CN112157271B - Method for preparing nano silver colloid from ageratum extract and application of nano silver colloid - Google Patents
Method for preparing nano silver colloid from ageratum extract and application of nano silver colloid Download PDFInfo
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Abstract
The invention discloses a method for preparing nano silver colloid from wrinkled giant hyssop extract and application thereof, which comprises the following specific steps: s1, taking fresh wrinkled giant hyssop stems and leaves, washing with double distilled water, airing, crushing, sieving with a 40-mesh sieve, and then mixing with ultra-pure water according to the proportion of 1: adding the extract into water according to the mass ratio of 10-300, carrying out ultrasonic water bath at 60-90 ℃ for 10-60 min, cooling, centrifugally filtering to obtain an ageratum water extract; s2, adding sodium dodecyl sulfate as a stable dispersing agent into the supernatant herba Agastaches water extract, wherein the final concentration is 0.5-50 mg/mL, then adding a silver nitrate solution to make the final concentration of silver ions be 1-100 mmol/mL, and then placing the mixture in a magnetic stirring reaction at 20-40 ℃ for 2-8 hours to obtain the nano silver colloid solution. The nano silver colloid of the invention has wide biological activity, not only has biological characteristics of nano silver such as broad-spectrum antibacterial activity, but also gathers biological activities of flavonoids, sterols and the like in ageratum, thus being capable of inhibiting tumor cell proliferation, inhibiting inflammation and the like.
Description
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 wrinkled giant hyssop herb water extract and application thereof.
Background
The nano silver colloid is widely focused on the small-size effect, the surface effect, the quantum effect, the macroscopic quantum tunneling effect, the antibacterial property and the like, and a series of substantial research results are obtained in the aspects of optics, heat, electricity, magnetism, mechanics, chemistry, food, medical treatment and the like. Can be used as high-end silver paste, conductive coating, catalytic material, antibacterial material and drug carrier, and has profound application prospect in the fields of photoelectric chip manufacture, medical health, green household appliances and the like. Moreover, the nano silver colloid has increasingly been applied in life science, and as the nano silver colloid has good biological activities of antibiosis, antivirus, anti-tumor and the like, the nano silver colloid has been used for developing various nano silver colloid products, is used in the fields of biological medicine and the like, and provides convenience for daily life of people.
The preparation method of the nano silver colloid is also various and mainly comprises chemical methods (such as oxidation and reduction, electrochemical methods, sol methods, microemulsion methods, template methods and microwave-assisted chemical vapor deposition methods), physical methods (such as physical vapor deposition methods, mechanical grinding methods, laser firing methods, ion etching methods and magnetron sputtering methods) and various biosynthesis methods (such as enzyme synthesis methods, microbial product synthesis methods and various plant extract synthesis methods).
Disclosure of Invention
The invention aims to provide a method for preparing nano silver colloid by using an ageratum water extract, which is simple, convenient, easy to implement, strong in operability and low in cost, and does not discharge toxic and harmful substances to the environment in the preparation process, and is relatively environment-friendly.
In order to achieve the above purpose, the invention adopts the following technical scheme:
1) Taking fresh wrinkled giant hyssop stems and leaves, washing with double distilled water, airing, crushing, sieving with a 40-mesh sieve, and then mixing with ultra-pure water according to the proportion of 1: adding the extract into water according to the mass ratio of 10-300, carrying out ultrasonic water bath at 60-90 ℃ for 10-60 min, centrifugally filtering, and cooling to obtain wrinkled gianthyssop leaf extract;
2) Firstly adding sodium dodecyl sulfate as a stable dispersing agent into the supernatant herba Agastaches water extract, wherein the final concentration is 0.5-50 mg/mL, then adding silver nitrate solution to make the final concentration of silver ions be 1-100 mmol/mL, and then placing the mixture in 20-40 ℃ for magnetic stirring reaction for 2-8h to obtain the nano silver colloid solution.
The invention also provides an application of the ageratum water extract in preparing antibacterial drugs.
The invention also provides an application of the ageratum water extract in preparing the anti-tumor medicine.
The invention also provides an application of the ageratum water extract in preparing the anti-inflammatory drugs.
The preparation method of the nano silver colloid by using the ageratum water extract is simple and easy, has strong operability, low cost and good stability, and the preparation process can not discharge toxic and harmful substances to the environment, so that the preparation method is a simple and environment-friendly nano silver preparation technology.
