A method for preparing nanosilver particles using rose extract, product and application

Abstract

The present invention discloses a method for preparing nanosilver particles using rose extract, a product, and an application thereof, belonging to the technical field of biosynthetic nanomaterials. The method comprises the following steps: (1) washing, drying, and crushing rose petals, mixing them with deionized water, stirring them evenly, letting them stand, and filtering them thoroughly to obtain a rose extract; (2) mixing the rose extract obtained in step (1) with an AgNO3 solution, reacting them in the dark to obtain a mixed solution, and centrifuging, washing, and vacuum freeze-drying the mixed solution to obtain the nanosilver particles. The nanosilver particles obtained by the method of the present invention have a small average particle size, uniform size, and stable structure, and can effectively inhibit the activity of Pseudomonas aeruginosa and prevent and control plant diseases caused by it. The nanosilver particles obtained by the present invention are used as agricultural fungicides in the prevention and control of bayberry wilt disease, and have a good prevention and control effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosynthetic nanomaterials, and particularly relates to a method for preparing nanosilver particles using rose extract, as well as a product and application thereof. Background Art

[0002] Nanosilver possesses unique antimicrobial properties, along with high surface activity, surface energy, and catalytic properties. It is currently widely used in food packaging, environmental technologies, agricultural antimicrobial agents, optical materials, and semiconductor materials. As an antimicrobial agent, nanosilver rarely develops or develops drug resistance, making it a long-lasting antimicrobial agent. The application of nanosilver materials can help address the potential health hazards and environmental pollution associated with chemical pesticides.

[0003] Currently, nanosilver is primarily prepared through physical, chemical, and microbiological methods. Physical methods primarily include sputtering, mechanical ball milling, and laser ablation, while chemical methods primarily include liquid-phase chemical reduction, microemulsion reduction, electrochemical reduction, and hydrothermal methods. While simple and feasible, physical and chemical methods require large amounts of energy, are prone to environmental pollution, and produce toxic byproducts during the synthesis process. Microbiological methods, while environmentally friendly, have long production cycles and demanding equipment requirements. Plant extract reduction is an environmentally friendly and sustainable method for preparing nanosilver. It offers advantages such as environmental friendliness, low cost, low energy consumption, and mild reaction conditions. It can address the large-scale production of nanosilver and has become a research hotspot in the field of nanosilver synthesis.

[0004] In the prior art, Chinese patent application publication number CN107671305A discloses a method for rapidly preparing a nanosilver antibacterial agent using Ligustrum lucidum fruit extract. The specific steps are: mixing an appropriate amount of AgNO3 solution with the Ligustrum lucidum fruit extract, and then subjecting the mixture to ultrasonic treatment and light irradiation to obtain the nanosilver antibacterial agent. The nanosilver antibacterial agent has antibacterial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas syringae, and others. Chinese patent application publication number CN106513707A discloses a process for preparing a green synthesis of a nanosilver antibacterial agent using blueberry leaf extract. The method involves uniformly stirring the blueberry leaf extract with an AgNO3 solution, followed by heating and refluxing to obtain the nanosilver antibacterial agent. The nanosilver antibacterial agent has good antibacterial activity against four aquatic pathogens: Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus, and Aeromonas punctata.

[0005] Different nano-silver antibacterial agents have different inhibitory effects on pathogens. In the field of agricultural technology, the phenomenon of infection by the pathogenic fungus of Pseudomonas aeruginosa is very common in various fruits such as bayberry, banana, strawberry, and various cash crops such as rubber trees, and the like. This pathogen easily causes huge losses to my country's agricultural economy. However, traditional chemical pesticides easily cause problems such as pathogen resistance and environmental pollution, and can cause potential harm to the human body. Nano-silver antibacterial agents prepared by plant extract reduction methods disclosed in the prior art do not clearly show an inhibitory effect on Pseudomonas aeruginosa. Therefore, it is even more necessary to develop a green and novel method for preventing and treating plant diseases caused by Pseudomonas aeruginosa. Summary of the Invention

[0006] The present invention provides a method for preparing nano-silver particles using rose extract. The method has low production cost, simple equipment, and is environmentally friendly. The prepared nano-silver particles have uniform size, good stability and dispersibility, and have a good inhibitory effect on plant pathogenic fungi.

