CN112471174A - Nano-silver/domiphen bromide composite antibacterial agent and preparation method and application thereof - Google Patents
Nano-silver/domiphen bromide composite antibacterial agent and preparation method and application thereof Download PDFInfo
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- 229960001859 domiphen bromide Drugs 0.000 title claims abstract description 62
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 34
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- OJIYIVCMRYCWSE-UHFFFAOYSA-M Domiphen bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CCOC1=CC=CC=C1 OJIYIVCMRYCWSE-UHFFFAOYSA-M 0.000 title claims abstract 15
- 238000000034 method Methods 0.000 claims abstract description 16
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000001035 drying Methods 0.000 claims description 8
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- 238000005303 weighing Methods 0.000 claims description 6
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- 239000003638 chemical reducing agent Substances 0.000 abstract description 4
- 238000006722 reduction reaction Methods 0.000 abstract description 3
- 239000001522 artemisia absinthium l. herb extract Substances 0.000 abstract description 2
- 229940119569 wormwood extract Drugs 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 53
- BHQQXAOBIZQEGI-UHFFFAOYSA-N methyl 2-chlorobutanoate Chemical compound CCC(Cl)C(=O)OC BHQQXAOBIZQEGI-UHFFFAOYSA-N 0.000 description 49
- 241000588626 Acinetobacter baumannii Species 0.000 description 43
- 239000000243 solution Substances 0.000 description 38
- 241000588724 Escherichia coli Species 0.000 description 36
- 241000191967 Staphylococcus aureus Species 0.000 description 35
- 241000222122 Candida albicans Species 0.000 description 25
- 229940095731 candida albicans Drugs 0.000 description 25
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- YXUPZGKORWTXID-UHFFFAOYSA-N domiphen Chemical group CCCCCCCCCCCC[N+](C)(C)CCOC1=CC=CC=C1 YXUPZGKORWTXID-UHFFFAOYSA-N 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
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- KFSLWBXXFJQRDL-UHFFFAOYSA-N Peracetic acid Chemical compound CC(=O)OO KFSLWBXXFJQRDL-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003214 anti-biofilm Effects 0.000 description 1
- 229940124350 antibacterial drug Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
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- -1 domiphen bromide compound Chemical class 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 1
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- 238000004659 sterilization and disinfection Methods 0.000 description 1
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- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/16—Heavy metals; Compounds thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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- A01N33/12—Quaternary ammonium compounds
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Abstract
The invention discloses a nano-silver/domiphen bromide composite antibacterial agent, a preparation method and application thereof. Firstly, a chemical reduction method is adopted, the wormwood extract is used as a reducing agent to prepare a nano silver solution with a certain concentration, then a domiphen bromide solution with a certain concentration is prepared, and the two solutions are respectively diluted and mixed to obtain the nano silver/domiphen bromide composite antibacterial agent. The invention combines the nano-silver and the domiphen bromide to have the synergistic antibacterial effect, greatly reduces the dosage of the medicament and simultaneously reduces the toxic and side effects of the medicament compared with the single medicament.
Description
Technical Field
The invention belongs to the technical field of nano antibacterial materials, and particularly relates to a nano silver/domiphen bromide composite antibacterial agent, and a preparation method and application thereof.
Background
The types of antibacterial agents circulating on the market are various, but most of the antibacterial agents contain toxic chemical agents, such as hypochlorous acid, peroxyacetic acid, glutaraldehyde and the like, and certain potential safety hazards exist in the use process. Under such circumstances, antibacterial agents which are naturally safe and have broad-spectrum antibacterial activity are highly demanded in the market.
Each of the antibacterial agents has different antibacterial effects on different kinds of bacteria, for example, a hydrophilic antibacterial agent has a high antibacterial efficiency against Escherichia coli, and a lipophilic antibacterial agent has a high antibacterial efficiency against Staphylococcus aureus. In order to exert broad-spectrum antibacterial effect, two or more than two antibacterial agents are compounded by means of physical blending or chemical reaction, so that the antibacterial agents can be used for the full-length, and the aims of improving antibacterial capability and reducing the dosage of the antibacterial agents are fulfilled. Therefore, the synergistic use of antimicrobial agents has been a focus and direction of attention.
