Preparation method and application of chlorophyll photosensitizer

Abstract

This invention provides a method for preparing and applying a chlorophyll-based photosensitizer, belonging to the field of novel photosensitizer preparation technology. The method includes dissolving sodium chlorophyll in water to a concentration of 0.5–8 mM, adding zinc oxide to the sodium chlorophyll solution, stirring in the dark, and then sequentially centrifuging, washing, and drying to obtain a sodium chlorophyll-zinc oxide complex. The combination of semiconductor materials and sodium chlorophyll significantly enhances the photostability of sodium chlorophyll and exhibits good antibacterial activity against plant pathogens.

Description

Technical Field

[0001] This invention belongs to the field of novel photosensitizer preparation technology, specifically a method for preparing and applying a chlorophyll-based photosensitizer. Background Technology

[0002] Currently, in practical applications in farmland, the control of plant bacterial infections mainly relies on copper-based fungicides. However, with the emergence of copper-resistant strains, even repeated spraying of copper-based fungicides is insufficient to effectively control plant bacterial infections. Furthermore, the long-term use of copper-based fungicides has led to a series of problems, including increasingly serious bacterial resistance, which severely threatens soil and water safety. To address these issues, there is an urgent need to develop practical, safe, and efficient antibacterial methods.

[0003] Photodynamic antimicrobial therapy (PACT) has attracted increasing attention due to its high efficiency and lack of drug resistance. In the field, PACT can utilize solar radiation, eliminating the need for artificial light to activate photosensitizers. The high irradiance and broad emission spectrum of solar radiation can activate different types of photosensitizers, regardless of whether they are excited by visible or ultraviolet radiation. Unlike localized applications in clinical settings, using PACT to control plant bacterial infections will require large-scale application of photosensitizers over a considerable land area. Therefore, the price and environmental safety of photosensitizers are important considerations. From the perspective of photodynamic therapy, our aim is to develop low-cost, application-friendly, highly efficient, and safe photosensitizers for the control of plant bacteria.

[0004] Sodium chlorophyll (Chl) is a photosensitizer derivative derived from chlorophyll, which is low in cost and easy to prepare. It has been approved as a food coloring agent in the EU and the US, exhibiting good safety. However, its poor antibacterial effect and moderate photostability limit its antibacterial applications in agriculture. Zinc oxide (ZnO) is a photocatalyst with good photostability, absorption under sunlight, low cost, and easy large-scale preparation, showing broad application prospects in agriculture. ZnO is a plant nutrient element and has been successfully used as a small-volume fertilizer for many crops, as well as an insecticide for pest control. Another characteristic of ZnO is its ability to penetrate plants and move within their tissues through the cuticle, epidermis, stomata, hydra, stigma, root tip, root bark, lateral cortex, root junction, bark, and several other plant surfaces. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the embodiments of the present invention is to provide a method for preparing chlorophyll-based photosensitizers and their applications.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A method for preparing a chlorophyll-based photosensitizer specifically includes the following steps:

[0008] Sodium chlorophyll was dissolved in water to a concentration of 0.5–8 mM. Zinc oxide was added to the sodium chlorophyll solution, and the mixture was stirred in the dark. Then, the mixture was centrifuged, washed, and dried in sequence to obtain the chlorophyll-based photosensitizer.

[0009] As a further embodiment of the present invention, the sodium chlorophyll salt is sodium magnesium chlorophyll or sodium zinc chlorophyll.

[0010] As a further embodiment of the present invention, the sodium chlorophyll salt is sodium zinc chlorophyll.

[0011] As a further embodiment of the present invention, the concentration of the sodium chlorophyll salt is 2 mM.

[0012] As a further embodiment of the present invention, the stirring time in the dark is 24 hours.

[0013] As a further embodiment of the present invention, the zinc oxide is prepared by the following method: zinc nitrate is dissolved in water, the pH is adjusted to 9, and the mixture is stirred and then centrifuged;

[0014] The precipitate was washed with distilled water and resuspended until the pH of the supernatant was neutral.

