A method for preparing nano-silver particles by using coleus extract, and products and applications thereof

Abstract

The application discloses a method for preparing nano-silver particles by using coleus extraction liquid and products and application thereof, and belongs to the technical field of green biosynthesis of nanometer materials, and comprises the following steps: (1) after pretreatment, the coleus is crushed, mixed with deionized water, stirred uniformly, and then allowed to stand, and the coleus extraction liquid is obtained after filtration; (2) the coleus extraction liquid obtained in step (1) is mixed with an AgNO3 solution to obtain a mixed solution, and then the nano-silver particles are obtained after further centrifugation, washing and vacuum freeze-drying. The nano-silver particles obtained by the method have small average particle size, uniform size and stable structure, can effectively inhibit the activity of multiple plant pathogenic bacteria such as Pseudomonas syringae pv. tomato, can destroy the cell structure to make the endosolutes exosmose, has good bactericidal effect, and can be used for preventing and treating bacterial black spot disease of cruciferous vegetables.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of green biosynthesis of nanomaterials, and particularly relates to a method for preparing nanosilver particles using coleus extract, products and applications. BACKGROUND

[0002] Nanosilver has unique physical and chemical properties and strong antibacterial ability, and does not produce drug resistance, and is widely used in chemical industry, medicine, food packaging, environmental technology, sensing and other fields, and has great market development potential. Among them, the strong antibacterial performance of nanosilver is due to its large specific surface area, small particle size, and early research reports that Ag has potential antibacterial activity and can inhibit Staphylococcus aureus, Escherichia coli, etc.

[0003] At present, the traditional methods for synthesizing nanosilver mainly include physical method, chemical method, microbial method, etc. Although the microbial method is green and environmentally friendly, it has a long production cycle and high requirements for equipment. The physical method and the chemical method are simple and feasible, but they require a large amount of energy during the synthesis process, are easy to cause environmental pollution, and are easy to produce toxic by-products. Therefore, the above-mentioned synthesis methods are greatly limited in large-scale application.

[0004] In order to solve the problem of large-scale production of nanosilver, the biosynthesis method of green synthesis of nanometallic materials using plant extracts has become a research hotspot at present. The main principle of synthesizing nanometallic materials is that the phenolic compounds, terpenoids, alkaloids and coenzymes contained in the plant extract can be used as reducing agents and stabilizing agents to prepare nanometer particles. Because the synthesis process is environmentally friendly and efficient, the green synthesis of nanosilver using plants has become an environmentally friendly and promising production method to replace traditional chemical, physical and microbial synthesis.

[0005] In the prior art, the Chinese patent document with publication number CN114769611A discloses a method for preparing nanosilver using pine needle extract, the Chinese patent document with publication number CN106513707A discloses a preparation process for green synthesis of nanosilver bacteriostatic agent using blueberry leaf extract, the Chinese patent document with publication number CN107671305A discloses a method for efficiently preparing nanosilver bacteriostatic agent using privet fruit extract, and the Chinese patent document with publication number CN103949658A discloses a method for green synthesis of nanosilver using eucommia bark water extract. However, in the above-mentioned patent documents, the antibacterial effect of the nanosilver bacteriostatic agent obtained by some methods has not been studied, and the antibacterial effect of the nanosilver obtained by some methods is poor. In addition, in the prior art, there are few types of plants that can be used to synthesize nanosilver particles. In view of this problem, there is an urgent need to develop a method that is efficient, green and can prepare nanosilver particles with excellent antibacterial effect.

[0006] In addition, a large number of studies have shown that cruciferous and solanaceous crops are very common to be infected by Pseudomonas syringae pathogen, which can easily cause huge losses to China's agricultural economy. However, traditional chemical pesticides can easily cause bacterial resistance, environmental pollution and other problems, and can also cause potential harm to the human body. Therefore, it is more necessary to develop a green new method for preventing and treating bacterial black spot of cruciferous vegetables. SUMMARY

[0007] The application provides a method for preparing nano-silver particles by using coleus extract, which has simple process, low preparation cost, relatively small energy consumption, and the obtained nano-silver particles have small average particle size, uniform size, stable structure, can effectively inhibit the activity of Pseudomonas syringae pv tomato and other plant pathogenic bacteria, destroy the cell structure to make the endosolutes exosmosis, and has good bactericidal effect, and can be used for preventing and treating bacterial black spot of cruciferous vegetables and other plant diseases.

