pH / cellulase dual-responsive carvacol nanoparticle, preparation method and application thereof

Abstract

This invention provides pH / cellulase dual-responsive carvacrol nanoparticles, their preparation method, and applications. Preparation method: First, mesoporous silica (MSN) nanoparticles are prepared using a surfactant-templating method with cetyltrimethylammonium bromide as a template; MSN-NH2 is prepared via grafting using 3-aminopropyltriethoxysilane as an amination reagent. Second, carvacrol@MSN-NH2 is prepared via electrostatic adsorption using carvacrol as the loading active substance. Finally, carboxymethyl cellulose (CMC) is used as a coating material, and carvacrol@MSN-NH2@CMC is obtained through an acylation reaction. The prepared carvacrol@MSN-NH2@CMC can achieve precise delivery and intelligent release of carvacrol at the target site of the disease by responding to changes in pH and cellulase release in the microenvironment of the lesion after infection with *Brassica napus*. Carvacrol@MSN-NH2@CMC has a significant effect on the prevention and control of soft rot in Chinese cabbage. Its preparation method is simple, the material cost is low, and the reaction conditions do not require high temperature and high pressure, which can provide a new solution for the green prevention and control of soft rot in Chinese cabbage.

Description

Technical Field

[0001] This invention relates to the preparation of pH / cellulase dual-responsive nanoparticles, specifically a pH / cellulase dual-responsive carvacrol@MSN-NH2@CMC nanoparticle and its preparation method, as well as the application of the nanoparticles in resisting soft rot disease in Chinese cabbage. Background Technology

[0002] Chinese cabbage, belonging to the Brassicaceae family and the Brassica genus, is a widely cultivated leafy vegetable in my country, playing a vital role in the vegetable supply. Soft rot of Chinese cabbage, caused by *Pectobacterium carotovorum* subsp. *carotovorum* (Pcc), is characterized by high incidence and rapid spread. Once an outbreak occurs, it often leads to rapid decay of the cabbage tissue, large-scale yield reduction, and severe economic losses.

[0003] A paper published in *Shanxi Agricultural Sciences* by the inventors, titled "Control Effects of Three Plant-Derived Active Ingredients on Soft Rot of Chinese Cabbage," demonstrated that carvacrol has a certain control effect on soft rot of Chinese cabbage in a short period. However, subsequent research revealed limitations in its application due to the volatility and oxidizability of carvacrol, leading to low utilization rates and poor field persistence. When the treatment duration exceeded 5 days, the control effect significantly decreased; by day 10, the preventive effect of carvacrol was only 3.27%–6.42%, and the curative effect was only 10.69%–19.68%.

[0004] The carvacrol@MSN-NH2@CMC nanoparticles constructed in this invention utilize regularly ordered mesoporous silica (MSN) nanoparticles as a carrier. Carvacrol, an antibacterial active ingredient, is electrostatically adsorbed onto the MSN surface or within its pores via hydrogen bonding and van der Waals forces. MSN possesses a high specific surface area and abundant internal pores, effectively blocking light and oxygen, thus significantly reducing the volatilization and oxidative degradation of carvacrol. Since soft rot pathogens infecting cabbage alter the microenvironment's pH and release cellulase, this invention utilizes an acylation reaction to coat carboxymethyl cellulose onto carvacrol-loaded MSN, achieving a response of the carvacrol@MSN-NH2@CMC nanoparticles to pH and cellulase.

[0005] The carvacrol@MSN-NH2@CMC nanoparticles constructed in this invention can significantly improve the control effect of carvacrol on soft rot of Chinese cabbage, overcoming its defects of low utilization rate and poor field persistence. The dual-response characteristics of carvacrol@MSN-NH2@CMC nanoparticles can also realize the precise delivery and intelligent release of carvacrol in time and space, thereby meeting the needs of its efficient utilization. Summary of the Invention

[0006] The purpose of this invention is to improve the control effect of carvacrol on soft rot of Chinese cabbage, and to solve the problems of low drug utilization and poor field persistence of carvacrol in practical applications. This invention provides a pH / cellulase dual-responsive carvacrol@MSN-NH2@CMC nanoparticle, its preparation method, and the application of the nanoparticle in the control of soft rot of Chinese cabbage.