[0007] The specific technical solutions adopted are as follows:

[0008] A method for preparing nanosilver particles using rose extract comprises the following steps:

[0009] (1) Washing, drying, and crushing rose petals, mixing them with deionized water, stirring them evenly, letting them stand, and filtering them thoroughly to obtain a rose extract;

[0010] (2) The rose extract obtained in step (1) is mixed with the AgNO3 solution, and the mixture is reacted in the dark to obtain a mixed solution, and the mixed solution is centrifuged, washed, and vacuum freeze-dried to obtain the silver nanoparticles (AgNPs).

[0011] Rose (Rosa multiflora Thunb), a plant of the genus Rosa in the Rosaceae family, boasts a long flowering period, beautiful flowers, and diverse colors. It has a long history of cultivation and is native to China. The Compendium of Materia Medica states that the rose's flowers, roots, and leaves can all be used as medicine. They are warm in nature, sweet in taste, and have been shown to treat qi stagnation and blood stasis, promote blood circulation and regulate menstruation, and reduce swelling and detoxify.

[0012] Rose petals contain terpenoids, polysaccharides, anthocyanins, and phenolic acid compounds, which play an important role in the reduction of silver nitrate and can serve as reducing agents and stabilizers for the synthesis of nanosilver particles. Using rose extract to prepare nanosilver particles facilitates better extraction of nanosilver. Furthermore, the present method directly utilizes a water-extracted mixture of rose petals as the rose extract, simplifying the process, minimizing energy consumption, and preventing the destruction of chemical substances contained in the petals.

[0013] Preferably, the dried rose petals are crushed using a juicer, and the power of the juicer is 200W-300W.

[0014] Preferably, in step (1), the mass ratio of the crushed rose petals to deionized water is 1:10-20; and the standing time is 20-40 minutes.

[0015] In step (2), the rose extract and the AgNO3 solution are mixed and placed in a shaker at a temperature of 55-65°C and a rotation speed of 180-200 rpm to react in the dark. The reaction is terminated when the color of the mixed solution turns gray-green. Setting the shaker temperature or rotation speed too low will result in insufficient reaction and poor synthesis effect, which will ultimately affect the particle size, stability and uniformity of the nanosilver particles, thereby affecting their antibacterial effect. Setting the temperature or rotation speed too high will easily cause the synthetic product to deteriorate and cause unnecessary energy consumption.

[0016] Preferably, the concentration of the AgNO3 solution is 3-4 mM, and the volume ratio of the rose extract to the AgNO3 solution is 10-25: 100. Too high a content of the rose active ingredient will affect the formation of nanosilver particles, while too low a content will reduce the efficiency of the final product synthesis.

[0017] Preferably, the centrifugation conditions are 8000-14000 rpm, 8-15 min. If the centrifugation rate is too low or the time is too short, the supernatant will not be clear, resulting in a low content of nanosilver particles and a high content of impurities, which will affect the antibacterial effect of the product.

[0018] The present invention also provides nano-silver particles prepared by the method for preparing nano-silver particles using rose extract. Preferably, the particle size of the nano-silver particles is 90-110 nm.

[0019] Experiments have shown that the nanosilver particles are stable, nearly spherical, and uniform in size. They exhibit a strong inhibitory effect against the fungus Pseudomonas axyridis, significantly improving their effectiveness in controlling plant diseases caused by the pathogen, and have broad application prospects.

[0020] The present invention also provides the use of the nano silver particles in inhibiting Pestalotiopsis versicolor.

[0021] The present invention also provides the use of the nano-silver particles as an agricultural fungicide in preventing and treating plant diseases caused by Pseudomonas axylochaete, wherein the plant diseases include bayberry wilt.

[0022] The specific application method is to dissolve the nanosilver particles in water to prepare an AgNPs solution, and then evenly spray the AgNPs solution on the plants to be controlled. The AgNPs solution concentration is preferably 100-500 μg / ml. Within this preferred range, the agricultural fungicide has a strong inhibitory effect on Pseudomonas axylodiscus. Further increasing the antibacterial concentration does not significantly improve the fungicidal effect. Therefore, considering both fungicidal effect and cost, the AgNPs solution concentration is preferably 100-500 μg / ml.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The nanosilver particles obtained by the method of the present invention have a small average particle size, uniform size, and stable structure, and can effectively inhibit the activity of Pseudomonas axylodiscus, prevent and control plant diseases caused by it, and have a strong bactericidal effect;

[0025] (2) The preparation process of the present invention is simple, low-cost, consumes relatively little energy, and does not produce toxic by-products. No additional reducing agent or catalyst is required, which is very environmentally friendly and safe. It belongs to a green synthesis process and is suitable for large-scale production.

