CN112157271A - Method for preparing nano-silver colloid from ageratum thistle extracting solution and application - Google Patents

Abstract

The invention discloses a method for preparing nano-silver colloid by using ageratum thistle extracting solution and application thereof, which comprises the following specific steps: s1, cleaning fresh stem leaves of ageratum conyzoides with double distilled water, air drying, pulverizing, sieving with a 40-mesh sieve, mixing with ultra-clean water according to the weight ratio of 1: adding the mixture into water according to the mass ratio of 10-300, performing ultrasonic water bath at the temperature of 60-90 ℃ for 10-60 min, cooling, and performing centrifugal filtration to obtain a wrinkled gianthyssop herb and thistle water extracting solution; s2, adding sodium dodecyl sulfate serving as a stable dispersant into the upward clear ageratum water extract to obtain a final concentration of 0.5-50 mg/mL, then adding a silver nitrate solution to obtain a final concentration of silver ions of 1-100 mmol/mL, and then performing magnetic stirring reaction at 20-40 ℃ for 2-8h to obtain the nano silver colloid solution. The nano silver colloid of the present invention has wide bioactivity, and has the biological characteristics of nano silver, such as broad spectrum antibiotic activity, and the bioactivity of gathering flavone, sterol, etc. in ageratum thistle, so that it has the functions of inhibiting tumor cell proliferation, inhibiting inflammation, etc.

Description

Method for preparing nano-silver colloid from ageratum thistle extracting solution and application

Technical Field

The invention belongs to the technical field of nano material preparation, and particularly relates to a method for preparing nano silver colloid from ageratum thistle water extract and application of nano silver colloid

Background

The nano silver colloid is widely concerned due to small-size effect, surface effect, quantum effect, macroscopic quantum tunneling effect, antibacterial performance and the like, and obtains a series of substantive research results in the aspects of optics, thermophysics, electrics, magnetics, mechanics, chemistry, food, medical treatment and the like. Can be used as high-end silver paste, a conductive coating, a catalytic material, an antibacterial material and a drug carrier, and has a far-reaching application prospect in the fields of photoelectric chip manufacturing, medical health, green household appliances and the like. And the application of the nano-silver colloid in the aspect of life science is increasing day by day, and due to good biological activities of antibiosis, antivirus, antitumor and the like, the nano-silver colloid is used for developing various nano-silver colloid products and is used in the fields of biological medicine and the like, thereby providing convenience for daily life of people.

The preparation method of the nano-silver colloid is also diversified, and mainly comprises a chemical method (such as oxidation, an electrochemical method, a sol method, a microemulsion method, a template method and a microwave-assisted chemical vapor deposition method), a physical method (such as a physical vapor deposition method, a mechanical grinding method, a laser burning method, an ion etching method and a magnetron sputtering method) and various biosynthesis methods (such as an enzyme synthesis method, a microbial product synthesis method and a plant extract synthesis method).

The invention content is as follows:

the invention aims to provide a method for preparing nano-silver colloid by using ageratum conyzoides water extract, which is simple, convenient and easy to implement, strong in operability and low in cost, does not discharge toxic and harmful substances to the environment in the preparation process, and is relatively environment-friendly.

In order to achieve the purpose, the invention adopts the technical scheme that:

1) cleaning fresh stem and leaf of ageratum conyzoides with double distilled water, air drying, pulverizing, sieving with 40 mesh sieve, mixing with ultra-pure water according to a ratio of 1: adding the extract into water according to the mass ratio of 10-300, performing ultrasonic water bath at the temperature of 60-90 ℃ for 10-60 min, performing centrifugal filtration, and cooling to obtain a wrinkled gianthyssop herb and thistle leaf extract;

2) firstly, adding sodium dodecyl sulfate serving as a stable dispersant into the upward clear ageratum water extract, wherein the final concentration of the sodium dodecyl sulfate is 0.5-50 mg/mL, then adding a silver nitrate solution to enable the final concentration of silver ions to be 1-100 mmol/mL, and then placing the mixture at 20-40 ℃ for magnetic stirring reaction for 2-8h to obtain a nano silver colloid solution.

The invention also provides application of the ageratum thistle water extract in preparing nano-silver colloid in preparing antibacterial drugs.

The invention also provides application of the ageratum thistle water extract in preparing nano silver colloid in preparing antitumor drugs.

The invention also provides application of the ageratum thistle water extract in preparing the nano-silver colloid in preparing anti-inflammatory drugs.

