Cinnamic acid and chlorogenic acid co-modified carbon quantum dots, a preparation method thereof and application thereof in prevention and treatment of leek gray mold

Abstract

The application discloses cinnamic acid and chlorogenic acid co-modified carbon quantum dots, which comprise chitosan carbon quantum dots, cinnamic acid and chlorogenic acid, wherein the cinnamic acid and the chlorogenic acid are covalently connected with the amino group on the surface of the chitosan carbon quantum dots through amide bonds formed by carboxyl groups. The application further discloses a preparation method of the cinnamic acid and chlorogenic acid co-modified carbon quantum dots, which comprises the following steps: mixing cinnamic acid, chlorogenic acid, ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-succinimidyl and an ethanol aqueous solution uniformly in an inert gas atmosphere to obtain solution A; mixing the solution A with a chitosan carbon quantum dot aqueous dispersion in an inert gas atmosphere, performing reaction, dialysis and freeze-drying to obtain the cinnamic acid and chlorogenic acid co-modified carbon quantum dots. The application further discloses application of the cinnamic acid and chlorogenic acid co-modified carbon quantum dots in inhibition or killing of Plasmopara halstedii. The application has a good killing effect on Plasmopara halstedii.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and in particular to a carbon quantum dot co-modified with cinnamic acid and chlorogenic acid, its preparation method, and its application in the control of gray mold in leeks. Background Technology

[0002] Gray mold of chives, also known as white spot disease or white leaf blight, is a disease caused by Botrytis cinerea. It mainly affects the leaves and is a major disease of chives grown in greenhouses. It often causes the leaves to wither and die, and the chives to rot and become moldy. In severe cases, it can reduce yield by more than 30%.

[0003] Currently, gray mold in chives is mainly controlled by applying pesticides such as boscalid, pyrimethanil, iprodione, procymidone, carbendazim, and metalaxyl. However, the use of these pesticides can easily lead to problems such as excessive pesticide residues and toxic chives. Therefore, there is a need to provide more environmentally friendly and efficient pesticides. Summary of the Invention

[0004] Based on the technical problems existing in the background technology, this invention proposes a method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid, and its application in the prevention and control of gray mold in leeks. This invention co-modifies the surface of chitosan carbon quantum dots with cinnamic acid and chlorogenic acid, which can improve the stability of cinnamic acid and chlorogenic acid, improve the adhesion of the drug to the leaf surface, and the combination of the three can significantly improve the killing effect on Botrytis cinerea and the control effect on gray mold in leeks; and it will not cause problems such as excessive pesticide residues or toxic leeks.

[0005] This invention proposes a carbon quantum dot co-modified with cinnamic acid and chlorogenic acid, comprising: chitosan carbon quantum dots, cinnamic acid and chlorogenic acid, wherein the cinnamic acid and chlorogenic acid are covalently linked by amide bonds formed by the carboxyl groups and the amino groups on the surface of the chitosan carbon quantum dots.

[0006] Preferably, the particle size of the carbon quantum dots co-modified with cinnamic acid and chlorogenic acid is <10 nm.

[0007] This invention proposes a method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid, comprising the following steps: In an inert gas atmosphere, cinnamic acid, chlorogenic acid, ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-succinylhydroxyimide, and an aqueous ethanol solution are mixed to obtain solution A; In an inert gas atmosphere, solution A is mixed with a chitosan carbon quantum dot aqueous dispersion, reacted, dialyzed, and freeze-dried to obtain carbon quantum dots co-modified with cinnamic acid and chlorogenic acid.

[0008] This invention uses ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-succinylhydroxyimide to allow the carboxyl groups in cinnamic acid and chlorogenic acid to react with the amino groups on the surface of chitosan carbon quantum dots to form amide bonds, thereby jointly modifying the surface of chitosan carbon quantum dots with cinnamic acid and chlorogenic acid.

[0009] The inert gas mentioned above can be nitrogen, etc.

[0010] Preferably, the mixture is stirred at room temperature for 5-6 hours to obtain solution A.

[0011] Preferably, the ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 10-20%.

[0012] Preferably, the weight ratio of chitosan carbon quantum dots to cinnamic acid is 8-10:1.

[0013] Preferably, the weight ratio of cinnamic acid, chlorogenic acid, ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-succinylhydroxyimide is 1:0.2-0.3:1.8-2.2:0.7-0.8.

[0014] Preferably, the reaction is carried out at room temperature for 20-24 hours.

[0015] Preferably, dialysis is performed at room temperature for 40-48 hours, with the dialysis solution changed every 12 hours.