The invention has the innovation points and advantages that:
1. the invention uses wrinkled giant hyssop water extract as reducer, uses dodecyl sodium sulfate as stabilizer and dispersant, and reacts with silver nitrate to reduce silver nanometer particles completely. The formed nano silver colloid can be kept stable and not coagulated for a long time at normal temperature.
2. The preparation method of the silver nano particles adopts magnetic stirring to assist in synthesizing the silver nano particles, 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 invention has wide biological activity, not only has biological characteristics of nano silver such as broad-spectrum antibacterial activity, but also gathers biological activities of flavonoids, sterols and the like in ageratum, thus being capable of inhibiting tumor cell proliferation, inhibiting inflammation and the like.
Drawings
FIG. 1 is a transmission electron microscope image of silver nanoparticles in example 1;
FIG. 2 is an ultraviolet-visible light absorption spectrum of silver nanoparticles (AC-AgNPs) and Agastache rugosa aqueous extract (AC-extract) in example 1;
FIG. 3 is a graph showing the particle size and distribution of silver nanoparticles in example 1;
FIG. 4 is a graph showing the effect of nano-silver colloid on expression of TNF- α, a secreted cytokine of RAW264.7 stimulated by LPS in example 1;
FIG. 5 is a graph showing the effect of nano-silver colloid on LPS stimulated expression of RAW264.7 secretion cytokine IL-1β in example 1;
FIG. 6 is a graph showing the effect of nano-silver colloid on expression of LPS-stimulated RAW264.7 secretion cytokine IL-6 in example 1.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Examples
In this embodiment, the experiment of synthesizing nano silver particles is performed under the condition of different addition amounts of the herba Agastaches extract, and the method for preparing nano silver colloid by using the herba Agastaches extract provided by this embodiment specifically comprises the following steps:
(1) The herba Agastaches used in the experiment is collected from suburb of the urban area of the sea opening, the stem and leaf parts of the herba Agastaches are washed three times by double distilled water, naturally dried, dried and crushed, and sieved by a 40-mesh sieve to obtain herba Agastaches powder. Mixing herba Agastaches powder 3 g with 180 mL pure water in conical flask, placing the conical flask in ultrasonic bath at 80deg.C for 30 min, cooling, and centrifuging the mixed solution through 10000 g for 15 min to obtain herba Agastaches water extract.
(2) Taking 2 mL herba Agastaches extract liquid in 50 mL brown bottle, and placing under magnetic stirring;
(3) 0.1 mL of 100 mg/mL Sodium Dodecyl Sulfate (SDS) is added and stirred uniformly;
(4) Adding 4 mL of 100 mu mol/mL silver nitrate, adding ultra-pure water to complement 10 mL, and uniformly stirring;
(5) Adding the brown bottle mixed solution and magnetically stirring at 25 ℃;
(6) The magnetic stirring rotation speed is 150 rpm;
(7) The reaction time was 6 h;
(8) After the reaction is finished, 30-100 nm nanometer silver colloid is obtained, and the obtained dry powder is stored after freeze drying; and the physicochemical properties of the synthesized nano silver colloid can be determined by a nano particle size analyzer, an ultraviolet visible spectrophotometer, a transmission electron microscope and the like.
Examples
The present example is similar to example 1, except that the concentration of the herba Pogostemonis extract (step (1)) is different, and the mass ratio of herba Pogostemonis powder to water is 1:10, 1:20, 1:40, 1:60, 1:80, 1:150, and 1:300, respectively.
Examples
This example was similar to example 1, except that SDS was added at 0, 50, 100, 200, 400. Mu.L of 100 mg/mL, respectively, in different concentrations (step (3)).
Examples
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. Mu. Mol/mL.
Examples
This example is similar to example 1, except that the stirring temperature (step (5)) is 4, 10, 25, 30, 40, 50 ℃.
Examples
This example is similar to example 1, except that the stirring time (step (7)) is different from 0.5, 1, 2, 4, 6, 8 and h, respectively.
Examples
In the embodiment, the nano silver colloid is prepared from the ageratum extract and the application of the nano silver colloid in the bacteriostasis field, and the silver nano silver colloid obtained in the embodiment 1 is taken for bacteriostasis experiments.