[0008] The specific technical solutions are as follows:

[0009] A method for preparing nano-silver particles by using coleus extract, comprising the following steps:

[0010] (1) After pretreatment, the coleus is crushed and mixed with deionized water, stirred uniformly, and then placed, filtered to obtain coleus extract;

[0011] (2) The coleus extract obtained in step (1) is mixed with AgNO3 solution to obtain a mixed solution, and then the nano-silver particles (AgNPs) are obtained by further centrifugation, washing and vacuum freeze-drying.

[0012] Coleus belongs to the Labiatae herbaceous plant, and is a kind of ornamental leaf flower with high ornamental value, and its colorful leaves are deeply loved by people. The coleus extract contains rich plant pigments, alkaloids, tannins, flavonoids, saponins and terpenes, among which the red pigment has certain reduction resistance and oxidation resistance. At the same time, the coleus is rich in rosmarinic acid and has medicinal value, and has the effects of treating headache, abrasion, indigestion and ophthalmia. Rosmarinic acid is also a natural antioxidant, which is widely used in the fields of medicine, food, cosmetics and the like.

[0013] The red pigment and rosmarinic acid in the coleus extract have good antioxidant properties and stability, and thus play a key role in the preparation of silver nanoparticles. In the method of the present application, ornamental coleus is selected as the raw material, and the leaf extract is used to prepare AgNPs. The silver nanoparticles prepared from coleus are spherical in size, uniform in size, and stable in structure. The resistance of the silver nanoparticles to various plant pathogenic bacteria is detected by the double plate method, and it is found that the silver nanoparticles have inhibitory effect on Pestalotiopsis versicolor and Pseudomonas syringae pv. tomato DC3000 and other plant pathogenic bacteria, and can significantly inhibit the growth of Pseudomonas syringae pv. tomato DC3000.

[0014] In step (1), the pretreatment of coleus includes dust removal and drying. The drying process specifically involves placing the coleus in an oven at 55°C to dry the water in the leaves.

[0015] The pretreated coleus is crushed into powder using a juicer with a power of 200-300W. If the power is too low, the extraction efficiency of the plant material will be low, and if the power is too high, unnecessary energy consumption will be caused.

[0016] Preferably, in step (1), the mass ratio of the crushed coleus to deionized water is 1:10-20, and the standing time is 15-30min.

[0017] In step (2), the coleus extract and AgNO3 solution are mixed and reacted at 55-65°C in the dark until the color of the reaction system turns dark brown, and the reaction is terminated. If the reaction temperature is too low, the reaction will not be complete, the synthesis will not be good, and the particle size of the silver nanoparticles will be affected, as well as their stability and uniformity, thereby affecting their antibacterial effect. If the temperature is set too high, the synthesized product will deteriorate, and unnecessary energy consumption will be caused.

[0018] Optionally, the light-protected reaction requires continuous stirring, or the coleus extract and AgNO3 solution are placed on a shaker for light-protected reaction.

[0019] Preferably, the stirring rate is 170-200rpm, and the rotation speed of the shaker is 150-200rpm.

[0020] Preferably, the concentration of the AgNO3 solution is 3-4mM, and the volume ratio of the coleus extract to the AgNO3 solution is 10-25:100. If the content of the effective components in the coleus is too high, it will affect the synthesis of silver nanoparticles, and if the amount is too low, the efficiency of the final product synthesis will be reduced.

[0021] The centrifugal rate is 8000-14000 rpm, and the centrifugal time is 8-15 min. If the centrifugal rate is too low and the centrifugal time is too short, the supernatant will not be clear, the content of the finally obtained nano-silver particles is less, and the impurities are more, thereby affecting the antibacterial effect of the product.

[0022] The application further provides the nano-silver particles prepared by the method.

[0023] The application further provides application of the nano-silver particles in inhibiting plant pathogenic bacteria, preferably, the plant pathogenic bacteria include Pestalotiopsis versicolor and Pseudomonas syringae pv.tomato DC3000.