[0016] Preferably, the dialysate is water.

[0017] Preferably, the molecular weight cutoff of the dialysis bag is 1000 Da.

[0018] Preferably, in the preparation process of chitosan carbon quantum dots, a chitosan solution is subjected to a hydrothermal reaction, centrifuged, the supernatant is filtered, the filtrate is dialyzed, and then freeze-dried to obtain chitosan carbon quantum dots.

[0019] Preferably, in the preparation of chitosan carbon quantum dots, the solvent for the chitosan solution is an aqueous solution of glacial acetic acid with a mass fraction of 1-2 wt%.

[0020] Preferably, in the preparation of chitosan carbon quantum dots, the concentration of chitosan in the chitosan solution is 0.04-0.06 g / ml.

[0021] Preferably, in the preparation of chitosan carbon quantum dots, the hydrothermal reaction temperature is 175-185℃ and the time is 7-9h.

[0022] Preferably, during the preparation of chitosan carbon quantum dots, centrifugation is performed at 5000-6000 rpm for 10-15 min.

[0023] Preferably, during the preparation of chitosan carbon quantum dots, a filter membrane with a pore size of 0.22 μm is used for filtration.

[0024] Preferably, during the preparation of chitosan carbon quantum dots, dialysis is performed at room temperature for 60-72 hours, with the dialysis solution being changed every 12 hours.

[0025] Preferably, water is used as the dialysis solution in the preparation of chitosan carbon quantum dots.

[0026] Preferably, during the preparation of chitosan carbon quantum dots, the molecular weight cutoff of the dialysis bag is 1000 Da.

[0027] This invention also proposes the application of the above-mentioned carbon quantum dots co-modified with cinnamic acid and chlorogenic acid in inhibiting or killing Botrytis cinerea.

[0028] Preferably, its application in the prevention and control of gray mold in leeks.

[0029] The carbon quantum dots co-modified with cinnamic acid and chlorogenic acid can be stored away from light and at low temperatures; when using them, they can be dispersed and diluted with water and then sprayed onto the surface of chive leaves.

[0030] This invention utilizes chitosan to prepare chitosan carbon quantum dots, whose surface has multiple active amino, hydroxyl, and carboxyl groups. Cinnamic acid and chlorogenic acid are co-modified by forming amide bonds between the carboxyl groups and the amino groups on the surface of the chitosan carbon quantum dots. This improves the stability of cinnamic acid and chlorogenic acid, and the synergistic effect of the three compounds significantly enhances the control effect against gray mold in chives. The modification with chlorogenic acid and cinnamic acid introduces catechol groups into the carbon quantum dots, and the cinnamic acid introduces an appropriate amount of hydrophobic groups, improving the adhesion of the carbon quantum dots to the hydrophobic surface of chive leaves. This process avoids drug detachment, which would reduce the bactericidal and preventative effects. Furthermore, the acidic environment created when *Botrytis cinerea* infects and multiplies causes the remaining amino groups on the carbon quantum dots to become positively charged, allowing them to electrostatically adsorb onto the surface of *Botrytis cinerea*. This enriches cinnamic acid and chlorogenic acid on the bacterial surface, increasing their local concentration and significantly enhancing the killing effect. In addition, the nano-sized carbon quantum dots themselves, along with cinnamic acid and chlorogenic acid, can disrupt the integrity of the bacterial cell membrane, further improving the bactericidal effect. The synergistic effect of these three factors can significantly enhance the killing effect on *Botrytis cinerea*. Detailed Implementation

[0031] The technical solution of the present invention will now be described in detail through specific embodiments.

[0032] Example 1

[0033] A method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid includes the following steps: A chitosan solution with a concentration of 0.04 g / ml (solvent being a 1 wt% aqueous solution of glacial acetic acid) was subjected to a hydrothermal reaction at 185 °C for 7 h. After centrifugation at 6000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm pore size filter membrane. The filtrate was then dialyzed at room temperature for 72 h, with the dialysate being water, and the molecular weight cutoff of the dialysis bag was 1000 Da. The solution was then lyophilized to obtain chitosan carbon quantum dots for later use. 0.1 g cinnamic acid, 0.03 g chlorogenic acid, 0.22 g ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.08 g N-succinyl hydroxyimide were added to 100 ml of 20% ethanol aqueous solution and stirred at room temperature for 6 h under a nitrogen atmosphere to obtain solution A. In a nitrogen atmosphere, solution A was added to an aqueous dispersion of chitosan carbon quantum dots with a solid content of 1 wt% and mixed well so that the weight ratio of chitosan carbon quantum dots to cinnamic acid was 10:1. The mixture was reacted at room temperature for 24 h and then dialyzed at room temperature for 40 h, with the dialysate being changed every 12 h. The dialysate was water and the molecular weight cutoff of the dialysis bag was 1000 Da. The mixture was then lyophilized to obtain carbon quantum dots co-modified with cinnamic acid and chlorogenic acid.