The nano antibacterial material is a novel material developed on the basis of inorganic antibacterial materials and nano materials. The nano silver has the advantages of high antibacterial performance, good heat resistance, difficulty in generating drug resistance, high safety and the like, and is a hotspot of research and application in the field of antibacterial materials. Domiphen bromide (named as Xiaodianning) has the advantages of good sterilization effect, small dosage and low toxic and side effects, has the effect of killing gram-positive bacteria, gram-negative bacteria and fungi, and is a broad-spectrum type sterilizing agent with wide application range, convenient use, safety and effectiveness. The antibacterial agent which is combined by the nano silver and the domiphen bromide has not been reported.
Disclosure of Invention
The invention aims to provide a preparation method of a nano-silver/domiphen bromide composite antibacterial agent, which has simple steps.
The invention also aims to provide the composite antibacterial agent prepared by the method, which has a good antibacterial effect at a low dosage.
The invention also aims to provide the application of the composite antibacterial agent in the aspect of sanitary antibacterial products.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: a preparation method of a nano-silver/domiphen bromide composite antibacterial agent comprises the following steps:
(1) respectively weighing a certain amount of silver nitrate and a certain amount of folium artemisiae argyi extract, dissolving the silver nitrate and the folium artemisiae argyi extract in water, stirring the mixture in a water bath at the temperature of 95-100 ℃ for 30-60 min to prepare a nano silver solution with a certain concentration, and drying and storing the nano silver solution in the dark for later use;
(2) weighing a certain amount of domiphen bromide, dissolving the domiphen bromide in water to prepare a domiphen bromide solution with a certain concentration, and drying and storing the domiphen bromide solution in a dark place for later use;
(3) and (3) mixing the nano silver solution obtained in the step (1) and the domiphen bromide solution obtained in the step (2) in proportion to obtain the nano silver/domiphen bromide composite antibacterial agent.
Preferably, the concentration of the silver nitrate in the step (1) is 1.5-1.8 mg/mL, and the concentration of the folium artemisiae argyi extract is 1-3 mg/mL.
Preferably, the concentration of the nano silver solution in the step (3) is not lower than 0.5ppm, and the concentration of the domiphen bromide solution is not lower than 1 ppm.
Preferably, the volume ratio of the nano silver solution to the domiphen bromide solution in the step (3) is 1-99: 99 to 1.
The invention also provides the composite antibacterial agent prepared by the preparation method.
The invention also provides the application of the composite antibacterial agent in the aspect of sanitary antibacterial products.
The composite antibacterial agent prepared by the invention has obvious antibacterial effects on acinetobacter baumannii, staphylococcus aureus, escherichia coli and candida albicans, and can realize good antibacterial effect under the condition of lower dosage; and because the dosage is reduced, the toxic and side effects of the nano silver and the domiphen bromide are greatly reduced, and the composite antibacterial agent can be used for sanitary antibacterial products.
Compared with the prior art, the invention has the following beneficial effects:
1. the nano-silver and domiphen bromide compound drug has a synergistic antibacterial effect, so that compared with the single drug, the dosage of the drug is greatly reduced, and the toxic and side effects of the drug are reduced;
2. the composite antibacterial agent prepared by the invention has obvious antibacterial effects on acinetobacter baumannii, staphylococcus aureus, escherichia coli and candida albicans, and can realize good antibacterial effect under the condition of lower dosage.
Drawings
FIG. 1 shows the results of the antibacterial ring test of nano-silver and domiphen bromide: and the results of the bacteriostatic ring test of the normal saline Control group and the 60ppm nano silver group on (A) staphylococcus aureus, (B) escherichia coli and (C) acinetobacter baumannii. Results of the zone of inhibition tests on (D) Staphylococcus aureus, (E) Escherichia coli, and (F) Acinetobacter baumannii in the saline Control group and the 60ppm domiphen group. The right arrow indicates the 60ppm antimicrobial group and the left arrow indicates the physiological saline control group.
FIG. 2 shows MIC test results of (A) Acinetobacter baumannii, (B) Staphylococcus aureus, (C) Escherichia coli and (D) Candida albicans by Control group and nano-silver group with different concentrations.
FIG. 3 shows MIC test results of Acinetobacter baumannii (A), Staphylococcus aureus (B), Escherichia coli (C) and Candida albicans (D) in Control group and domiphen bromide group with different concentrations.