[0007] The method for preparing carvacrol@MSN-NH2@CMC nanoparticles provided by this invention includes the following steps:

[0008] (1) Weigh 0.9-1.30 g of hexadecyltrimethylammonium bromide and add it to 30-50 mL of ultrapure water. Stir the solution with a magnetic stirrer until it is clear and transparent. Take a fresh beaker and add 40-60 mL of anhydrous ethanol and 40-60 mL of ultrapure water to it to prepare a mixed solution. Then add 1.5-1.9 mL of ammonia water to the mixed solution and add the above-mentioned hexadecyltrimethylammonium bromide aqueous solution. Stir at 27°C-33°C for 20-40 min. Add 3-7 mL of tetraethyl orthosilicate dropwise to the above mixed solution in three portions, with each dropwise addition occurring 1 hour apart. After stirring magnetically at 300-500 r / min for 10-14 h at 27°C-33°C, the reaction solution is centrifuged at 7000-10000 r / min for 10-14 min. After pouring out the supernatant, the precipitate is washed 4 times each with ultrapure water and anhydrous ethanol, and then dried at 70-90°C to obtain MSN nanoparticles containing hexadecyltrimethylammonium bromide.

[0009] MSN nanoparticles containing hexadecyltrimethylammonium bromide were dispersed in a mixed solution of 1-3 mL hydrochloric acid and 80-120 mL anhydrous ethanol. The hexadecyltrimethylammonium bromide was removed by magnetic stirring at 50-70°C for 10-14 h. The resulting product was centrifuged at 7000-10000 r / min for 10-14 min. After discarding the supernatant, the precipitate was washed four times each with ultrapure water and anhydrous ethanol, and then dried at 70-90°C to obtain MSN nanoparticles.

[0010] (2) Disperse 100-300 mg of MSN nanoparticles obtained in the previous step in 60-140 mL of DMF, then add 200-300 µL of 3-aminopropyltriethoxysilane and stir magnetically at room temperature for 18-28 h. Centrifuge the reaction solution at 8000-10000 r / min for 13-17 min, pour off the supernatant, and wash the precipitate 4 times each with DMF, anhydrous ethanol and ultrapure water. After freeze-drying, MSN-NH2 nanoparticles are obtained.

[0011] (3) Mix MSN-NH2, carvacrol and chloroform in a ratio of 1:1:20 to 1:1:40, evaporate the chloroform in a fume hood, and repeat 2-4 times to obtain carvacrol@MSN-NH2 nanoparticles.

[0012] (4) Disperse 300-500 mg CMC in cold water at 2-6°C and stir until dissolved. Then add 50-100 mg EDC and stir at room temperature for 20-40 min. Then add 30-60 mg NHS and 300-500 mg carvacrol@MSN-NH2. After stirring the reaction solution at room temperature for 18-28 h, centrifuge the reaction solution at 7000-10000 r / min for 10-14 min. After pouring out the supernatant, wash the precipitate with ultrapure water 4 times and vacuum dry to obtain carvacrol@MSN-NH2@CMC nanoparticles.

[0013] In step (1), the preferred amount of hexadecyltrimethylammonium bromide is 1.1 g, added to 40 mL of ultrapure water. When preparing the mixed solution, the preferred amount of anhydrous ethanol is 50 mL, and the preferred amount of ultrapure water is 50 mL. The preferred amount of ammonia added to the mixed solution is 1.7 mL, the preferred reaction temperature is 30°C, and the preferred stirring time is 30 min. The preferred amount of tetraethyl orthosilicate is 5 mL, the preferred stirring speed is 400 r / min, and the preferred reaction time is 12 h. The preferred centrifugation speed is 8500 r / min, the preferred centrifugation time is 12 min, and the preferred drying temperature is 80°C. When removing the template, the preferred amount of hydrochloric acid is 2 mL, the preferred amount of anhydrous ethanol is 100 mL, the preferred stirring temperature is 60°C, the preferred stirring time is 12 h, and the preferred drying temperature is 80°C.

[0014] In step (2), the preferred amount of MSN nanoparticles is 200 mg, the preferred amount of DMF is 100 mL, the preferred amount of 3-aminopropyltriethoxysilane is 250 µL, the preferred stirring time is 24 h, the preferred centrifugation speed is 9000 r / min, and the preferred centrifugation time is 15 min.

[0015] In step (3), the preferred ratio of MSN-NH2, carvacrol and chloroform is 1:1:30, and the preferred number of volatilization cycles is 3.

[0016] In step (4), the preferred dosage of CMC is 400 mg, the preferred cold water temperature is 4°C, the preferred dosage of EDC is 75 mg, the preferred stirring time is 30 min, the preferred dosage of NHS is 45 mg, and the preferred dosage of carvacrol@MSN-NH2 is 400 mg. The preferred stirring time of the reaction solution is 24 h, the preferred centrifugation speed is 8500 r / min, and the preferred centrifugation time is 12 min.

