent, the relative toxicity of Pyr-ZIF-8 and Pyr-ZIF-8-TA to Botrytis cinerea was 2.41 times and 3.35 times that of pyraclostrobin original drug. The EC of Pyr-ZIF-8-TA to Botrytis cinerea 50 The smallest, indicating the highest antibacterial activity. Therefore, tannic acid interface modification can effectively improve the biological activity and enhance the antibacterial effect of pyraclostrobin.

[0046] Table 1. Toxicity analysis of Pyr, Pyr-ZIF-8 and Pyr-ZIF-8-TA to Botrytis cinerea

[0047]

[0048] 3: Reactive oxygen analysis

[0049] The DCFH-DA reactive oxygen species probe was used to evaluate the effects of Pyr-ZIF-8 and Pyr-ZIF-8-TA on reactive oxygen species in Botrytis cinerea. Botrytis cinerea was inoculated into 10 mL potato dextrose medium (HKM / Huankai, 021053) and cultured at 28°C with shaking at 180 r / min for 2 days. Pyr-ZIF-8 and Pyr-ZIF-8-TA were added with an effective final concentration of 100 ng / mL of pyraclostrobin, and an equal amount of sterile deionized water was used instead of pyraclostrobin as a control group (CK). The cells were incubated for 6 h and 12 h, respectively. Then, an appropriate amount of DCFH-DA reactive oxygen species fluorescent probe was added to each sample to a final concentration of 100 ng / mL. 2μM, co-cultured in the dark for 30 minutes, centrifuged at 25°C, 3000rpm for 10 minutes to collect the mycelia, washed three times with sterile PBS (10mM, pH7.4), and a portion of the mycelia was observed using a fluorescence microscope to observe the distribution of active oxygen in the mycelia; the other portion of the washed mycelia was filtered and weighed freshly, and the mycelia were dispersed in 1mL sterile PBS. A fluorescence spectrophotometer was used to measure the fluorescence value of active oxygen in the mycelia at an excitation wavelength of 480nm and an emission wavelength scanning range of 500nm to 800nm. Figure 4 As shown in Figures A, 4B, and 4C, the fluorescence produced by reactive oxygen species in Botrytis cinerea in the Pyr-ZIF-8 and Pyr-ZIF-8-TA treatment groups was enhanced compared to the blank control group. Further quantitative analysis using a fluorescence spectrophotometer revealed that after incubation of Pyr-ZIF-8 and Pyr-ZIF-8-TA with Botrytis cinerea for 6 hours, the ROS fluorescence values ​​in Botrytis cinerea were 3.08 times and 3.91 times higher than those in the blank control group, respectively. The ROS content in the cells increased significantly, and the effect was time-dependent ( Figure 4 D and 4E). The results showed that tannic acid modified at the interface of pyraclostrobin metal-organic framework nanocomposites can induce increased ROS production in Botrytis cinereaT bacteria. Excessive ROS leads to intracellular oxidative damage, impaired hyphal cell membrane integrity, and ultimately cell apoptosis. Tannic acid, with its polyphenolic functional groups, can synergistically enhance anti-inflammatory, antibacterial, and antiviral efficacy. Based on the hydrophobic, hydrogen-bonding, and electrostatic interactions between tannic acid and metal ions, proteins, and polysaccharides, tannic acid-modified pyraclostrobin metal-organic framework nanocomposites can significantly enhance their antifungal efficacy.

[0050] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite, characterized in that: The following steps are involved: (1) dissolving zinc acetate and glacial acetic acid in deionized water to prepare solution A; (2) dissolving pyraclostrobin and 2-methylimidazole in methanol to prepare solution B; (3) adding solution A dropwise to solution B, stirring magnetically for 1 to 12 hours, collecting the precipitate by centrifugation, and washing the precipitate with deionized water to obtain a pyraclostrobin metal organic framework nanocomposite; (4) The pyraclostrobin metal organic framework nanocomposite is dispersed in 10-100 mL Tris-HCl buffer, and then 0.005-0.05 g tannic acid is added, magnetic stirring is performed, and the precipitate is collected by centrifugation. The precipitate is washed with deionized water to obtain a tannic acid-modified pyraclostrobin metal organic framework nanocomposite.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of zinc acetate in solution A is 50 to 500 mmol / L, and the concentration of glacial acetic acid in solution A is 0.005 to 0.05% by volume.

3. The preparation method according to claim 1, characterized in that: In step (2), the molar ratio of pyraclostrobin to 2-methylimidazole is 1:(1-20).

4. The preparation method according to claim 1, characterized in that: In step (3), the time for adding solution A dropwise to solution B is 1-20 min.

5. The preparation method according to claim 1, characterized in that: The speed of the magnetic stirring is 300-600 rpm; The centrifugation is carried out at 8000-11000 rpm for 15-60 min. 6 . The pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite prepared by the preparation method according to claim 1 .

7. The pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite according to claim 6, characterized in that: The pH response range of the tannic acid modified pyraclostrobin metal organic framework nanocomposite is from acidic to neutral.

8. Use of the pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite according to claim 6 in the preparation of antifungal drugs.

9. The use according to claim 8, characterized in that: The fungi include Botrytis cinerea.

10. An antifungal drug, characterized in that: The invention comprises an effective amount of the pH-responsive tannic acid-modified pyraclostrobin metal-organic framework nanocomposite according to claim 6 as an active ingredient.

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