[0024] The application further provides application of the nano-silver particles as an agricultural bactericide in preventing and treating plant diseases caused by plant pathogenic bacteria, and the plant diseases include red bayon wilt and cruciferous vegetable bacterial black spot disease. Experiments show that the nano-silver particles prepared by the method have strong antibacterial effect on Pseudomonas syringae pv.tomato DC3000, Pestalotiopsis versicolor and other plant pathogenic bacteria, thereby being capable of controlling cruciferous vegetable bacterial black spot and red bayon wilt and other diseases caused by the plant pathogenic bacteria.

[0025] The specific application method for preventing and treating plant diseases is that the nano-silver particles are dissolved in water to prepare an AgNPs suspension, and the AgNPs suspension is uniformly sprayed on the plants to be prevented and treated.

[0026] Preferably, the concentration of the nano-silver particles in the AgNPs suspension is 25-100 μg / ml. The AgNPs suspension with the concentration in the range has good prevention and treatment effect on cruciferous vegetable bacterial black spot, and the antibacterial agent concentration is further improved, and the effect is not improved much.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] (1) The application provides a method for preparing nano-silver particles by using a coleus caninus extract, and the obtained nano-silver particles have small average particle size, uniform size and stable structure, and can effectively inhibit the activity of red bayon wilt pathogenic fungi and Pseudomonas syringae pv.tomato DC3000 and other plant pathogenic bacteria, especially Pseudomonas syringae pv.tomato DC3000, destroy the cell structure, make the cell membrane wrinkle, and cause the exosmosis of endosolutes, thereby having good bactericidal effect.

[0029] (2) The method of the present invention has mild reaction conditions, simple process, low preparation cost, relatively low energy consumption and no toxic by-products, no need to add additional reducing agent and catalyst, which is very environmentally friendly and safe. It belongs to green synthesis process and is suitable for large-scale production.

[0030] (3) The nano-silver particles obtained in this invention are used as agricultural antibacterial agents to prevent and control plant diseases such as bacterial black spot disease of cruciferous vegetables. They have a good control effect. This plant-derived agricultural fungicide will not only not cause potential harm to the human body, but also will not pollute the environment. The application method is simple and easy to implement. It has a promising prospect and broad application in the field of agricultural production. Attached Figure Description

[0031] Figure 1 In Example 1, A is an optical image showing the color change of silver nanoparticles prepared using Coleus extract, and B is the UV-Vis spectrum of silver nanoparticles synthesized at different reaction times.

[0032] Figure 2 In this diagram, A is the infrared spectrum of the silver nanoparticles prepared in Example 1, B is the transmission electron microscope image of the silver nanoparticles, and C is the X-ray diffraction pattern of the silver nanoparticles.

[0033] Figure 3 This is a scanning electron microscope image of the silver nanoparticles prepared in Example 1;

[0034] Figure 4 The graph shows the inhibitory effects of different concentrations of AgNPs suspension on plant pathogens. In the graph, A is the graph showing the change in the diameter of the inhibition zone, and B is the graph showing the change in absorbance. In the graph, a, b, c, and d indicate significant differences between different treatments at the 5% level, and bc and cd indicate significant differences between different treatments at the 1% level.

[0035] Figure 5 In the figure, A is the release curve of AgNPs suspension in PBS solution, and B is the effect of AgNPs solution on the contact time with plant pathogens on the population activity of plant pathogens; in the figure, a, b, c, d, and e indicate significant differences among different treatments at the 5% level, and bc indicate significant differences among different treatments at the 1% level.

[0036] Figure 6 The images show the fluorescence staining of plant pathogens before and after treatment with AgNPs suspension. In the images, A is the negative control without AgNPs treatment, and B is the plant pathogen after treatment with AgNPs suspension.

[0037] Figure 7 In the image, A and C are TEM and SEM images of the untreated plant pathogens, respectively, while B and D are TEM and SEM images of the plant pathogens treated with AgNPs suspension.

[0038] Figure 8 This is a statistical graph showing the extravasation of intracellular solutes after treatment with different concentrations of AgNPs suspensions on plant pathogens. In the graph, a, b, c, and d indicate significant differences among different treatments at the 5% level. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to the embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] Pseudomonas syringae pv. tomato DC3000, a pathogenic strain of tomato, is deposited in the Biobw Culture Collection with accession number bio-103914.