[0015] The precipitate was dispersed in secondary water and then boiled, centrifuged, dried, ground, and calcined to obtain zinc oxide.

[0016] This invention also provides an application of chlorophyll-based photosensitizers in the preparation of products for preventing bacterial infections in plants.

[0017] Compared with the prior art, the beneficial effect of the present invention is that the photosensitizer complex provided by the present invention, after the semiconductor material is combined with sodium chlorophyll salt, can significantly improve the photostability of sodium chlorophyll salt.

[0018] The chlorophyll-based photosensitizer prepared by this invention has good antibacterial activity against plant pathogens and can effectively inhibit the growth of a variety of plant bacteria.

[0019] The chlorophyll-based photosensitizer prepared by this invention has a good preventive effect against plant bacterial infections under simulated sunlight irradiation conditions, and can be used to control plant bacterial diseases such as tomato spot disease and citrus canker. Attached Figure Description

[0020] Figure 1Figure (a) shows the preparation process of sodium chlorophyll salt-zinc oxide (Chl-ZnO); Figure (b) shows the bactericidal and inhibitory effects of ZnO loaded with 4.73% MgChl on *Escherichia coli*; Figure (c) shows the bactericidal and inhibitory effects of ZnO loaded with 23.43% MgChl on *Escherichia coli*; and Figure (d) shows the bactericidal and inhibitory effects of ZnO loaded with 57.75% MgChl. Figure (e) shows the bactericidal and inhibitory effects of ZnO loaded with MgChl on Escherichia coli. Figure (f) shows the bactericidal and inhibitory effects of ZnO loaded with 21.75% ZnChl on Escherichia coli. Figure (g) shows the bactericidal and inhibitory effects of ZnO loaded with 58.95% ZnChl on Escherichia coli.

[0021] Figure 2 (a) shows the XRD pattern of the ZnO and Chl-ZnO photosensitizer composite material; (b) shows the UV-Vis spectrum of the ZnO and chlorophyll-ZnO photosensitizer composite material; (c) shows the scanning electron microscope (SEM) image of ZnO; (d) shows the scanning electron microscope (SEM) image of MgChl-ZnO; (e) shows the scanning electron microscope (SEM) image of ZnChl-ZnO; (f) shows the high-magnification SEM image of ZnO; (g) shows the high-magnification SEM image of MgChl-ZnO; and (h) shows the high-magnification SEM image of ZnChl-ZnO.

[0022] Figure 3 Figure (a) shows the inhibitory effect of different components (ZnChl, ZnO, or ZnChl-ZnO) on *X. tomato spot pathogen (XJ1602)*. Figure (b) shows the inhibitory effect of different components (ZnChl, ZnO, or ZnChl-ZnO) on *X. citrus canker pathogen (Xcc29-1)*. Figure (c) shows the inhibitory effect of different components (ZnChl, ZnO, or ZnChl-ZnO) on *X. citrus canker pathogen (BT0505)*. Figure (d) shows the quantitative analysis of the antibacterial effects of ZnChl, ZnO, or ZnChl-ZnO on the three plant pathogens.

[0023] Figure 4Figure (a) shows a comparison of total reactive oxygen species (ROS) generated by ZnO, ZnChl, and ZnChl-ZnO; Figure (b) shows a comparison of •OH generated by ZnO and ZnChl-ZnO; Figure (c) shows the morphology of XJ1602 in the untreated control group; Figure (d) shows the morphology of XJ1602 in the ZnChl-treated group; Figure (e) shows the morphology of XJ1602 in the ZnO-treated group; and Figure (f) shows the morphology of XJ1602 in the ZnChl-ZnO-treated group.

[0024] Figure 5 A comparison of the photostability of ZnChl-ZnO photosensitizer composite materials.