(1) Experiment of inhibition zone
The nano silver colloid in example 1 was dispersed in ultra-pure water to prepare a dispersion with a concentration of 10 mg/mL. Preparing LB plate culture medium, sucking different bacterial suspensions such as colibacillus, staphylococcus aureus, klebsiella pneumococcus, methicillin-resistant staphylococcus aureus, bacillus subtilis, bacillus proteus, pseudomonas aeruginosa and the like, coating on the sterilized plate culture medium, and controlling the bacterial concentration of the bacterial suspension to be about 10 5 cfu, soaking 20 mu l of the dispersion liquid in a 6 mm filter paper sheet, and after sterile air drying, sequentially placing the filter paper sheet in a flat plate culture medium of escherichia coli, staphylococcus aureus, klebsiella pneumoniae, methicillin-resistant staphylococcus aureus, bacillus subtilis, proteus, pseudomonas aeruginosa and the like.
Each pathogen was cultured at 37℃for 18 to 24℃and h (in this example, the time period was 18 to h).
The inhibition zone of 7 pathogenic bacteria is shown in Table 1.
(2) Minimum inhibitory concentration
The nano silver colloid provided by the invention is tested for the Minimum Inhibitory Concentration (MIC) of different indication pathogenic bacteria such as escherichia coli, staphylococcus aureus, klebsiella pneumoniae, methicillin-resistant staphylococcus aureus, bacillus subtilis, bacillus proteus, pseudomonas aeruginosa and the like. The MIC was determined by 2-fold dilution and the experimental procedure was as follows:
picking out monoclonal colony from the culture dish, inoculating into sterilized 50 mL triangular flask containing 10 mL LB liquid medium, and shake culturing in shaking table at 37deg.C and 220 rpm for 6 h;
160 mu L of LB liquid medium is added to each well of 96 empty plates. Then 160 mu L of LB liquid medium with the concentration of 250 mu g/mL of nano silver colloid is added into all No. 1 spaces, 160 mu L to No. 2 spaces are sucked after uniform mixing, and the holes No. 12 are sucked and discarded by analogy. Then 40. Mu.L of the solution containing 10 was added to each well 4 The cultured bacterial solutions of cfu are uniformly mixed, and the bacterial solution is not added in a blank group.
After the treated 96-well plate is placed in a biochemical incubator at 37 ℃ for incubation of 12 h, the OD value of each well bacterial liquid is measured under an enzyme-labeled instrument.
The records were made and the Minimum Inhibitory Concentration (MIC) was calculated as shown in table 1, wherein the smaller the MIC, the better the bacteriostatic effect against this pathogen.
TABLE 1 inhibition of silver colloid nanoparticles on different pathogenic bacteria
Example 8
In the embodiment, the nano silver colloid is prepared from the ageratum extract and the application of the nano silver colloid in the anti-tumor field, and the anti-tumor activity test is performed by taking the nano silver colloid obtained in the embodiment 1.
The MTT method is adopted to resist the tumor activity of the nano silver colloid. The operation steps are as follows:
collecting tumor cells in logarithmic growth phase, and adjusting cell suspension concentration to 1×10 4 Per mL, go to100 mu L of complete medium is added into each well of a 96-well plate, 200 mu L of complete medium is added in a blank way, and CO is placed 2 In an incubator, 37 ℃ and 5% CO 2 Culture 4 h. 100 mu L of nano silver colloid culture medium with different concentrations is added into each hole, 6 gradients are arranged, the final concentration of each hole is 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 100 mu L of whole culture medium is added into the negative control. 3 replicates were set for each gradient, and the culture was continued for 24. 24h and removed. After the completion of the drug action, 40. Mu.L of MTT at a concentration of 5 mg/mL was added to each well, and 4. 4h was cultured. The culture was terminated and the in-well culture solution was gently aspirated. 150. Mu.L of DMSO was added to each well. The shaker was oscillated at low speed for 10min. The OD was then measured by a microplate reader and the absorbance of each well at 490 nm (A). Calculation of cell inhibition ratio: inhibition ratio = (experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value) ×100%. The concentration of the sample at 50% inhibition was IC 50 . IC50 values were calculated and the results are expressed as mean ± standard deviation.
The nano silver colloid of the invention has inhibition effect in activity test of 5 cancer cells, wherein, IC 50 The smaller the value is, the better the inhibition effect of the nano silver colloid provided by the invention on the cancer cells is. The test results are shown in Table 2.