[0026] (3) The nano silver particles obtained by the present invention are used as agricultural fungicides to prevent and treat bayberry wilt disease, which has a good prevention and control effect. The agricultural fungicide derived from this plant not only does not bring potential harm to the human body, but also does not pollute the environment. The application method is also simple and easy, and it has promotion prospects and broad applications in the field of agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram showing the color change of the solution when AgNPs were synthesized using rose extract in Example 1.

[0028] Figure 2 This is the UV-visible spectrum of the AgNPs synthesized in Example 1.

[0029] Figure 3 This is the Fourier transform infrared spectrum of AgNPs synthesized in Example 1.

[0030] Figure 4 is the X-ray diffraction pattern of AgNPs synthesized in Example 1.

[0031] Figure 5 This is a transmission electron microscopy image of AgNPs synthesized in Example 1.

[0032] Figure 6 This is a scanning electron micrograph of the AgNPs synthesized in Example 1.

[0033] Figure 7 This is the EDS image of AgNPs synthesized in Example 1.

[0034] Figure 8 The graph shows the inhibitory effect of different concentrations of AgNPs solution on the growth of the pathogenic fungus Polytrichomonas spp. in Example 1, where A is the control group, which is not treated with the AgNPs solution; B, C, and D are the experimental groups treated with 500, 250, and 100 μg / mL AgNPs solution, respectively; and E is a statistical graph of colony diameters.

[0035] Figure 9 Figures 2 and 3 show the different conditions of the detached bayberry leaves inoculated with Pseudomonas axylodiscus treated with different concentrations of AgNPs solution in Example 1. Figure A shows a healthy detached bayberry leaf; Figure B shows a diseased detached bayberry leaf; Figures C, D, and E show detached bayberry leaves sprayed with 100, 250, and 500 μg / mL AgNPs solution, respectively. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the examples and accompanying drawings. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The operating methods in the following examples where no specific conditions are specified are generally performed under conventional conditions or as recommended by the manufacturer.

[0037] The pathogen Pestalotiopsis versicolor used in the examples was isolated from diseased plants in Xianju County, Taizhou City, Zhejiang Province.

[0038] Example 1

[0039] (1) Freshly picked rose petals were washed, dried, and crushed with a 200W juicer. 5 g of the petals were weighed and mixed with 50 ml of deionized water. The mixture was stirred evenly with a glass rod and allowed to stand for 20 minutes. During this process, the mixture was stirred intermittently with a glass rod. The mixture was then double-filtered through cotton cloth and filter paper to ensure that all solid impurities were removed, thereby obtaining a rose extract.

[0040] (2) 20 ml of the rose extract obtained in step (1) was mixed with 200 ml of a 4 mM AgNO3 solution, and the mixture was placed in a shaker at a temperature of 65° C. and a rotation speed of 180 rpm. The mixture was allowed to react overnight in the dark. The reaction was terminated when the color of the mixture changed from pink to gray-green. After the mixture was taken out, it was centrifuged at a speed of 14000 rpm for 8 minutes, the supernatant was removed, and the precipitate was washed and freeze-dried in vacuum to obtain powdered silver nanoparticles (AgNPs).

[0041] Figure 1This is a color change diagram of the solution synthesized AgNPs using rose extract in this example. It was found that after the rose extract was added to the transparent AgNO3 solution and placed on a shaker for reaction, its color changed from the initial pink to gray-green.

[0042] The structural characteristics and physicochemical properties of the AgNPs were further evaluated using ultraviolet-visible absorption spectroscopy (UV-VIS), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectrometry (EDS), and X-ray diffraction (XRD).

[0043] UV-visible spectrum results ( Figure 2 ) shows that the absorption value of the AgNPs at 490nm is the strongest, indicating that the synthesized AgNPs are relatively stable; Fourier transform infrared spectrum ( Figure 3 ) indicates that the AgNPs have been successfully synthesized.

[0044] Figure 4 The X-ray diffraction pattern of AgNPs shows characteristic peaks of (100), (45.7), (22.5), and (22.2) at 2θ=38.11°, 44.30°, 64.44°, and 77.40°, respectively, which are consistent with the characteristic peaks of the AgO powder diffraction pattern, proving that stable AgNPs have been synthesized.

[0045] Figure 5 The transmission electron microscopy image of AgNPs shows that the AgNPs are nearly spherical particles; Figure 6 This is a scanning electron microscope image of AgNPs. It can be seen that the AgNPs are uniform in size and the particle size range is 90-110nm.