[0017] The carvacrol@MSN-NH2@CMC nanoparticles prepared in this invention have a significant preventive effect against soft rot disease in Chinese cabbage.

[0018] The advantages of this invention compared to the prior art are as follows:

[0019] Under conditions of pH=5 or pH=9, the cumulative release rate of carvacrol@MSN-NH2@CMC was significantly higher than that at pH=7. During the 144-hour release period, the cumulative release rate of carvacrol@MSN-NH2@CMC in the presence of cellulase was 91.28%±1.48%, which was significantly higher than that under enzyme-free conditions, indicating that the prepared carvacrol@MSN-NH2@CMC had good responsiveness to pH changes and cellulase release. The preventive effect of carvacrol@MSN-NH2@CMC on soft rot of Chinese cabbage on day 10 was 63.06%–82.78%, which was better than the preventive effect of carvacrol by 3.27%–6.42%. The curative effect of carvacrol@MSN-NH2@CMC on day 10 was 66.68%–86.48%, which was better than the curative effect of carvacrol by 10.69%–19.68%. This indicates that the prepared dual-response nanoparticles greatly improved the prevention and control effect of carvacrol on soft rot of Chinese cabbage.

[0020] It is evident that the carvacrol@MSN-NH2@CMC nanoparticles rapidly respond to pH changes and cellulase release in the lesion microenvironment, achieving precise delivery and intelligent release of carvacrol at the target site. This demonstrates significant preventative and therapeutic effects against soft rot in Chinese cabbage, solving the problems of low drug utilization and poor field persistence encountered in practical applications. Furthermore, the carvacrol@MSN-NH2@CMC preparation process involved in this invention is simple, mild, and the materials are safe, promising to provide a highly efficient new solution for the green control of soft rot in Chinese cabbage. Attached Figure Description

[0021] Figure 1 MSN, MSN-NH2, carvacrol, carvacrol@MSN-NH2, carvacrol@MSN-NH2@CMC infrared spectrum

[0022] Figure 2 Wide-angle X-ray diffraction pattern of MSN

[0023] Figure 3 MSN, MSN-NH2, carvacrol@MSN-NH2, carvacrol@MSN-NH2@CMC small angle diffraction pattern

[0024] Figure 4 Scanning electron micrographs of MSN and carvacrol@MSN-NH2@CMC

[0025] Figure 5Transmission electron microscopy images of MSN and carvacrol@MSN-NH2@CMC

[0026] Figure 6 Regression curve and fitting equation of carvacrol

[0027] Figure 7 pH response behavior measurement results

[0028] Figure 8 Cellulase response behavior assay results Detailed Implementation

[0029] Examples 1, 2, and 3 describe the preparation of carvacrol@MSN-NH2@CMC nanoparticles; Examples 4, 5, and 6 describe the preventive effects of carvacrol@MSN-NH2@CMC nanoparticles; Examples 7, 8, and 9 describe the therapeutic effects of carvacrol@MSN-NH2@CMC nanoparticles.

[0030] Example 1

[0031] (1) Weigh 0.9 g of hexadecyltrimethylammonium bromide and add it to 30 mL of ultrapure water. Stir the solution with a magnetic stirrer until it is clear and transparent. Take a new beaker and add 40 mL of anhydrous ethanol and 40 mL of ultrapure water to it to make a mixed solution. Then add 1.5 mL of ammonia water to the mixed solution and add the above hexadecyltrimethylammonium bromide aqueous solution to it. Stir at 27°C for 20 min. Add 3 mL of tetraethyl orthosilicate dropwise to the above mixed solution in three portions, with an interval of 1 hour between each addition. After stirring magnetically at 300 r / min for 10 h at 27°C, centrifuge the reaction solution at 7000 r / min for 10 min. After pouring out the supernatant, wash the precipitate with ultrapure water and anhydrous ethanol 4 times each, and dry it at 70°C to obtain MSN nanoparticles containing hexadecyltrimethylammonium bromide.

[0032] To obtain porous MSN nanoparticles, MSN nanoparticles containing hexadecyltrimethylammonium bromide were dispersed in a mixed solution of 1 mL hydrochloric acid and 80 mL anhydrous ethanol. The hexadecyltrimethylammonium bromide was removed by magnetic stirring at 50°C for 10 h. The resulting product was centrifuged at 7000 r / min for 10 min. After discarding the supernatant, the precipitate was washed four times each with ultrapure water and anhydrous ethanol, and then dried at 70°C to obtain the MSN nanoparticles.