FIG. 4 shows the MBC test results of Control group and nano-silver group with different concentrations for (A) Acinetobacter baumannii, (B) Staphylococcus aureus, (C) Escherichia coli and (D) Candida albicans.
FIG. 5 shows the MBC test results of (A) Acinetobacter baumannii, (B) Staphylococcus aureus, (C) Escherichia coli and (D) Candida albicans in Control group and domiphen bromide groups with different concentrations.
FIG. 6 shows the results of the Control group and the nano-silver groups with different concentrations on the inhibition of the activity of the biological membranes of (A) Acinetobacter baumannii, (C) Staphylococcus aureus, (E) Escherichia coli and (G) Candida albicans.
FIG. 7 shows the results of the Control group and the domiphen bromide groups at different concentrations on the biofilm activity inhibition of Acinetobacter baumannii, Staphylococcus aureus (D), Escherichia coli (F) and Candida albicans (H).
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
The starting materials and reagents used in the following examples are all commercially available products unless otherwise specified.
Example 1: preparation of Nano silver and domiphen bromide solution
1.1 preparation of nano silver solution: weighing silver nitrate 8.5mg and folium Artemisiae Argyi extract 10mg, dissolving in 5mL distilled water, boiling water bath at 100 deg.C for 40min to obtain 1000ppm nano silver solution, and drying and storing in dark for use.
The prepared 1000ppm nano silver solution is filtered by a 0.22 mu m microporous filter membrane by adopting a chemical reduction method and a wormwood extract as a reducing agent, and the particle size is about 100nm measured by a particle size analyzer.
Preparation of 1.2 degree mirifene solution: weighing 10mg of domiphen bromide, pouring the domiphen bromide into a 10mL volumetric flask, fixing the volume with distilled water, preparing a domiphen bromide solution with the concentration of 1000ppm, and drying and storing the solution in dark for later use.
Example 2: antibacterial performance test of combined use of nano silver and domiphen bromide
2.1 bacteriostatic Ring test
The bacteriostat is continuously dissolved and diffused by the agar to form different concentration gradients so as to show the bacteriostasis. The experiment judges whether the bacteriostatic ability is possessed or not according to the size of the bacteriostatic ring.
1) Preparing the bacteriostatic tablets: a sterile and dried filter paper sheet was taken. Dripping 20 μ L of nano silver/domiphen bromide solution with different concentrations into each piece, placing the filter paper piece in a clean sterile plate, opening the cover, and baking in a warm box (37 ℃), or naturally drying at room temperature for later use.
5mm in diameter and no more than 4mm in thickness, one set of every 4 pieces.
2) Preparation of negative control sample: and (4) taking a sterile dry filter paper sheet, dripping 20 mu L of sterile distilled water into each sheet, and drying for later use.
And (3) preparing a negative control sample into a sample sheet with the same size as the test group by taking a sample which is made of the same material and does not contain nano silver.
3) Inoculation of test bacteriaThe method comprises the following steps: dipping with sterile cotton swab to 5 × 10 concentration5CFU/mL～5×106CFU/mL Acinetobacter baumannii suspension is evenly smeared on the surface of a nutrient agar culture medium plate for 3 times. For each application 1 time, the plate should be rotated 60 °, and finally the cotton swab is applied around the edge of the plate for one revolution. The plate was covered and dried at room temperature for 5 min.
4) And (3) pasting and placing an antibacterial agent sample piece: each test is stuck with 1 infectious bacterium plate, and each plate is stuck with 4 test sample plates and 1 negative control sample plate, and the number of the negative control sample plates is 5. A sample was taken with sterile forceps and placed on the surface of the plate. The distance between the centers of the various pieces is more than 25mm, and the distance between the centers of the various pieces and the periphery of the flat plate is more than 15 mm. After the sample is placed, the sample is lightly pressed by using sterile tweezers to be tightly attached to the surface of the flat plate. The plate is covered, the plate is placed in an incubator at 37 ℃, and the result is observed after the plate is cultured for 16 to 18 hours. The diameter of the antibacterial ring (including the patch) was measured with a vernier caliper and recorded.
When the inhibition zone is measured, the inhibition zone which is uniform and completely aseptically grows is selected for measurement. The diameter of the catheter is measured by taking the outer edge of the bacteriostatic ring as a boundary.