[0034] Example 2

[0035] A method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid includes the following steps: A chitosan solution with a concentration of 0.06 g / ml (solvent being a 2 wt% aqueous solution of glacial acetic acid) was subjected to a hydrothermal reaction at 175 °C for 9 h. After centrifugation at 5000 rpm for 15 min, the supernatant was filtered through a 0.22 μm pore size filter membrane. The filtrate was dialyzed at room temperature for 60 h, with the dialysate being water every 12 h. The molecular weight cutoff of the dialysis bag was 1000 Da. The solution was then lyophilized to obtain chitosan carbon quantum dots for later use. 0.1g cinnamic acid, 0.02g chlorogenic acid, 0.18g ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.07g N-succinylhydroxyimide were added to 100ml of 10% ethanol aqueous solution and stirred at room temperature for 5h under a nitrogen atmosphere to obtain solution A. In a nitrogen atmosphere, solution A was added to an aqueous dispersion of chitosan carbon quantum dots with a solid content of 1 wt% and mixed well, so that the weight ratio of chitosan carbon quantum dots to cinnamic acid was 8:1. The reaction was carried out at room temperature for 20 h, followed by dialyzing at room temperature for 48 h, with the dialysate being changed every 12 h. The dialysate was water, and the molecular weight cutoff of the dialysis bag was 1000 Da. The mixture was then lyophilized to obtain carbon quantum dots co-modified with cinnamic acid and chlorogenic acid.

[0036] Example 3

[0037] A method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid includes the following steps: A chitosan solution with a concentration of 0.05 g / ml (solvent being a 1 wt% aqueous solution of glacial acetic acid) was subjected to a hydrothermal reaction at 180℃ for 8 h. After centrifugation at 5500 rpm for 12 min, the supernatant was filtered through a 0.22 μm pore size filter membrane. The filtrate was dialyzed at room temperature for 72 h, with the dialysate being water, and the molecular weight cutoff of the dialysis bag was 1000 Da. The solution was then lyophilized to obtain chitosan carbon quantum dots for later use. 0.1 g cinnamic acid, 0.025 g chlorogenic acid, 0.2 g ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.075 g N-succinyl hydroxyimide were added to 100 ml of 15% ethanol aqueous solution and stirred at room temperature for 5.5 h under a nitrogen atmosphere to obtain solution A. In a nitrogen atmosphere, solution A was added to an aqueous dispersion of chitosan carbon quantum dots with a solid content of 1 wt% and mixed well, so that the weight ratio of chitosan carbon quantum dots to cinnamic acid was 9:1. The reaction was carried out at room temperature for 24 h, followed by dialyzing at room temperature for 48 h, with the dialysate being changed every 12 h. The dialysate was water, and the molecular weight cutoff of the dialysis bag was 1000 Da. The mixture was then lyophilized to obtain carbon quantum dots co-modified with cinnamic acid and chlorogenic acid.

[0038] Comparative Example 1 Chitosan carbon quantum dots prepared according to the method in Example 3.

[0039] Comparative Example 2 Without adding chlorogenic acid, and otherwise the same as in Example 3, cinnamic acid-modified carbon quantum dots were prepared.

[0040] Comparative Example 3 Without adding cinnamic acid, and otherwise the same as in Example 3, chlorogenic acid-modified carbon quantum dots were prepared.

[0041] The products of Example 3 and Comparative Examples 1-3 were used to test their effects on inhibiting Botrytis cinerea infection of leek plants. The specific steps are as follows: The products of Example 3 and Comparative Examples 1-3 were diluted with sterile water to prepare diluents of different concentrations (0.5, 1, and 2 mg / ml) for later use. Using chives at the same growth stage and with good growth as experimental materials, wounds were created on the surface of chive leaves by needle pricking. Then, 20 μl of a 20 μg / ml suspension of *Botrytis cinerea* was sprayed onto the leaves. After culturing for 24 h, different concentrations of diluted products from Examples 3 and Comparative Examples 1-3 were sprayed as treatment groups. Chives sprayed with only sterile water served as the control group, and chives not infected with *Botrytis cinerea* and not treated in any way served as the healthy control group. All chives in each group were grown under the same conditions for 7 days. The number of leaves with lesions in each group was counted, and the inhibition rate of each group was calculated. The results are shown in Table 1.