[0033] (2) Disperse 100 mg of MSN nanoparticles obtained in the previous step in 60 mL of DMF, then add 200 µL of 3-aminopropyltriethoxysilane and stir magnetically at room temperature for 18 h. Centrifuge the reaction solution at 8000 r / min for 13 min, pour off the supernatant, and wash the precipitate 4 times each with DMF, anhydrous ethanol and ultrapure water. After freeze-drying, MSN-NH2 nanoparticles are obtained.

[0034] (3) Mix MSN-NH2, carvacrol and chloroform in a ratio of 1:1:20, evaporate the chloroform in a fume hood, and repeat twice to obtain carvacrol@MSN-NH2 nanoparticles.

[0035] (4) Disperse 300 mg CMC in cold water at about 2°C and stir until dissolved. Then add 50 mg EDC and stir at room temperature for 20 min. Then add 30 mg NHS and 300 mg carvacrol@MSN-NH2. After stirring the reaction solution at room temperature for 18 h, centrifuge the reaction solution at 7000 r / min for 10 min. After pouring out the supernatant, wash the precipitate with ultrapure water 4 times and vacuum dry to obtain carvacrol@MSN-NH2@CMC nanoparticles.

[0036] Example 2

[0037] (1) Weigh 1.30 g of hexadecyltrimethylammonium bromide and add it to 50 mL of ultrapure water. Stir the solution with a magnetic stirrer until it is clear and transparent. Take a new beaker and add 60 mL of anhydrous ethanol and 60 mL of ultrapure water to it to make a mixed solution. Then add 1.9 mL of ammonia water to the mixed solution and add the above hexadecyltrimethylammonium bromide aqueous solution to it. Stir at 33°C for 40 min. Add 7 mL of tetraethyl orthosilicate to the above mixed solution slowly in three portions, with an interval of 1 hour between each addition. After stirring magnetically at 500 r / min at 33°C for 14 h, centrifuge the reaction solution at 10000 r / min for 14 min. After pouring out the supernatant, wash the precipitate with ultrapure water and anhydrous ethanol 4 times each, and dry it at 90°C to obtain MSN nanoparticles containing hexadecyltrimethylammonium bromide.

[0038] MSN nanoparticles containing hexadecyltrimethylammonium bromide were dispersed in a mixture of 3 mL hydrochloric acid and 120 mL anhydrous ethanol. The hexadecyltrimethylammonium bromide was removed by magnetic stirring at 70°C for 14 h. The resulting product was centrifuged at 10000 r / min for 14 min. After discarding the supernatant, the precipitate was washed four times each with ultrapure water and anhydrous ethanol, and then dried at 90°C to obtain MSN nanoparticles.

[0039] (2) Disperse 300 mg of MSN nanoparticles obtained in the previous step in 140 mL of DMF, then add 300 µL of 3-aminopropyltriethoxysilane and stir magnetically at room temperature for 28 h. Centrifuge the reaction solution at 10000 r / min for 17 min, pour off the supernatant, and wash the precipitate 4 times each with DMF, anhydrous ethanol and ultrapure water. After freeze-drying, MSN-NH2 nanoparticles are obtained.

[0040] (3) Mix MSN-NH2, carvacrol and chloroform in a ratio of 1:1:40, evaporate the chloroform in a fume hood, and repeat 4 times to obtain carvacrol@MSN-NH2 nanoparticles.

[0041] (4) Disperse 500 mg CMC in cold water at about 6°C and stir until dissolved. Then add 100 mg EDC and stir at room temperature for 40 min. Then add 60 mg NHS and 500 mg carvacrol@MSN-NH2. After stirring the reaction solution at room temperature for 28 h, centrifuge the reaction solution at 10000 r / min for 14 min. After pouring out the supernatant, wash the precipitate with ultrapure water 4 times and vacuum dry to obtain carvacrol@MSN-NH2@CMC nanoparticles.

[0042] Example 3

[0043] (1) Weigh 1.10 g of hexadecyltrimethylammonium bromide and add it to 40 mL of ultrapure water. Stir the solution with a magnetic stirrer until it is clear and transparent. Take a new beaker and add 50 mL of anhydrous ethanol and 50 mL of ultrapure water to it to make a mixed solution. Then add 1.7 mL of ammonia water to the mixed solution and add the above hexadecyltrimethylammonium bromide aqueous solution to it. Stir at 30°C for 30 min. Add 5 mL of tetraethyl orthosilicate dropwise to the above mixed solution in three portions, with an interval of 1 hour between each addition. After stirring magnetically at 400 r / min at 30°C for 12 h, centrifuge the reaction solution at 8500 r / min for 12 min. After pouring out the supernatant, wash the precipitate with ultrapure water and anhydrous ethanol 4 times each, and dry it at 80°C to obtain MSN nanoparticles containing hexadecyltrimethylammonium bromide.