If the diameter of the bacteriostatic ring is larger than 7mm, the bacteriostatic ring is judged to have bacteriostatic action; if the diameter of the antibacterial ring is less than or equal to 7mm, judging that the antibacterial ring has no antibacterial effect; the negative control group should have no bacteriostatic ring, otherwise the test is invalid.
The MIC test of nano-silver for Staphylococcus aureus (ATCC 6538), Escherichia coli (8099), Acinetobacter baumannii (ATCC19606) and the MIC test of domiphen for the above three bacteria were performed as described above.
TABLE 1 diameters of nanosilver and domiphen bromide inhibiting rings
As can be seen from fig. 1, compared with the Control group, the bacteriostatic rings of the 60ppm nano-silver group on staphylococcus aureus (ATCC 6538), escherichia coli (8099) and acinetobacter baumannii (ATCC19606) are significantly increased, which indicates that the bacteriostatic effect of the 60ppm nano-silver on staphylococcus aureus (ATCC 6538), escherichia coli (8099) and acinetobacter baumannii (ATCC19606) is significant; in addition, compared with the Control group, the 60ppm domiphen bromide group has larger inhibition ring to staphylococcus aureus (ATCC 6538), escherichia coli (8099) and acinetobacter baumannii (ATCC19606), which shows that the 60ppm domiphen bromide has obvious inhibition effect to staphylococcus aureus (ATCC 6538), escherichia coli (8099) and acinetobacter baumannii (ATCC 19606).
The results of the diameter measurement of the inhibition ring are shown in table 1, and the diameters of the inhibition ring of the 60ppm nano silver group to staphylococcus aureus (ATCC 6538), escherichia coli (8099) and acinetobacter baumannii (ATCC19606) are respectively 19 +/-2 mm, 16 +/-1 mm and 13 +/-1 mm; the diameters of the inhibition rings of the 60ppm domiphen bromide group to staphylococcus aureus (ATCC 6538), escherichia coli (8099) and acinetobacter baumannii (ATCC19606) are respectively 12 +/-3 mm, 10 +/-3 mm and 11 +/-3 mm.
The experimental results show that the nano-silver and the domiphen bromide have good antibacterial effects on common pathogenic bacteria such as staphylococcus aureus (ATCC 6538), escherichia coli (8099), acinetobacter baumannii (ATCC19606) and the like.
2.2 Single drug susceptibility test
The Minimum Inhibitory Concentration (MIC) of the nano-silver is determined according to a method prepared by the Clinical and Laboratory Standards Institute (CLSI), and the method comprises the following steps:
(1) preparation of 0.5 McLeod turbidimetry (bacterial concentration of about 1X 10)8CFU/mL) of Acinetobacter baumannii (ATCC19606) solution, diluted 100-fold with sterile distilled water (suspension concentration of about 1X 10)6CFU/mL)；
(2) Preparing 7 sterilized test tubes and numbering, adding 4mL of MueLLER-Hinton broth (MHB) into a No. 1 tube, equivalently adding MHB1mL into a No. 2-7 test tube, adding 64 mu L of 1000ppm nano-silver solution into the No. 1 tube, shaking and uniformly mixing, taking 1mL of the No. 1 test tube, adding the 1mL of the No. 2 test tube into the No. 6 test tube, diluting the mixture to the No. 6 test tube by the method in a multiple ratio, sucking 1mL of the No. 6 test tube, discarding the 1mL of the No. 1-7 test tube, and taking the No. 7 test tube as a negative control tube;
(3) respectively adding 50 mu L of bacterial liquid into the 7 test tubes, and putting the test tubes into a constant-temperature incubator at 37 ℃ for culturing for 24 hours;
(4) comparing the turbid conditions of the liquid in each test tube, wherein the lowest nano-silver concentration without bacterial growth (obvious clarity) is the MIC of the nano-silver acting on the acinetobacter baumannii;
the MIC test of nano-silver against Staphylococcus aureus (ATCC 6538), Escherichia coli (8099), Candida albicans (ATCC10231) and the MIC test of domiphen against the above four test bacteria were performed as described above.
The results of determining MIC of nano-silver and domiphen bromide to Acinetobacter baumannii (ATCC19606), Staphylococcus aureus (ATCC 6538), Escherichia coli (8099) and Candida albicans (ATCC10231) by microdilution method are judged according to CLSI standard, and are all in quality control range.