[0042] Gray mold incidence (%) = (Number of infected leaves / Total number of leaves) 100%.

[0043] Inhibition rate (%) = (Incidence rate in control group - Incidence rate in treatment group) / Incidence rate in control group 100%.

[0044] Table 1. Results of antibacterial rate

[0045] As can be seen from Table 1, the carbon quantum dots co-modified with cinnamic acid and chlorogenic acid described in this invention have a good control effect on gray mold of leeks caused by Botrytis cinerea infection.

[0046] The products of Example 3 and Comparative Examples 1-3 were diluted with sterile water to the same concentration, and then sprayed onto the surface of detached leek leaves. After the leaves dried, sterile water was sprayed onto the leaf surface for 40 minutes. The drug residue rate on the leaf surface was detected, and the results are shown in Table 2.

[0047] Table 2 Residual Rate Results

[0048] As can be seen from Table 2, the present invention can improve the adhesion of the drug to the surface of leek leaves, thereby improving the efficacy.

[0049] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A carbon quantum dot co-modified with cinnamic acid and chlorogenic acid, characterized in that, include: Chitosan carbon quantum dots, cinnamic acid, and chlorogenic acid are used, wherein cinnamic acid and chlorogenic acid are covalently linked by amide bonds formed by carboxyl groups on the surface of chitosan carbon quantum dots.

2. The carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to claim 1, characterized in that, The particle size of carbon quantum dots co-modified with cinnamic acid and chlorogenic acid is <10 nm.

3. A method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid as described in claim 1 or 2, characterized in that, The process includes the following steps: In an inert gas atmosphere, cinnamic acid, chlorogenic acid, ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-succinylhydroxyimide, and an aqueous ethanol solution are mixed to obtain solution A; In an inert gas atmosphere, solution A is mixed with a chitosan carbon quantum dot aqueous dispersion, reacted, dialyzed, and lyophilized to obtain carbon quantum dots co-modified with cinnamic acid and chlorogenic acid.

4. The method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to claim 3, characterized in that, Mix at room temperature for 5-6 hours to obtain solution A; preferably, the ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 10-20%.

5. The method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to claim 3 or 4, characterized in that, The weight ratio of chitosan carbon quantum dots to cinnamic acid is 8-10:1; preferably, the weight ratio of cinnamic acid, chlorogenic acid, ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-succinyl hydroxyimide is 1:0.2-0.3:1.8-2.2:0.7-0.

8.

6. The method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to any one of claims 3-5, characterized in that, The reaction is carried out at room temperature for 20-24 hours; preferably, dialysis is carried out at room temperature for 40-48 hours, with the dialysis solution changed every 12 hours; preferably, the dialysis solution is water; preferably, the molecular weight cutoff of the dialysis bag is 1000 Da.

7. The method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to any one of claims 3-6, characterized in that, In the preparation of chitosan carbon quantum dots, a chitosan solution is subjected to a hydrothermal reaction, centrifuged, the supernatant is filtered, the filtrate is dialyzed, and then freeze-dried to obtain chitosan carbon quantum dots.

8. The method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to claim 7, characterized in that, In the preparation of chitosan carbon quantum dots, the solvent of the chitosan solution is an aqueous solution of glacial acetic acid with a mass fraction of 1-2 wt%; preferably, in the preparation of chitosan carbon quantum dots, the concentration of chitosan in the chitosan solution is 0.04-0.06 g / ml; preferably, in the preparation of chitosan carbon quantum dots, the hydrothermal reaction temperature is 175-185℃ and the time is 7-9 h.

9. The method for preparing carbon quantum dots co-modified with cinnamic acid and chlorogenic acid according to claim 7, characterized in that, In the preparation of chitosan carbon quantum dots, centrifugation is performed at 5000-6000 rpm for 10-15 min; preferably, filtration is performed using a filter membrane with a pore size of 0.22 μm; preferably, dialysis is performed at room temperature for 60-72 h, with the dialysis solution changed every 12 h; preferably, the dialysis solution is water; preferably, the molecular weight cutoff of the dialysis bag is 1000 Da.

10. The application of carbon quantum dots co-modified with cinnamic acid and chlorogenic acid as described in claim 1 or 2 in inhibiting or killing Botrytis cinerea; preferably, the application in the prevention and control of gray mold in leeks.

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