[0025] Figure 6 (a) shows the spots on the leaf surface of tomato plants 21 days after spraying with XJ1602 (blank control), quinoline copper treatment group (1.42mM), and ZnChl-ZnO (8μg / mL). (b) shows the quantitative statistics of the number of tomato spot diseases. (c) shows the canker lesions formed on the leaf surface 30 days after spraying with Xcc29-1 (blank control), quinoline copper treatment group (1.42mM), and ZnChl-ZnO (8μg / mL). (d) shows the quantitative statistics of the number of citrus canker lesions. Detailed Implementation

[0026] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0027] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0028] Two photosensitizers, chlorophyll (Chl) and zinc chlorophyll (ZnChl), were dissolved in 1 mL of deionized water at final concentrations of 0.5 mM, 2 mM, and 8 mM, respectively. Then, 10 mg of ZnO semiconductor material was added. The mixture was stirred in the dark at room temperature for 24 h to prepare ZnO with high, medium, and low photosensitizer loadings. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the precipitate was washed with deionized water repeatedly until the supernatant was colorless. The precipitate was then dried at 60°C for 24 h. The three loadings of the two photosensitizers (PS1, PS2) on ZnO were calculated by subtracting the amount of photosensitizer in all washing solutions from the initial photosensitizer dose. PS1 is sodium magnesium chlorophyll (MgChl), and PS2 is sodium zinc chlorophyll (ZnChl).

[0029] The preparation steps of ZnO are as follows: 0.05 mol of Zn(NO3)2·6H2O is completely dissolved in 20 mL of deionized water and stirred at 400 rpm for 1 h at room temperature. Ammonium hydroxide is then slowly added dropwise to the Zn(NO3)2·6H2O aqueous solution, gradually producing a large amount of white precipitate until no more white precipitate forms, at which point the addition of ammonium hydroxide is stopped. The pH of the solution is then adjusted to 9.0. After stirring at 400 rpm for 3 h at room temperature, the solution is centrifuged at 10,000 rpm for 10 min. After centrifugation, the supernatant is discarded, and the precipitate is washed with distilled water and resuspended until the pH of the supernatant reaches approximately 7.0. The precipitate is dispersed in a certain amount of deionized water and then boiled in a heater for 5 min. After boiling, the solution is centrifuged at 10,000 rpm, and the resulting precipitate is dried at 80 °C for 3 h. After drying, the ZnO is ground evenly in a mortar and pestle and then calcined at 500 °C for 2 h to obtain ZnO.

[0030] like Figure 1 As shown, the ordinate represents the logarithmic value of bacterial count. A higher ordinate value indicates a weaker antibacterial effect. The ZnChl-ZnO prepared from a 2mM ZnChl solution contained a ZnChl mass fraction of 21.75 wt%, the ZnChl-ZnO prepared from a 0.5mM ZnChl solution contained a ZnChl mass fraction of 4.62 wt%, and the ZnChl-ZnO prepared from an 8mM ZnChl solution contained a ZnChl mass fraction of 58.95 wt%. The MgChl-ZnO prepared from the MgChl solution of M had a MgChl mass fraction of 4.73 wt%, the MgChl-ZnO prepared from the 2 mM MgChl solution had a MgChl mass fraction of 23.43 wt%, and the MgChl-ZnO prepared from the 8 mM MgChl solution had a MgChl mass fraction of 57.75 wt%. Among them, the ZnChl-ZnO loaded with a moderate concentration of sodium zinc chlorophyll (ZnChl) showed good antibacterial effects against plant bacteria.