[0046] Figure 7 This is the EDS element distribution diagram of AgNPs. The results show that the AgNPs are mainly composed of Ag, C and O. The C element is inevitably introduced during the experiment.

[0047] Example 2

[0048] (1) Freshly picked rose petals were washed, dried, and crushed with a 250W juicer. 5 g of the petals were weighed and mixed with 75 ml of deionized water. The mixture was stirred evenly with a glass rod and allowed to stand for 20 minutes. During this process, the mixture was stirred intermittently with a glass rod. The mixture was then double-filtered through cotton cloth and filter paper to ensure that the solid impurities were completely removed, thereby obtaining a rose extract.

[0049] (2) 20 ml of the rose extract obtained in step (1) was mixed with 100 ml of a 3.5 mM AgNO3 solution, and the mixture was placed in a shaker at a temperature of 60°C and a rotation speed of 180 rpm. The mixture was allowed to react overnight in the dark. The reaction was terminated when the color of the mixture changed from pink to gray-green. After the mixture was taken out, it was centrifuged at a rotation speed of 10,000 rpm for 10 minutes, the supernatant was removed, and the precipitate was washed and vacuum freeze-dried to obtain powdered nanosilver particles.

[0050] Example 3

[0051] (1) Freshly picked rose petals were washed, dried, and crushed with a 300W juicer. 5 g of the petals were weighed and mixed with 100 ml of deionized water. The mixture was stirred evenly with a glass rod and allowed to stand for 20 minutes. During this process, the mixture was stirred intermittently with a glass rod. The mixture was then double-filtered through cotton cloth and filter paper to ensure that all solid impurities were removed, thereby obtaining a rose extract.

[0052] (2) 25 ml of the rose extract obtained in step (1) was mixed with 100 ml of a 3 mM AgNO3 solution, and the mixture was placed in a shaker at a temperature of 55° C. and a rotation speed of 180 rpm. The mixture was allowed to react overnight in the dark. The reaction was terminated when the color of the mixture changed from pink to gray-green. After the mixture was taken out, it was centrifuged at a rotation speed of 8000 rpm for 15 minutes, the supernatant was removed, and the precipitate was washed and freeze-dried in vacuum to obtain powdered nanosilver particles.

[0053] Sample analysis (I) Effect of AgNPs concentration on antibacterial effect

[0054] In order to evaluate the activity of AgNPs in inhibiting Polytrichomonas axylodiscus, the AgNPs powder prepared in Example 1 was prepared into solutions with concentrations of 100, 250, and 500 μg / mL with water, and the antibacterial properties of different AgNPs concentrations were evaluated by measuring the mycelial growth inhibition rate.

[0055] Depend on Figure 8As shown in the AE, 1 ml of AgNPs solution at concentrations of 1000, 2500, and 5000 μg / mL was added dropwise to 9 ml of PDA solution. The mixture was then poured into a petri dish and allowed to condense and air-dry. A piece of Pseudomonas axylodiscus was then inoculated in the center of the plate. After the plates were incubated under suitable conditions for 72 hours, the colony diameters on the plates containing AgNPs decreased with increasing silver nanoparticle concentration compared to the control group. After treatment with AgNPs solution at concentrations of 100, 250, and 500 μg / mL, the growth inhibition rates were 52.34%, 57.48%, and 68.22%, respectively. This indicates that the higher the concentration of the AgNPs solution, the better the antibacterial effect. However, when the concentration was further increased from 500 μg / mL, the inhibition rate did not increase much, and the antibacterial effect was not significantly improved.

[0056] Depend on Figure 8 It can be seen that the higher the concentration of the AgNPs solution, the better the antibacterial effect. The AgNPs synthesized using rose can significantly inhibit the growth of the pathogenic fungus Polytrichomonas syringae.

[0057] (2) Antibacterial effect of AgNPs

[0058] To further demonstrate the inhibitory effect of AgNPs on the growth of P. axyridis, the AgNPs powder synthesized in Example 1 was diluted with water to prepare AgNPs solutions at concentrations of 100, 250, and 500 μg / ml. 2 ml of each AgNP solution at different concentrations was sprayed onto detached bayberry leaves inoculated with 6 mm diameter P. axyridis discs. This treatment was repeated ten times. Diseased bayberry leaves inoculated with 6 mm diameter P. axyridis discs served as the positive control (denoted as CK diseased), while healthy bayberry leaves not inoculated with the discs served as the negative control (denoted as CK healthy).