The method for preparing the nano-silver colloid by using the ageratum thistle water extract is simple, convenient and easy to implement, strong in operability, low in cost and good in stability, does not discharge toxic and harmful substances to the environment in the preparation process, and is a simple and environment-friendly nano-silver preparation technology.

The innovation points and advantages of the invention are as follows:

1. the invention uses ageratum thistle water extract as a reducing agent, sodium dodecyl sulfate as a stabilizing agent and a dispersing agent to react with silver nitrate, so that silver ions are completely reduced into silver nano particles. The formed nano silver colloid can be kept stable for a long time at normal temperature without coagulation.

2. The preparation method of the silver nanoparticles adopts magnetic stirring to assist in synthesizing the silver nanoparticles, has simple preparation process, low cost and short reaction time, and avoids the use of toxic reducing agents.

3. The nano silver colloid of the present invention has wide bioactivity, and has the biological characteristics of nano silver, such as broad spectrum antibiotic activity, and the bioactivity of gathering flavone, sterol, etc. in ageratum thistle, so that it has the functions of inhibiting tumor cell proliferation, inhibiting inflammation, etc.

Description of the drawings:

FIG. 1 is a transmission electron micrograph of silver nanoparticles of example 1;

FIG. 2 is a UV-VIS absorption spectrum of silver nanoparticles (AC-AgNPs) and an extract of ageratum water (AC-extract) in example 1;

FIG. 3 is a graph showing the particle size and particle size distribution of silver nanoparticles in example 1;

FIG. 4 is the effect of nanosilver on the expression of TNF- α, a cytokine secreted by RAW264.7 stimulated by LPS in example 1;

FIG. 5 is the effect of nanosilver on the expression of LPS-stimulated RAW264.7 secreting cytokine IL-1 β in example 1;

FIG. 6 is the effect of nanosilver on the expression of LPS-stimulated RAW264.7 cytokine IL-6 in example 1.

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

In this example, a nano silver particle synthesis experiment is performed under different adding amounts of ageratum herb extract, and the method for preparing nano silver colloid from ageratum herb extract provided in this example includes the following specific steps:

(1) the ageratum thistle used in the experiment is collected from the suburbs in the urban area of Haikou, the stem and leaf parts of the ageratum thistle are washed for three times by double distilled water, dried naturally, dried and crushed, and screened by a 40-mesh sieve to obtain ageratum thistle powder. Stirring 3g of ageratum thistle powder and 180mL of pure water uniformly in an erlenmeyer flask, putting the erlenmeyer flask into an ultrasonic pool, performing ultrasonic water bath at 80 ℃ for 30min, cooling, and centrifuging the mixed solution for 15min by 10000g to obtain an ageratum thistle water extract.

(2) Placing 2mL of ageratum extract solution in a 50mL brown bottle, and magnetically stirring;

(3) adding 0.1mL of 100mg/mL Sodium Dodecyl Sulfate (SDS), and uniformly stirring;

(4) adding 4mL of silver nitrate with the concentration of 100 mu mol/mL, adding ultra-clean water to supplement 10mL, and uniformly stirring;

(5) adding the mixed solution in a brown bottle, and magnetically stirring at 25 ℃;

(6) the magnetic stirring speed is 150 rpm;

(7) the reaction time is 6 h;

(8) after the reaction is finished, obtaining 30-100 nm of nano-silver colloid, and freeze-drying the nano-silver colloid to obtain dry powder for storage; and the physicochemical properties of the synthesized nano-silver colloid can be determined by a nano-particle analyzer, an ultraviolet-visible spectrophotometer, a transmission electron microscope and the like.

Example 2

This example was the same as example 1 except that the concentration of the extract solution of ageratum conyzoides (step (1)) was different, and the mass ratio of ageratum conyzoides powder to water was 1: 10; 1: 20; 1: 40; 1: 60; 1: 80; 1: 150; 1:300.

Example 3

This example was similar to example 1, except that the concentration of SDS (step (3)) was different, and 0, 50, 100, 200, and 400. mu.L of 100mg/mL SDS were added.

Example 4

This example is substantially the same as example 1 except that the concentration of silver nitrate (step (4)) is different so that the final silver ion concentration reaches 10, 20, 40, 60, 80, 100 μmol/mL.

Example 5

This example was similar to example 1 except that the stirring temperature (step (5)) was changed to 4, 10, 25, 30, 40 and 50 ℃.

Example 6

This example was similar to example 1 except that the stirring time (step (7)) was 0.5, 1, 2, 4, 6, and 8 hours.

Example 7

In this embodiment, the agastache rugosa extract is used to prepare the nano-silver colloid and the application thereof in the bacteriostatic field, and the nano-silver colloid obtained in example 1 is used for bacteriostatic experiments.