[0031] like Figure 2 As shown, Figure 2(a) shows the XRD pattern of the synthesized ZnO. It can be seen that the diffraction peaks corresponding to the (100), (002), and (101) crystal planes of ZnO appear at 31.8°, 34.4°, and 36.2°, respectively. These results are consistent with the data from the hexagonal magnesium aluminum oxide (JCPDS-36-1451) standard card. Furthermore, other crystal planes of ZnO, including (102), (110), (103), (201), (112), and (200), also conform to the data from the standard card. These results indicate that the synthesized ZnO possesses a hexagonal wurtzite structure. Further analysis shows that the crystal planes observed in the XRD patterns of the MgChl-ZnO and ZnChl-ZnO photosensitizer composites are consistent with the crystal planes of ZnO. This indicates that the incorporation of the photosensitizer did not affect the crystal plane structure of ZnO. Furthermore, the UV-Vis absorption changes of ZnO, MgChl-ZnO, and ZnChl-ZnO were determined by liquid-phase UV-Vis spectroscopy. All three samples exhibited a distinct characteristic absorption peak of ZnO at approximately 357 nm. The MgChl-ZnO and ZnChl-ZnO photosensitizer composites also showed characteristic absorption peaks of chlorophyll-based photosensitizers at 410 nm and 630 nm, respectively, while these peaks were not observed in the pure ZnO sample. Figure 2 (b) As shown in the figure. This result further confirms that both photosensitizers were successfully loaded onto ZnO.

[0032] The morphology of ZnO, MgChl-ZnO, and ZnChl-ZnO was observed using SEM. ZnO had a relatively smooth surface, was rod-shaped with sharp ends, and resembled a grain of rice. Figure 2 As shown in Figure (c), after loading two different photosensitizers onto the ZnO surface, it can be observed that the surfaces of MgChl-ZnO and ZnChl-ZnO become rougher compared to pure ZnO. Figure 2 (d) Figure and Figure 2 As shown in Figure (e), this result indicates that the photosensitizer has been successfully loaded onto the ZnO surface. It can be observed that the synthesized ZnO has a size on the micrometer scale, such as... Figure 2 (f) As shown in the figure. Furthermore, it is noteworthy that the loading of MgChl and ZnChl did not affect the size of ZnO, as... Figure 2 (g) diagram and Figure 2 (h) As shown in the figure.

[0033] like Figure 3As shown, a comparative experiment was conducted on the plant bacterial infection prevention effect of the novel sodium chlorophyll photosensitizer. According to the results, ZnChl-ZnO loaded with 21.75 wt% ZnChl exhibited good antibacterial efficacy against *Escherichia coli* luminescentis. The final ZnChl-ZnO prepared from a 2 mM ZnChl solution contained 21.75 wt% ZnChl. To further evaluate its bactericidal effect against plant pathogens, the antibacterial properties of ZnChl-ZnO (21.75 wt% ZnChl) against three plant pathogen strains were studied. The final concentration of ZnChl-ZnO was 8 μg / mL, and the concentrations of *Tomato Spot Fever* (XJ1602), *Citrus Canker Fever* (Xcc29-1), or *Tomato Canker* (BT0505) were 10 μg / mL. 6 CFU / mL. The bacterial suspension was mixed with ZnChl-ZnO and incubated for 10 minutes, followed by irradiation under simulated sunlight (100 mW / cm²) for 5 minutes. 2 Light dose 30 J / cm 2 The results showed that using only ZnChl or ZnO for 5 minutes of irradiation (30 J / cm²) was effective. 2 After irradiation, only a portion of the bacteria were killed, with an antibacterial rate of less than 50%. In contrast, under irradiation conditions, ZnChl-ZnO showed antibacterial efficacy exceeding 99% against XJ1602 and Xcc29-1, and nearly 4 log (99.99%) against BT0505. The colony count results clearly demonstrate the differences in antibacterial efficacy between ZnChl, ZnO, and ZnChl-ZnO against these three plant pathogens. Figure 3 (d) presents the quantitative analysis of the antimicrobial results. These findings indicate that ZnChl-ZnO exhibits excellent antimicrobial activity against plant pathogens, particularly against strain BT0505.