[0044] To obtain porous MSN nanoparticles, MSN nanoparticles containing hexadecyltrimethylammonium bromide were dispersed in a mixed solution of 2 mL hydrochloric acid and 100 mL anhydrous ethanol. The hexadecyltrimethylammonium bromide was removed by magnetic stirring at 60°C for 12 h. The resulting product was centrifuged at 8500 r / min for 12 min. After discarding the supernatant, the precipitate was washed four times each with ultrapure water and anhydrous ethanol, and then dried at 80°C to obtain the MSN nanoparticles.

[0045] (2) Disperse 200 mg of MSN nanoparticles obtained in the previous step in 100 mL of DMF, then add 250 µL of 3-aminopropyltriethoxysilane and stir magnetically at room temperature for 24 h. Centrifuge the reaction solution at 9000 r / min for 15 min, pour off the supernatant, and wash the precipitate 4 times each with DMF, anhydrous ethanol and ultrapure water. After freeze-drying, MSN-NH2 nanoparticles are obtained.

[0046] (3) Mix MSN-NH2, carvacrol and chloroform in a ratio of 1:1:30, evaporate the chloroform in a fume hood, and repeat 3 times to obtain carvacrol@MSN-NH2.

[0047] (4) Disperse 400 mg CMC in cold water at about 4°C and stir until dissolved. Then add 75 mg EDC and stir at room temperature for 30 min. Then add 45 mg NHS and 400 mg carvacrol@MSN-NH2. After stirring the reaction solution at room temperature for 24 h, centrifuge the reaction solution at 8500 r / min for 12 min. After pouring out the supernatant, wash the precipitate with ultrapure water 4 times and vacuum dry to obtain carvacrol@MSN-NH2@CMC nanoparticles.

[0048] The characterization results of carvacrol@MSN-NH2@CMC nanoparticles are shown below. Infrared spectroscopy characterization results show that MSN exhibits high activity at 811 cm⁻¹. -1 The absorption peak at [value] is the symmetric stretching vibration peak of Si-O-Si; after loading carvacrol, the antisymmetric stretching vibration peak of the methyl group of carvacrol appears at 2960 cm⁻¹ in the carvacrol@MSN-NH2 spectrum, indicating that carvacrol has been successfully loaded into the MSN-NH2 support; after CMC coating, a new absorption peak appears at 1655 cm⁻¹ in the carvacrol@MSN-NH2@CMC spectrum, which is not present in the MSN-NH2 and carvacrol spectra, directly proving that the carboxyl group on the CMC molecular chain has covalently bonded to the amino group on the MSN-NH2 surface, and that CMC has been successfully coated on the MSN-NH2 surface. Figure 1 Wide-angle XRD patterns showed that MSN exhibited only a broad, dome-shaped peak near 23.5°, without any sharp diffraction peaks, confirming it as amorphous MSN. Figure 2 Small-angle XRD showed that the broad diffraction peak at approximately 1.4° was a characteristic peak of the mesoporous structure of MSN, indicating that the prepared MSN had ordered mesoporous channels. Small-angle XRD also showed that the diffraction peak intensity of MSN was the highest, and the peak intensity decreased sequentially after amino modification, loading with carvacrol, and coating with CMC. Figure 3Scanning electron microscopy revealed that MSN nanoparticles had smooth surfaces and uniform sizes. Compared to MSN, carvacrol@MSN-NH2@CMC nanoparticles exhibited a blurred coating layer on their surface, displaying a rough texture similar to ground glass, indicating that CMC was successfully coated onto the surface of MSN-NH2. Figure 4 Transmission electron microscopy revealed that MSNs were spherical in shape, uniform in size, with clear boundaries, and good dispersion. Compared to MSNs with clear boundaries, carvacrol@MSN-NH2@CMC exhibited a darker color in the center and a lighter color at the edges, and was also coated with a lighter-colored substance, indicating that CMC had successfully formed a coating on the surface of MSN-NH2. Figure 5 ).Depend on Figure 6 It can be seen that carvacrol exhibits a good linear relationship within the concentration range of 7.8125 µg / mL to 500 µg / mL (R0). 2 =0.99997), the fitted equation is y=15.301x+23.296, and the drug loading of carvacrol in the nanoparticles was calculated to be 23.90%±0.44% through the fitted equation. pH response results show that the amide bond connecting MSN-NH2 and CMC can be hydrolyzed under acidic or alkaline conditions, causing CMC to detach from the carrier surface, thus realizing the pH response of carvacrol@MSN-NH2@CMC. Figure 7 Cellulase response results showed that the cumulative release rate of carvacrol@MSN-NH2@CMC in the presence of cellulase was 91.28% ± 1.48%, significantly higher than that under enzyme-free conditions, confirming that carvacrol@MSN-NH2@CMC is responsive to cellulase stimulation. Figure 8 ).