(1) Zone of inhibition experiment

The nano-silver colloid in the example 1 is dispersed in ultra-clean water to prepare the nano-silver colloid with the concentration of 10mg/mLAnd (3) dispersing the mixture. Preparing LB plate culture medium, sucking different bacterial suspensions such as Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Proteus, copper green pseudomonas and the like to the sterilized plate culture medium, and coating the bacterial suspensions with the bacterial concentration of about 10⁵cfu, 20. mu.l of the dispersion was soaked in 6mm filter paper, aseptically air-dried, and then sequentially placed in a plate medium containing Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Proteus, Pseudomonas aeruginosa, etc.

Each pathogenic bacterium was cultured at 37 ℃ for 18 to 24 hours (in this example, 18 hours).

The inhibition zones of 7 pathogenic bacteria are shown in Table 1.

(2) Minimum inhibitory concentration

The Minimum Inhibitory Concentration (MIC) determination of the nano-silver colloid of the invention on different indication pathogenic bacteria such as escherichia coli, staphylococcus aureus, Klebsiella pneumoniae, methicillin-resistant staphylococcus aureus, bacillus subtilis, proteus, copper green pseudomonas and the like is tested. The MIC of the strain is determined by adopting a 2-fold dilution method, and the experimental steps are as follows:

picking out a monoclonal colony from a culture dish, inoculating the colony into a sterilized 50mL triangular flask filled with 10mL LB liquid culture medium, and placing the flask in a shaking table at 37 ℃ and 220rpm for shake culture for 6 h;

each well of the 96-well plate was filled with 160. mu.L of LB liquid medium. And adding 160 mu L of LB liquid culture medium with the concentration of 250 mu g/mL of nano-silver colloid into all the No. 1 cavities, uniformly mixing, sucking 160 mu L to the No. 2 cavities, and repeating the steps, sucking 160 mu L from the No. 12 cavity and discarding. Then 40. mu.L of a solution containing 10 per well was added⁴And (5) uniformly mixing the cultured bacterial liquid of the cfu, and adding no bacterial liquid in a blank group.

And placing the treated 96-well plate in a biochemical incubator at 37 ℃ for incubation for 12h, and then measuring the OD value of bacterial liquid in each well under an enzyme-labeling instrument.

The Minimum Inhibitory Concentration (MIC) is calculated and recorded as shown in Table 1, wherein the smaller the MIC is, the better the inhibitory effect on the pathogenic bacteria is.

TABLE 1 inhibitory Effect of Nano-silver colloid on different pathogenic bacteria

Example 8

In the embodiment, the ageratum thistle extract is used for preparing the nano-silver colloid and the application thereof in the anti-tumor field, and the anti-tumor activity test is carried out on the nano-silver colloid obtained in the embodiment 1.

The MTT method is adopted to carry out the anti-tumor activity on the nano-silver colloid. The method comprises the following operation steps:

collecting tumor cells in logarithmic growth phase, and adjusting cell suspension concentration to 1 × 10⁴Per/mL, add 100. mu.L/well of 96-well plate, blank 200. mu.L complete medium, put in CO₂In an incubator, 37 ℃ and 5% CO₂And culturing for 4 h. Add 100. mu.L of nanosilver medium with different concentrations into each well, set up 6 gradients to make the final concentration of each well divided into 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL and 15.63. mu.g/mL, and add 100. mu.L of complete medium for the negative control. Each gradient is provided with 3 repeats, and the culture is continued for 24 hours and then taken out. After the drug action was completed, 40. mu.L of MTT was added to each well at a concentration of 5mg/mL and cultured for 4 hours. The culture was terminated and the culture medium in the wells was gently aspirated. Add 150. mu.L of DMSO per well. Shaking table at low speed for 10 min. Then, OD was measured by a microplate reader, and the OD value was the absorbance value (A) of each well at 490 nm. Calculated inhibition of cell inhibition: the inhibition rate is (experimental OD value-blank OD value)/(negative OD value-blank OD value) × 100%. The concentration of the sample at which the inhibition rate is 50% is IC₅₀. IC50 values were calculated and the results are expressed as mean ± standard deviation.

The nano-silver colloid of the invention shows inhibition effect in activity test of 5 cancer cells, wherein, IC₅₀The smaller the value, the better the inhibition effect of the nano-silver colloid on the cancer cells. The test results are shown in table 2.