As shown in fig. 2A, the acinetobacter baumannii bacterial liquid is in a turbid state when no nano silver is added, and is obviously clear when the concentration of the nano silver solution is 2ppm or more, so that it can be seen that the lowest nano silver concentration of 2ppm without bacterial growth (obviously clear) is the MIC of the acinetobacter baumannii acted by nano silver; by analogy with this method, as shown in fig. 2B-2D, MICs of nanosilver acting on staphylococcus aureus, escherichia coli, and candida albicans are 2ppm, and 4ppm, respectively; as shown in FIGS. 3A-3D, the MICs of domiphen bromide against Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Candida albicans were 2ppm, 4ppm, 8ppm, and 4ppm, respectively.
2.2 Combined susceptibility testing
The method for determining the synergistic MIC of the nano-silver and domiphen bromide combination by using a micro chessboard dilution method comprises the following specific steps:
(1) preparation of 0.5 McLeod turbidimetry (bacterial concentration of about 1X 10)8CFU/mL) of Acinetobacter baumannii bacterial liquid, and diluting the Acinetobacter baumannii bacterial liquid by 100 times by using distilled water;
(2) diluting the nano silver and domiphen bromide with sterile MHB at a ratio of 8 times to the maximum MIC concentration, taking 6 dilutions, respectively taking 50 mu L of the dilutions and respectively arranging the 50 mu L of the dilutions on rows and columns of a sterile 96-well plate, and then adding 100 mu L of bacterial liquid into the sterile 96-well plate to ensure that the final bacterial inoculation amount is 5 multiplied by 105CFU/mL, the final drug concentration is 2 times MIC, and the mixture is placed into a constant-temperature incubator at 37 ℃ for 24 hours;
(3) the lowest drug concentration without bacterial growth (clearly clear) was MIC';
(4) by calculating partial bacteriostatic concentration index
The FIC is less than or equal to 0.5, the FIC is less than or equal to 1 when the FIC is less than or equal to 0.5, the FIC is less than or equal to 1 and the FIC is less than 2 and the FIC is less than or equal to 2 respectively represent synergy, addition, irrelevance and antagonism.
The steps of the synergistic MIC test of staphylococcus aureus, escherichia coli and candida albicans are the same as above.
TABLE 2 Combined susceptibility testing of nanosilver and domiphen bromide
The chessboard microdilution method is adopted to determine the combined drug sensitivity test of the nano silver and the domiphen under different concentrations to four strains, and FIC judgment results are expressed as four effects of synergy, addition, antagonism and independence, and specific data are shown in Table 2.
As shown in Table 2, when the nano-silver and domiphen bromide are used in combination, the synergistic effect (FIC is less than or equal to 0.5) is achieved on the four bacteria, and the synergistic effect on killing staphylococcus aureus (ATCC 6538), escherichia coli (8099) and candida albicans (ATCC10231) is particularly prominent. The results show that when the nano-silver and the domiphen bromide are jointly applied, the nano-silver and the domiphen bromide have synergistic effect on the antibacterial effects of acinetobacter baumannii (ATCC19606), staphylococcus aureus (ATCC 6538), escherichia coli (8099) and candida albicans (ATCC10231), can enhance the antibacterial effect of antibacterial drugs, and not only can reduce the drug dosage, but also can reduce the toxic and side effects of the drugs.
2.3 minimum bactericidal concentration test (MBC)
(1) Preparation of 0.5 McLeod (bacterial suspension concentration about 1X 10)8CFU/mL) of Acinetobacter baumannii (ATCC19606) solution, diluted 100-fold with sterile distilled water (suspension concentration of about 1X 10)6CFU/mL)；
(2) Setting the concentration of the test drug according to the MIC of bacteria, preparing 8 sterilized test tubes and numbering, adding 2mL of MueLLER-Hinton broth (MHB) into a No. 1 tube, equivalently adding 1mL of MHB into a No. 2-8 test tube, and adding an appropriate amount of 1000ppm nano-silver solution into the No. 1 tube;
(3) after shaking and mixing uniformly, taking 1mL from the test tube No. 1, adding the 1mL into the test tube No. 2, diluting to the test tube No. 6 according to the method by a multiple ratio, sucking 1mL by the test tube No. 7, discarding, putting the test tube No. 1-8 with the nano-silver concentrations of 64MIC, 32MIC, 16MIC, 8MIC, 4MIC, 2MIC, MIC and 0ppm into a constant-temperature incubator at 37 ℃ for incubation for 18-24 h;
(4) after 18-24 h, dipping a proper amount of solution in the test tube by using a sterile cotton swab, uniformly smearing the solution on a culture dish containing a TSA culture medium, and putting the culture dish into a constant-temperature incubator at 37 ℃ for culturing for 18-24 h;
(5) after 18h to 24h, the number of colonies in the petri dish representing different drug concentrations was observed, with a minimum concentration of less than 5 colonies as MBC.