[0041] Pestalotiopsis versicolor was isolated from the branches of a Chinese bayberry plant suffering from wilt disease in Xianju, Zhejiang.

[0042] Example 1

[0043] (1) Wash and dry the freshly picked leaves of Coleus, crush them with a juicer with a power of 250W, weigh 5g and mix with 100ml of deionized water, stir well and let stand for 20 minutes, filter to remove solid impurities, and obtain Coleus extract.

[0044] (2) Take 20 ml of the Coleus extract obtained in step (1) and mix it with 100 ml of 4 mM AgNO3 solution. Place the mixture in a shaker at 55°C and 180 rpm. After reacting in the dark, the color changes from pale yellow-green to dark brown. Remove the mixture (the specific color change is as follows). Figure 1 As shown in A, after adding coleus extract to a colorless and transparent AgNO3 solution, it eventually turns dark brown. The mixture is then centrifuged at 14,000 rpm for 8 minutes to remove the supernatant. The precipitate is washed, and then freeze-dried under vacuum to obtain powdered silver nanoparticles.

[0045] The structural characteristics and physicochemical properties of silver nanoparticles were evaluated using ultraviolet-visible absorption spectroscopy (UV-VIS), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD).

[0046] Figure 1 B in the figure represents the UV-Vis spectra of silver nanoparticles synthesized using Coleus extract at different reaction times. The results show that the absorption value at 428 nm is the strongest with increasing reaction time, indicating that the synthesized silver nanoparticles are relatively stable.

[0047] Figure 2 A in FIG. 1 is a Fourier transform infrared spectrogram of the nano-silver particles obtained by reacting the Coleus extract solution with AgNO3 in this embodiment, and from the figure it can be seen that the nano-silver particles have been successfully synthesized; Figure 2 B in FIG. 2 is a transmission electron microscope image of the product nano-silver particles, and it can be seen that the synthesized nano-silver particles are spherical particle materials; Figure 2 C in FIG. 3 is an X-ray diffraction pattern of the nano-silver particles, and characteristic peaks of (111), (200), (220), (311) respectively appear at 2θ = 27.831°, 32.243°, 46.233° and 54.828°, which are consistent with the characteristic peaks of the AgO powder diffraction pattern, proving that stable AgNPs have been synthesized.

[0048] Figure 3 FIG. 4 is an SEM image of the nano-silver particles prepared in this embodiment, and from the figure it can be seen that the nano-silver particles synthesized in this embodiment are mostly spherical and uniform in size, with a particle size range of 30-50 nm; the compositions of the nano-silver particles synthesized from different plants are different, and from the EDS test results it can be seen that the nano-silver synthesized in this embodiment is mainly composed of Ag and Cl elements, and the Cl element is derived from the Coleus extract solution.

[0049] Example 2

[0050] (1) freshly picked Coleus leaves were washed, dried, crushed with a juicer with a power of 200 W, 10 g of which was mixed with 100 ml of deionized water, stirred uniformly, and then left to stand for 25 minutes, after which the solid impurities were removed by filtration to obtain a Coleus extract solution;

[0051] (2) 15 ml of the Coleus extract solution obtained in step (1) was mixed with 100 ml of a 4 mM AgNO3 solution, and placed in a shaking bed with a temperature of 60°C and a rotation speed of 180 rpm, and left to react overnight in the dark, after which the color changed from light yellow-green to dark brown, and the mixture was then centrifuged at a rotation speed of 10,000 rpm for 10 minutes, after which the supernatant was removed, and the precipitate was washed, vacuum freeze-dried to obtain a powder of nano-silver particles.