TABLE 2 inhibition of different tumor cells by nano silver colloid
Examples
In the embodiment, the nano silver colloid is prepared from the ageratum extract and the application of the nano silver colloid in the anti-inflammatory field, and the influence of the nano silver colloid obtained in the embodiment 1 on the secretion of inflammatory factors by RAW264.7 stimulated by bacterial endotoxin lipopolysaccharide LPS is taken.
RAW264.7 is stimulated by bacterial endotoxin Lipopolysaccharide (LPS), and the influence of nano silver colloid on the expression of inflammatory factors is detected from the protein level by using an enzyme-linked immunosorbent assay (ELISA) experiment.
Sample preparation and cell experiments were as follows:
(a) Macrophage RAW264.7 is spread in a 6-hole plate, and five groups of NC (blank control), LPS group, low nano silver colloid (1.0 mug/mL), medium nano silver colloid (2.0 mug/mL) group and high nano silver colloid (4.0 mug/mL) group are arranged.
(b) LPS group and each nanosilver gel group were incubated with cells for two hours.
(c) The cells in the LPS group and each nanosilver gel group were stimulated with 1. Mu.g/ml bacterial Lipopolysaccharide (LPS).
(d) After 24h of stimulation, cell supernatants were collected and assayed for TNF- α, IL-6, IL-1β content in different groups of RAW264.7 cells according to ELISA kit protocol.
The foregoing is a further detailed description of the invention in connection with specific embodiments, and it is not intended that the invention be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the invention.
Claims (2)
1. The application of the ageratum nano silver colloid in preparing an anti-tumor medicament is characterized by comprising the following specific steps:
s1, taking fresh wrinkled giant hyssop stems and leaves, washing with double distilled water, airing, crushing, sieving with a 40-mesh sieve, and then mixing with ultra-pure water according to the proportion of 1: adding the extract into water according to the mass ratio of 10-300, carrying out ultrasonic water bath at 60-90 ℃ for 10-60 min, cooling, centrifugally filtering to obtain an ageratum water extract;
s2, adding sodium dodecyl sulfate as a stable dispersing agent into the supernatant herba Agastaches water extract, wherein the final concentration is 0.5-50 mg/mL, then adding a silver nitrate solution to make the final concentration of silver ions be 1-100 mu mol/mL, and then placing the solution in a 20-40 ℃ magnetic stirring reaction for 2-8h to obtain herba Agastaches nano silver colloid solution;
in the step S1, the mass ratio of the herba Agastaches to the ultra-pure water in the herba Agastaches extract is 1:10 to 300 percent;
the final concentration of the added silver nitrate in the step S2 is 1-100 mu mol/mL;
the final concentration of SDS added in the step S2 is 0.5-50 mg/mL;
the nano silver colloid inhibited tumor comprises melanoma, gastric cancer and cervical cancer.
2. The application of ageratum nano silver colloid in preparing antibacterial drugs is characterized in that: the method comprises the following specific steps:
s1, taking fresh wrinkled giant hyssop stems and leaves, washing with double distilled water, airing, crushing, sieving with a 40-mesh sieve, and then mixing with ultra-pure water according to the proportion of 1: adding the extract into water according to the mass ratio of 10-300, carrying out ultrasonic water bath at 60-90 ℃ for 10-60 min, cooling, centrifugally filtering to obtain an ageratum water extract;
s2, adding sodium dodecyl sulfate as a stable dispersing agent into the supernatant herba Agastaches water extract, wherein the final concentration is 0.5-50 mg/mL, then adding a silver nitrate solution to make the final concentration of silver ions be 1-100 mu mol/mL, and then placing the solution in a 20-40 ℃ magnetic stirring reaction for 2-8h to obtain herba Agastaches nano silver colloid solution;
in the step S1, the mass ratio of the herba Agastaches to the ultra-pure water in the herba Agastaches extract is 1:10 to 300 percent;
the final concentration of the added silver nitrate in the step S2 is 1-100 mu mol/mL;
the final concentration of SDS added in the step S2 is 0.5-50 mg/mL;
the pathogenic bacteria inhibited by the nano silver colloid comprise escherichia coli, staphylococcus aureus, klebsiella pneumoniae, methicillin-resistant staphylococcus aureus, bacillus subtilis, bacillus proteus and pseudomonas aeruginosa.
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