The MBC test of nano-silver on staphylococcus aureus (ATCC 6538), escherichia coli (8099) and candida albicans (ATCC10231) and the MBC test of domiphen on the above four bacteria are performed in the same manner.
And (3) uniformly smearing the solution in the test tube with the MIC larger than that of the test tube on a culture dish containing the TSA culture medium, and incubating for 24h to obtain test results as shown in figures 4 and 5, wherein the minimum concentration of colony count smaller than 5 or sterile colony growth is MBC.
As shown in FIG. 4A, the Control group had a large amount of Acinetobacter baumannii growth, and the culture dishes treated by the 8ppm nanosilver-treated group grew aseptically, compared to the Control group, and thus, the MBC of nanosilver to Acinetobacter baumannii was 8 ppm; by analogy with this method, as shown in fig. 4B-4D, the MBC of the nanosilver acting on staphylococcus aureus, escherichia coli, and candida albicans was 32ppm, and 32ppm, respectively; as shown in FIGS. 5A-5D, the MBC of domiphen bromide, Staphylococcus aureus, Escherichia coli, Candida albicans was 8ppm, 16ppm, 256ppm, and 512ppm, respectively.
Therefore, the nano silver and the domiphen bromide can generate good killing effect on the acinetobacter baumannii, and the killing effect on staphylococcus aureus, escherichia coli and candida albicans is inferior.
2.4 anti-biofilm formation mechanism of Nano silver in combination with domiphen bromide
2.4.1 biofilm eradication concentration-2 test (BEC-2)
Biofilm eradication concentration-2: minimum concentration of compound that results in a 2-fold decrease in biofilm metabolic activity.
(1) Preparation of 0.5 McLeod (bacterial suspension concentration about 1X 10)8CFU/mL) of Acinetobacter baumannii solution, diluted 100-fold with MHB (suspension concentration of about 1X 10)6CFU/mL)；
(2) Adding 100 mu L of Acinetobacter baumannii diluted bacterial liquid into an aseptic 96-well plate experimental hole, putting the diluted bacterial liquid into a constant-temperature incubator at 37 ℃ for incubation for 1h, and enabling the biofilm to be adhered to the hole;
(3) absorbing and removing bacteria liquid, and washing with PBS to remove floating bacteria;
(4) diluting the nano-silver by using sterile MHB multiple ratio from 4 times of MIC concentration at most, taking 7-9 dilutions, sequentially adding 100 mu L of the diluted nano-silver into a sterile 96-well plate according to the drug concentration from small to large, and putting the diluted nano-silver into a constant-temperature incubator at 37 ℃ for incubation for 18-24 h;
(5) absorbing the nano silver solution, washing with PBS to remove residual nano silver solution, and adding 100 μ L methanol for fixation for 15 min;
(6) absorbing methanol, air drying, and dyeing crystal violet for 10 min;
(7) absorbing the crystal violet solution, and washing for 3 times by PBS;
(8) measuring OD value by an enzyme-labeling instrument 595nm, wherein the minimum nano-silver concentration of 1/2 which is less than the OD value of the negative control column is that the nano-silver acts on BEC-2 of the acinetobacter baumannii;
the BEC-2 test of the nano-silver on staphylococcus aureus, escherichia coli and candida albicans and the BEC-2 test of the domiphen bromide on the four bacteria have the same steps.