[0059] Figure 9 A in the figure indicates that the healthy detached bayberry leaves are in normal condition. Figure 9 B in the figure shows that all the detached leaves of bayberry inoculated with the fungus Pseudomonas axylodiscus became diseased. Figure 9 As shown in Figure C, the number of diseased leaves and the diameter of lesions of detached bayberry leaves treated with 100 μg / mL AgNPs were significantly reduced. Figure 9 As can be seen from D in the figure, only two detached leaves of bayberry treated with 250 μg / mL AgNPs became diseased. Figure 9 As shown in Figure E, only one detached leaf of bayberry treated with 500 μg / mL AgNPs became diseased, indicating that AgNPs can inhibit the growth of P. axyridis and thus prevent and control the plant diseases caused by it.

[0060] The disease incidence and disease index were obtained by grading as follows: 0: no lesions; 1: brown necrotic spots; 2: obvious brown necrotic spots; 3: the diameter of the necrotic spots was larger than 6 mm in diameter and close to the leaf edge; 4: the necrotic spots reached the leaf edge. The results are shown in Table 1.

[0061] Table 1 Statistical results of the incidence and disease index of detached bayberry leaves under different treatments

[0062]

[0063] As shown in the table above, the incidence and disease index of detached bayberry leaves gradually decreased with increasing AgNPs concentration. After spraying 500 μg / mL AgNPs, the incidence dropped to 10%, and the disease index decreased from 87.50 to 2.50. This indicates that the silver nanoparticles can significantly inhibit the growth of P. axylodiscus, demonstrating their effectiveness in preventing and controlling bayberry wilt.

[0064] Application Examples

[0065] 5 g of the AgNPs powder obtained in Example 1 was weighed and dissolved in 10 L of water to prepare a 500 μg / ml AgNPs solution, which was evenly sprayed on bayberry crops in the greenhouse or field that may be infected by the pathogen Polytrichomoniasis. Observations showed that the crops sprayed with the AgNPs solution grew better than those not sprayed, indicating that the AgNPs solution can effectively prevent and control diseases in crops.

[0066] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of nano silver particles in inhibiting Polytrichomonas axylodiscus is characterized in that: The particle size of the nano silver particles is 90-110 nm; The nano silver particles are prepared using rose extract, and the preparation method comprises the following steps: (1) Washing, drying, and crushing rose petals, mixing them with deionized water, stirring them evenly, letting them stand, and filtering them thoroughly to obtain a rose extract; (2) mixing the rose extract obtained in step (1) with an AgNO3 solution, reacting in the dark to obtain a mixed solution, and centrifuging, washing, and vacuum freeze-drying the mixed solution to obtain the nanosilver particles; In step (2), the rose extract and the AgNO3 solution are mixed and reacted in a light-proof environment at a temperature of 55-65°C and a rotation speed of 180-200 rpm. The reaction is terminated when the color of the mixed solution turns gray-green; the concentration of the AgNO3 solution is 3-4 mM, and the volume ratio of the rose extract to the AgNO3 solution is 10-25:

100.

2. Application of nanosilver particles as agricultural fungicides in preventing and controlling plant diseases caused by Pseudomonas axylochaetes, characterized in that: The plant disease includes bayberry wilt; the particle size of the nanosilver particles is 90-110 nm; The nano silver particles are prepared using rose extract, and the preparation method comprises the following steps: (1) Washing, drying, and crushing rose petals, mixing them with deionized water, stirring them evenly, letting them stand, and filtering them thoroughly to obtain a rose extract; (2) mixing the rose extract obtained in step (1) with an AgNO3 solution, reacting in the dark to obtain a mixed solution, and centrifuging, washing, and vacuum freeze-drying the mixed solution to obtain the nanosilver particles; In step (2), the rose extract and the AgNO3 solution are mixed and reacted in a light-proof environment at a temperature of 55-65°C and a rotation speed of 180-200 rpm. The reaction is terminated when the color of the mixed solution turns gray-green; the concentration of the AgNO3 solution is 3-4 mM, and the volume ratio of the rose extract to the AgNO3 solution is 10-25:

100.

3. The use according to claim 1 or 2, characterized in that In step (1), the mass ratio of the crushed rose petals to deionized water is 1:10-20.

4. The use according to claim 1 or 2, characterized in that In step (1), the standing time is 20-40 minutes.

5. The use according to claim 1 or 2, characterized in that: The centrifugal conditions are 8000-14000 rpm, 8-15 min.

6. The use according to claim 2, characterized in that The specific application method is: dissolving the nano silver particles in water to prepare an AgNPs solution, and then evenly spraying the AgNPs solution on the plants to be controlled.

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