[0052] Example 3

[0053] (1) freshly picked Coleus leaves were washed, dried, crushed with a juicer with a power of 300 W, 5 g of which was mixed with 100 ml of deionized water, stirred uniformly, and then left to stand for 20 minutes, after which the solid impurities were removed by filtration to obtain a Coleus extract solution;

[0054] (2) Take 10 ml of the extract of Tradescantia fluminensis obtained in step (1) and mix with 100 ml of AgNO3 solution with a concentration of 4 mM. Place in a shaking table at a temperature of 65°C and a rotation speed of 180 rpm, and place overnight in the dark to react. After the color changes from light yellow green to dark brown, take out, further centrifuge the mixture at a rotation speed of 8000 rpm for 15 minutes, remove the supernatant, and take the precipitate to wash, vacuum freeze dry, and obtain powdered nano-silver particles.

[0055] Sample analysis

[0056] Evaluation of antibacterial effect:

[0057] (1) Effect of nano-silver concentration on antibacterial effect

[0058] In order to evaluate the activity of the nano-silver prepared by the method of the present application in inhibiting plant pathogenic bacteria, the nano-silver particles prepared in Example 1 are respectively prepared into AgNPs suspensions with concentrations of 25, 50, 75, and 100 μg / mL, and the antibacterial performance of the AgNPs suspensions with different concentrations on Pseudomonas syringae pv. tomato is evaluated by measuring the absorbance of the bacterial solution and the diameter of the antibacterial circle on the plate.

[0059] As can be seen from A in Table 1, Figure 4 , after 24 h of culture, the antibacterial circle on the plate with the AgNPs suspension added can reach a maximum of 11.17 mm compared with the control group; as can be seen from B in Table 1, Figure 4 , after 24 h of culture under suitable conditions, the growth inhibition rates of the AgNPs suspensions with concentrations of 25, 50, 75, and 100 μg / mL on Pseudomonas syringae pv. tomato are 34.02%, 39.50%, 47.41%, and 51.52% respectively, and it can be seen that the higher the concentration, the better the antibacterial effect, but when the concentration is further increased on the basis of 100 μg / mL, the antibacterial rate does not increase much, and the antibacterial effect does not increase significantly.

[0060] As can be seen from Table 1, Figure 4 , the higher the concentration of the AgNPs suspension, the better the antibacterial effect, and the nano-silver particles synthesized by Tradescantia fluminensis can significantly inhibit the growth of Pseudomonas syringae pv. tomato.

[0061] (2) Effect of contact time of nano-silver and plant pathogenic bacteria on antibacterial effect

[0062] To study whether the inhibition effect of AgNPs synthesized in Example 1 on plant pathogens is related to the contact reaction time between them, considering the cost and the antibacterial effect, the AgNPs suspension with a concentration of 100 μg / mL is added to the Pseudomonas syringae pv. tomato bacteria liquid, the OD value is measured by controlling the reaction time between them, and the OD value of the Pseudomonas syringae pv. tomato bacteria liquid with the same volume is taken as a blank control. In addition, the release amount of silver nanoparticles in the PBS solution is measured by the point sampling experiment to further verify.

[0063] Figure 5 A in the figure is the release curve of the AgNPs suspension in the PBS solution, and it can be seen from the figure that the release amount gradually increases with the increase of time, and thus the antibacterial effect is also enhanced with the increase of time.

[0064] Figure 5 B in the figure is the influence diagram of the contact time between the AgNPs suspension and Pseudomonas syringae pv. tomato on the population viability, and it can be seen from the figure that the number of cell survival significantly decreases compared with the control group after the contact for 0 h, 24 h and 48 h, and the inhibition rates are 11.76%, 48.48% and 62.94% respectively. It can be seen that the longer the contact time between them is, the better the antibacterial effect of silver nanoparticles is.

[0065] In summary, the inhibition effect of the silver nanoparticles synthesized by the method of the application on Pseudomonas syringae pv. tomato is related to the contact time between them, and the longer the contact time is, the better the antibacterial effect is.