TABLE 2 inhibitory Effect of Nanosilver on various tumor cells

Example 9

In the embodiment, nano-silver colloid prepared from ageratum thistle extracting solution and application thereof in the anti-inflammatory field are used, and the influence of the nano-silver colloid obtained in the embodiment 1 on the secretion of inflammatory factors of RAW264.7 stimulated by Lipopolysaccharide (LPS) in bacterial endotoxin is taken.

Stimulating RAW264.7 with bacterial endotoxin Lipopolysaccharide (LPS), and detecting the effect of the nano-silver colloid on the expression of the inflammatory factor from the protein level by using an enzyme-linked immunosorbent assay (ELISA) experiment.

Sample preparation and cell assay procedures were as follows:

(a) macrophage RAW264.7 was plated in a 6-well plate, and five groups, including NC (blank control), LPS group, low-nm silver colloid (1.0. mu.g/mL) group, medium-nm silver colloid (2.0. mu.g/mL) group, and high-nm silver colloid (4.0. mu.g/mL) group, were set.

(b) The LPS group and each nanosilver group were incubated with the cells for two hours.

(c) Cells in LPS group and each nanosilver group were stimulated with 1. mu.g/ml bacterial Lipopolysaccharide (LPS).

(d) After 24h of stimulation, cell supernatants are collected, and the contents of TNF-alpha, IL-6 and IL-1 beta in different groups of RAW264.7 cells are detected according to the operation flow of an ELISA kit.

The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.

Claims (7)

1. A method for preparing nano-silver colloid by using ageratum thistle extracting solution is characterized by comprising the following specific steps:

s1, cleaning fresh stem leaves of ageratum conyzoides with double distilled water, air drying, pulverizing, sieving with a 40-mesh sieve, mixing with ultra-clean water according to the weight ratio of 1: adding the mixture into water according to the mass ratio of 10-300, performing ultrasonic water bath at the temperature of 60-90 ℃ for 10-60 min, cooling, and performing centrifugal filtration to obtain a wrinkled gianthyssop herb and thistle water extracting solution;

s2, adding sodium dodecyl sulfate serving as a stable dispersant into the upward clear ageratum water extract to obtain a final concentration of 0.5-50 mg/mL, then adding a silver nitrate solution to obtain a final concentration of silver ions of 1-100 mmol/mL, and then performing magnetic stirring reaction at 20-40 ℃ for 2-8h to obtain the nano silver colloid solution.

2. The method for preparing nano-silver colloid by using the ageratum thistle extracting solution as claimed in claim 1, wherein the method comprises the following steps: in the step S1, the mass ratio of the ageratum leaves in the ageratum leaves extract to the ultra-pure water is 1:10 to 300.

3. The method for preparing nano-silver colloid by using the ageratum thistle extracting solution as claimed in claim 1, wherein the method comprises the following steps: in the step S2, the final concentration of silver nitrate added is 1-100 mg/mL.

4. The method for preparing nano-silver colloid by using the ageratum thistle extracting solution as claimed in claim 1, wherein the method comprises the following steps: in step S2, SDS is added to a final concentration of 0.5-50 mg/mL.

5. The use of the nanosilver as prepared in claim 1 in antitumor drugs, characterized in that: the tumor includes melanoma, gastric cancer, and breast cancer.

6. The use of nanosilver in the preparation of a microorganism resistant medicament according to claim 1, wherein the microorganism is a pathogenic bacterium such as escherichia coli, staphylococcus aureus, klebsiella pneumoniae, methicillin resistant staphylococcus aureus, bacillus subtilis, proteus, pseudomonas aeruginosa.

7. The application of the nano-silver colloid prepared according to the claim 1 in anti-inflammatory drugs is characterized by comprising the following specific steps:

stimulating RAW264.7 with bacterial endotoxin Lipopolysaccharide (LPS), and detecting the effect of the nano-silver colloid on the expression of the inflammatory factor from the protein level by using an enzyme-linked immunosorbent assay (ELISA) experiment.

(a) Macrophage RAW264.7 was plated in a 6-well plate, and five groups, including NC (blank control), LPS group, low-nm silver colloid (1.0. mu.g/mL) group, medium-nm silver colloid (2.0. mu.g/mL) group, and high-nm silver colloid (4.0. mu.g/mL) group, were set.

(b) The LPS group and each nanosilver group were incubated with the cells for two hours.

(c) Cells in LPS group and each nanosilver group were stimulated with 1. mu.g/ml bacterial Lipopolysaccharide (LPS).

(d) After 24h of stimulation, cell supernatants are collected, and the contents of TNF-alpha, IL-6 and IL-1 beta in different groups of RAW264.7 cells are detected according to the operation flow of an ELISA kit.

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