[0034] To investigate the efficacy of ZnChl-ZnO in preventing bacterial infections in plants, a preventative infection experiment was conducted on tomato plants under simulated sunlight conditions (light dose of 60 J / cm²). Figure 6 As shown, an 8 μg / mL concentration of ZnChl-ZnO was used in the experiment, with quinoline copper reagent (approximately 1.42 mM) as a positive control. In a 21-day preventative infection experiment, the preventative effect of ZnChl-ZnO (8 μg / mL) against XJ1602 infection was evaluated by monitoring the area of ​​infected leaves daily. In the blank control group, tomato leaves showed dark brown to black circular lesions accompanied by leaf yellowing, and the infection was almost complete. Compared with the blank control group, the quinoline copper treatment group showed a certain preventative effect against XJ1602 infection, with a reduced degree of infection on tomato leaves, such as... Figure 6As shown in (a), the ZnChl-ZnO photosensitizer combined treatment group showed a more effective preventive effect, with the lowest infection level on tomato leaves. Statistical analysis of the preventive efficacy of each group revealed that the quinoline copper preparation had a prevention rate of 65.85% against XJ1602, while ZnChl-ZnO showed a superior preventive effect, with a disease inhibition rate as high as 98.20%. Figure 6 (b) shows that these results indicate that ZnChl-ZnO can effectively prevent tomato plants from being infected with tomato spot disease.

[0035] Furthermore, under simulated sunlight conditions (light dose of 60 J / cm²), 2 The study also investigated its preventive effect on citrus plants. ZnChl-ZnO was applied at the same concentration (8 μg / mL), with quinoline copper as a positive control (approximately 1.42 mM). In a 30-day preventive infection experiment, the efficacy of ZnChl-ZnO (8 μg / mL) in preventing Xccc29-1 infection was evaluated by monitoring the number of lesions appearing on citrus leaves daily. Results showed that oily, yellowish lesions with raised, crater-like patterns appeared on leaves infected with citrus canker. Compared to the blank control group, the number of lesions in the quinoline copper treatment group was significantly reduced. The ZnChl-ZnO treatment group had the fewest lesions (…). Figure 6 c). Statistical analysis of the number of lesions in each group showed that the control group had 5.22 lesions / cm². 2 The quinoline copper treatment group had 1.81 lesions / cm². 2 The ZnChl-ZnO treatment group had only 0.75 lesions / cm². 2 ,like Figure 6 As shown in (d), the bar charts represent the standard error of the mean. These results indicate that the ZnChl-ZnO treatment group has a good preventive effect against Xccc29-1 infection, and its efficacy is superior to that of quinoline copper reagent.

[0036] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0037] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a chlorophyll-based photosensitizer, characterized in that, Specifically, the following steps are included: Sodium chlorophyll was dissolved in water to a concentration of 0.5–8 mM. Zinc oxide was added to the sodium chlorophyll solution, and the mixture was stirred in the dark. Then, the mixture was centrifuged, washed, and dried in sequence to obtain a sodium chlorophyll and zinc oxide complex.

2. The method for preparing a chlorophyll-based photosensitizer according to claim 1, characterized in that, The sodium chlorophyll salt is sodium magnesium chlorophyll or sodium zinc chlorophyll.

3. The method for preparing a chlorophyll-based photosensitizer according to claim 2, characterized in that, The sodium chlorophyll salt is sodium zinc chlorophyll.

4. The method for preparing a chlorophyll-based photosensitizer according to claim 1, characterized in that, The concentration of the sodium chlorophyll salt is 2 mM.

5. The method for preparing a chlorophyll-based photosensitizer according to claim 1, characterized in that, Stirring in the dark for 24 hours.

6. The method for preparing a chlorophyll-based photosensitizer according to claim 1, characterized in that, The zinc oxide is prepared by the following method: zinc nitrate is dissolved in water, the pH is adjusted to 9, and the mixture is stirred and then centrifuged. The precipitate was washed with distilled water and resuspended until the pH of the supernatant was neutral. The precipitate was dispersed in secondary water and then boiled, centrifuged, dried, ground, and calcined to obtain zinc oxide.

7. The use of a chlorophyll-based photosensitizer prepared by any one of claims 1-6 in the preparation of products for preventing bacterial infections in plants.

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