[0049] Example 4

[0050] Carvacrol@MSN-NH2@CMC nanoparticles were prepared according to the method in Example 3. One-month-old Chinese cabbage seedlings were prepared, and wounds (without penetration) were made on the surface of the leaf petioles using 10 bundled sterile needles. 50 mL of carvacrol@MSN-NH2@CMC and carvacrol test solution at a concentration of 25 μg / mL were sprayed onto the Chinese cabbage seedlings, while a 3% DMSO aqueous solution was sprayed as a negative control. After 1 day and 5 days, 100 µL of Pcc bacterial suspension (OD200) was added... 600 =0.6) was injected into the wound. The treated cabbage seedlings were placed in a light incubator and cultured for 10 days at 28℃ with a photoperiod of 14h / 10h. The preventive effect of cabbage was calculated according to formula (1). The results showed (Table 1) that on the 10th day, the preventive effect of carvacrol@MSN-NH2@CMC test solution was 63.06%, which was greater than the preventive effect of carvacrol (3.27%).

[0051] Prevention effect = (Disease index of prevention control group - Disease index of prevention experimental group) / Disease index of prevention control group × 100% Formula (1)

[0052] Table 1. Preventive effect of 25 μg / mL carvacrol@MSN-NH2@CMC nanoparticles on soft rot pathogens in Chinese cabbage.

[0053] Group preventive effect <![CDATA[Carvacrol@MSN-NH2@CMC]]> 63.06% carvacrol 3.27%

[0054] Example 5

[0055] Carvacrol@MSN-NH2@CMC nanoparticles were prepared according to the method in Example 3. One-month-old Chinese cabbage seedlings were prepared, and wounds (without penetration) were made on the surface of the leaf petioles using 10 bundled sterile needles. 50 mL of carvacrol@MSN-NH2@CMC and carvacrol test solution at a concentration of 50 μg / mL were sprayed onto the Chinese cabbage seedlings, while a 3% DMSO aqueous solution was sprayed as a negative control. After 1 day and 5 days, 100 µL of Pcc bacterial suspension (OD200) was added... 600 =0.6) was injected into the wound. The treated cabbage seedlings were placed in a light incubator and cultured for 10 days at 28℃ with a photoperiod of 14h / 10h. The preventive effect of cabbage was calculated according to formula (1). The results showed (Table 2) that on the 10th day, the preventive effect of carvacrol@MSN-NH2@CMC test solution was 72.85%, which was greater than the preventive effect of carvacrol (5.42%).

[0056] Table 2. Preventive effect of 50 μg / mL carvacrol@MSN-NH2@CMC nanoparticles on soft rot pathogens in Chinese cabbage.

[0057] Group preventive effect <![CDATA[Carvacrol@MSN-NH2@CMC]]> 72.85% carvacrol 5.42%

[0058] Example 6

[0059] Carvacrol@MSN-NH2@CMC nanoparticles were prepared according to the method in Example 3. One-month-old Chinese cabbage seedlings were prepared, and wounds (without penetration) were made on the surface of the leaf petioles using 10 bundled sterile needles. 50 mL of 100 μg / mL carvacrol@MSN-NH2@CMC and carvacrol test solution were sprayed onto the Chinese cabbage seedlings, while a 3% DMSO aqueous solution was sprayed as a negative control. After 1 day and 5 days, 100 µL of Pcc bacterial suspension (OD200) was added. 600=0.6) was injected into the wound. The treated cabbage seedlings were placed in a light incubator and cultured for 10 days at 28℃ with a photoperiod of 14h / 10h. The preventive effect of cabbage was calculated according to formula (1). The results showed (Table 3) that on the 10th day, the preventive effect of carvacrol@MSN-NH2@CMC test solution was 82.78%, which was greater than the preventive effect of carvacrol (6.42%).

[0060] Table 3. Preventive effect of 100 μg / mL carvacrol@MSN-NH2@CMC nanoparticles on soft rot pathogens in Chinese cabbage.