[0066] (3) Antibacterial effect of silver nanoparticles

[0067] To further prove that the silver nanoparticles can cause the death of bacteria by destroying the cell structure of the bacteria, thereby playing a good antibacterial effect, the AgNPs powder synthesized in Example 1 is prepared into a suspension with a concentration of 100 μg / mL. After the obtained AgNPs suspension is contacted with Pseudomonas syringae pv. tomato for 8 h, observation is carried out under a fluorescence microscope by staining, and the bacteria liquid sample without AgNPs treatment is taken as a negative control. Green fluorescence indicates that the cell has activity and complete structure, and red fluorescence indicates that the cell is dead or loses activity due to damage or other reasons. The obtained staining diagram shows that the untreated cells are in normal survival state (A in the figure, most of which are green fluorescence), and the cells treated by 100 μg / mL AgNPs are significantly reduced (B in the figure, most of which are red fluorescence), which indicates that the AgNPs can inhibit the growth of cells. Figure 6 Figure 6

[0068] In addition, the scanning electron microscope and the transmission electron microscope can more directly show the degree of cell damage. Figure 7 ​​B and D in the figure are TEM and SEM photos of P. syringae pv. tomato cells treated by AgNPs suspension with a concentration of 100 μg / ml in Example 1, while A and C are photos of P. syringae pv. tomato cells without treatment. From A and C, it can be seen that the cells are complete and normal, while B and D show that after treatment by AgNPs, the cell membrane of the bacteria cells is wrinkled and the AgNPs enter the cells, making the cells abnormal.

[0069] (4) Effect of AgNPs on exosmosis of P. syringae pv. tomato

[0070] The bactericidal ability of the synthesized AgNPs was evaluated by measuring the amount of endosmosis of P. syringae pv. tomato treated by AgNPs suspension with different concentrations. The contents of RNA and DNA of P. syringae pv. tomato treated by AgNPs suspension with different concentrations were measured at OD260 nm and OD280 nm, and the results are shown in Figure 8 .

[0071] As shown in Figure 8 , when the concentration of AgNPs suspension is 100 μg / ml, the amount of endosmosis is the largest, indicating that the damage to the cell structure of P. syringae pv. tomato is the largest. With the decrease of the concentration of AgNPs suspension, the contents of RNA and DNA of endosmosis gradually decrease, indicating that the damage to the cell structure of P. syringae pv. tomato decreases. That is, AgNPs have an effect on the endosmosis of P. syringae pv. tomato, thereby indicating that AgNPs can damage the cell structure of the pathogenic bacteria.

[0072] Application Example

[0073] In a greenhouse or a field, 1 g of AgNPs powder obtained in Example 1 was dissolved in 10 L of water to prepare AgNPs suspension with a concentration of 100 μg / ml, which was uniformly sprayed on the tomato plants where bacterial black spot disease might occur. It was observed that the tomato plants sprayed with the AgNPs suspension grew better than the tomato plants without spraying, which indicates that the AgNPs suspension can effectively prevent and treat diseases of the crops.

[0074] The above examples have described the technical solutions of the present application in detail. It should be understood that the above examples are only specific embodiments of the present application and are not used to limit the present application. Any modification, supplement or similar replacement within the principle range of the present application should be included in the protection range of the present application.

Claims

1. Use of nano-silver particles for inhibiting plant pathogenic fungi, characterized in that, The plant pathogenic bacteria is Pseudomonas syringae pv. tomato DC3000 ; The nano-silver particles are prepared by using the Coleus extraction solution, and the method comprises the following steps: (1) The pretreated Coleus is crushed into powder, mixed with deionized water, stirred uniformly, and then placed for a period of time, filtered to obtain the Coleus extraction solution; (2) The Coleus extraction solution obtained in step (1) is mixed with AgNO3 solution to obtain a mixed solution, and then the mixed solution is further centrifuged, washed, and vacuum freeze-dried to obtain the nano-silver particles; After the Coleus extraction solution is mixed with the AgNO3 solution, the mixture is reacted at 55-65 DEG C in the dark, and the reaction is terminated when the color of the reaction system turns to dark brown; the reaction in the dark requires continuous stirring; the concentration of the AgNO3 solution is 3-4 mM, and the volume ratio of the Coleus extraction solution to the AgNO3 solution is 10-25:100; the centrifugal speed is 8000-14000 rpm, and the centrifugal time is 8-15 min.

2. Use according to claim 1, characterized in that, In the process of preparing the nano-silver particles by using the Coleus extraction solution, a juicer is used to crush the pretreated Coleus into powder, and the power of the juicer is 200-300 W.

3. Use according to claim 1, characterized in that, In step (1), the mass ratio of the crushed Coleus to deionized water is 1:10-20, and the standing time is 15-30 min.

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