[0061] Group preventive effect <![CDATA[Carvacrol@MSN-NH2@CMC]]> 82.78% carvacrol 6.42%

[0062] Example 7

[0063] Carvacrol@MSN-NH2@CMC nanoparticles were prepared according to the method in Example 3. One-month-old Chinese cabbage seedlings were prepared, and wounds (without penetration) were made on the surface of the leaf petioles using 10 bundled sterile needles. At days 0 and 5, 100 µL of Pcc bacterial suspension (OD200) was added. 600 =0.6) was injected into the wound. One day later, 50 mL of carvacrol@MSN-NH2@CMC and carvacrol test solution with a concentration of 25 μg / mL were sprayed onto the cabbage seedlings, and 3% DMSO aqueous solution was sprayed as a negative control. The treated cabbage seedlings were placed in a light incubator and cultured at 28℃ with a photoperiod of 14h / 10h for 10 days. The therapeutic effect of the cabbage was calculated according to formula (2). The results showed (Table 4) that on the 10th day, the therapeutic effect of carvacrol@MSN-NH2@CMC was 66.68%, which was greater than the therapeutic effect of carvacrol (10.69%).

[0064] Treatment effect = (Disease index of treatment control group - Disease index of treatment experimental group) / Disease index of treatment control group × 100% Formula (2)

[0065] Table 4. Therapeutic effects of 25 μg / mL carvacrol@MSN-NH2@CMC nanoparticles on soft rot pathogens of Chinese cabbage.

[0066] Group Treatment effect <![CDATA[Carvacrol@MSN-NH2@CMC]]> 66.68% carvacrol 10.69%

[0067] Example 8

[0068] Carvacrol@MSN-NH2@CMC nanoparticles were prepared according to the method in Example 3. One-month-old Chinese cabbage seedlings were prepared, and wounds (without penetration) were made on the surface of the leaf petioles using 10 bundled sterile needles. At days 0 and 5, 100 µL of Pcc bacterial suspension (OD200) was added. 600=0.6) was injected into the wound. One day later, 50 mL of carvacrol@MSN-NH2@CMC and carvacrol test solution with a concentration of 50 μg / mL were sprayed onto the cabbage seedlings, and 3% DMSO aqueous solution was sprayed as a negative control. The treated cabbage seedlings were placed in a light incubator and cultured at 28℃ with a photoperiod of 14h / 10h for 10 days. The therapeutic effect of the cabbage was calculated according to formula (2). The results showed (Table 5) that on the 10th day, the therapeutic effect of carvacrol@MSN-NH2@CMC was 75.84%, which was greater than the therapeutic effect of carvacrol by 14.10%.

[0069] Table 5. Therapeutic effects of 50 μg / mL carvacrol@MSN-NH2@CMC nanoparticles on soft rot pathogens of Chinese cabbage.

[0070] Group Treatment effect <![CDATA[Carvacrol@MSN-NH2@CMC]]> 75.84% carvacrol 14.10%

[0071] Example 9

[0072] Carvacrol@MSN-NH2@CMC nanoparticles were prepared according to the method in Example 3. One-month-old Chinese cabbage seedlings were prepared, and wounds (without penetration) were made on the surface of the leaf petioles using 10 bundled sterile needles. At days 0 and 5, 100 µL of Pcc bacterial suspension (OD200) was added. 600 =0.6) was injected into the wound. One day later, 50 mL of carvacrol@MSN-NH2@CMC and carvacrol test solution with a concentration of 100 μg / mL were sprayed onto the cabbage seedlings, and 3% DMSO aqueous solution was sprayed as a negative control. The treated cabbage seedlings were placed in a light incubator and cultured at 28℃ with a photoperiod of 14h / 10h for 10 days. The therapeutic effect of the cabbage was calculated according to formula (2). The results showed (Table 6) that on the 10th day, the therapeutic effect of carvacrol@MSN-NH2@CMC was 86.48%, which was greater than the therapeutic effect of carvacrol (19.68%).

[0073] Table 6. Therapeutic effects of 100 μg / mL carvacrol@MSN-NH2@CMC nanoparticles on soft rot pathogens of Chinese cabbage.

[0074] Group Treatment effect <![CDATA[Carvacrol@MSN-NH2@CMC]]> 86.48% carvacrol 19.68%

Claims

1. A method for preparing a pH / cellulase dual-responsive carvacol nanoparticle, characterized by, Includes the following steps: (1) Weigh 0.9-1.30 g of hexadecyltrimethylammonium bromide and add it to 30-50 mL of ultrapure water. Stir the solution with a magnetic stirrer until it is clear and transparent. Take a beaker and add 40-60 mL of anhydrous ethanol and 40-60 mL of ultrapure water to it to make a mixed solution. Then add 1.5-1.9 mL of ammonia water to the mixed solution and add the above-mentioned hexadecyltrimethylammonium bromide aqueous solution to it. Stir at 27°C-33°C for 20-40 min; slowly add 3-7 mL of tetraethyl orthosilicate to the above mixed solution in three portions, with an interval of 1 hour between each addition; after stirring magnetically at 300-500 r / min at 27°C-33°C for 10-14 h, centrifuge the reaction solution at 7000-10000 r / min for 10-14 min, pour off the supernatant, wash the precipitate 4 times each with ultrapure water and anhydrous ethanol, and dry at 70-90°C to obtain mesoporous silica (MSN) nanoparticles containing hexadecyltrimethylammonium bromide; MSN nanoparticles containing hexadecyltrimethylammonium bromide were dispersed in a mixed solution of 1-3 mL hydrochloric acid and 80-120 mL anhydrous ethanol. The solution was magnetically stirred at 50-70°C for 10-14 h to remove hexadecyltrimethylammonium bromide. The resulting product was centrifuged at 7000-10000 r / min for 10-14 min. After discarding the supernatant, the precipitate was washed four times each with ultrapure water and anhydrous ethanol, and then dried at 70-90°C to obtain MSN nanoparticles. (2) Disperse 100-300 mg of MSN nanoparticles obtained in the previous step in 60-140 mL of DMF, then add 200-300 µL of 3-aminopropyltriethoxysilane and stir magnetically at room temperature for 18-28 h. Centrifuge the reaction solution at 8000-10000 r / min for 13-17 min, pour off the supernatant, and wash the precipitate 4 times each with DMF, anhydrous ethanol and ultrapure water. After freeze-drying, MSN-NH2 nanoparticles are obtained. (3) Mix MSN-NH2, carvacrol and chloroform in a ratio of 1:1:20-40, evaporate the chloroform in a fume hood, and repeat 2-4 times to obtain carvacrol@MSN-NH2 nanoparticles; (4) Disperse 300-500 mg of carboxymethyl cellulose (CMC) in cold water at 2-6°C and stir until dissolved. Then add 50-100 mg of EDC and stir at room temperature for 20-40 min. Then add 30-60 mg of NHS and 300-500 mg of carvacrol@MSN-NH2. After stirring the reaction solution at room temperature for 18-28 h, centrifuge the reaction solution at 7000-10000 r / min for 10-14 min. After pouring out the supernatant, wash the precipitate with ultrapure water 4 times and vacuum dry to obtain carvacrol@MSN-NH2@CMC nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step (1), the amount of hexadecyltrimethylammonium bromide is 1.1 g, which is added to 40 mL of ultrapure water; when preparing the mixed solution, the amount of anhydrous ethanol is 50 mL and the amount of ultrapure water is 50 mL; the amount of ammonia added to the mixed solution is 1.7 mL, the reaction temperature is 30°C, and the stirring time is 30 min; the amount of tetraethyl orthosilicate is 5 mL, the stirring speed is 400 r / min, and the reaction time is 12 h; the centrifugation speed is 8500 r / min, the centrifugation time is 12 min, and the drying temperature is 80°C; when removing the template, the amount of hydrochloric acid is 2 mL, the amount of anhydrous ethanol is 100 mL, the stirring temperature is 60°C, the stirring time is 12 h, and the drying temperature is 80°C.

3. The preparation method according to claim 1, characterized in that, In step (2), the MSN nanoparticles are 200 mg, DMF is 100 mL, 3-aminopropyltriethoxysilane is 250 µL, the stirring time is 24 h, the centrifugation speed is 9000 r / min, and the centrifugation time is 15 min.

4. The preparation method according to claim 1, characterized in that, In step (3), the ratio of MSN-NH2, carvacrol and chloroform is 1:1:30, and the chloroform volatilization is repeated 3 times.

5. The preparation method according to claim 1, characterized in that, In step (4), CMC is 400 mg, cold water temperature is 4°C, EDC is 75 mg, stirring time is 30 min, NHS is 45 mg, carvacrol@MSN-NH2 is 400 mg; the reaction solution is stirred for 24 h, centrifuged at 8500 r / min, and centrifuged for 12 min.

6. Carvacrol@MSN-NH2@CMC nanoparticles prepared by any one of the methods described in claims 1-5.

7. The application of carvacrol@MSN-NH2@CMC nanoparticles as described in claim 6 in the prevention and control of soft rot in Chinese cabbage.

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