As shown in fig. 6A, the activity of the nano-silver treated group biofilm at a concentration of 1MIC and below was hardly changed compared to the Control group, while the activity of the nano-silver treated group biofilm at a concentration of 2MIC and above was significantly reduced to 50% or less, and thus it can be seen that BEC-2 of nano-silver to acinetobacter baumannii (the minimum concentration of the compound causing a 2-fold reduction in the metabolic activity of the biofilm) was 2 MIC; by analogy with this method, as shown in fig. 6C, 6E, and 6G, BEC-2 of nano-silver against staphylococcus aureus, escherichia coli, and candida albicans is 2MIC, 1/4MIC, and 1MIC, respectively; as shown in FIGS. 7B, 7D, 7F, and 7H, BEC-2 of domiphen bromide to Acinetobacter baumannii, Staphylococcus aureus, Escherichia coli, and Candida albicans was 2MIC, 1/2MIC, 1MIC, and 1MIC, respectively.
2.4.2 combination of BEC-2
In order to determine the synergistic interaction between nano-silver and domiphen bromide on the influence of the metabolic activity of the biological capsule, the test method is improved on the basis of the reference.
(1) Preparation of 0.5 McLeod (bacterial suspension concentration about 1X 10)8CFU/mL) of Acinetobacter baumannii solution, diluted 100-fold with MHB (suspension concentration of about 1X 10)6CFU/mL)；
(2) Adding 100 mu L of Acinetobacter baumannii diluted bacterial liquid into a sterile 96-well plate, and putting the diluted bacterial liquid into a constant-temperature incubator at 37 ℃ for incubation for 1h to enable the biofilm to be adhered to the bottom of the well;
(3) absorbing and removing bacteria liquid, and washing with PBS to remove floating bacteria;
(4) diluting the nano silver and the domiphen bromide with sterile MHB at the highest concentration of 4 times BEC-2, taking 6 dilutions, respectively taking 50 mu L of the dilutions, respectively arranging the dilutions on rows and columns of a sterile 96-pore plate, and putting the dilutions into a 37 ℃ constant-temperature incubator for culturing for 24 hours;
(5) absorbing the nano silver solution, washing with PBS to remove residual nano silver solution, and adding 100 μ L methanol for fixation for 15 min;
(6) absorbing methanol, air drying, and dyeing crystal violet for 10 min;
(7) absorbing the crystal violet solution, and washing for 3 times by PBS;
(8) measuring OD value by an enzyme-labeling instrument 595nm, wherein the minimum nano-silver concentration of 1/2 which is less than the OD value of the negative control column, namely BEC-2 of the Acinetobacter baumannii acted by nano-silver is marked as BEC-2' of the Acinetobacter baumannii acted by the drug;
(9) by calculating partial bacteriostatic concentration index
The FIC is less than or equal to 0.5, the FIC is less than or equal to 1 when the FIC is 1 and the FIC is 2 when the FIC is 2 and less than or equal to 2 respectively represent synergy, addition, irrelevance and antagonism;
the synergic BEC-2 test procedure of Staphylococcus aureus, Escherichia coli and Candida albicans is the same as above.
TABLE 3 inhibition of biofilm metabolic activity by nanosilver and domiphen bromide
As shown in Table 3, when the nano-silver and domiphen bromide are used in combination, the synergistic inhibition effect on the biofilms of the above four bacteria is remarkable, and the synergistic inhibition effect on the biofilms of Acinetobacter baumannii (ATCC19606), Escherichia coli (8099) and Candida albicans (ATCC10231) is particularly remarkable.
Example 3: nano silver/domiphen bromide composite antibacterial agent
Diluting the nano-silver solution prepared in the example 1 to 0.5ppm, diluting the domiphen bromide solution to 1ppm, and then mixing the nano-silver solution and the domiphen bromide solution according to the volume ratio of 1-99: 99-1, and obtaining the nano silver/domiphen bromide composite antibacterial agent. Under the concentration, the composite antibacterial agent has remarkable antibacterial effects on acinetobacter baumannii, staphylococcus aureus, escherichia coli and candida albicans.
Claims (5)
1. A preparation method of a nano-silver/domiphen bromide composite antibacterial agent is characterized by comprising the following steps:
(1) respectively weighing a certain amount of silver nitrate and a certain amount of folium artemisiae argyi extract, dissolving the silver nitrate and the folium artemisiae argyi extract in water, stirring the mixture in a water bath at the temperature of 95-100 ℃ for 30-60 min to prepare a nano silver solution with a certain concentration, and drying and storing the nano silver solution in the dark for later use;
(2) weighing a certain amount of domiphen bromide, dissolving the domiphen bromide in water to prepare a domiphen bromide solution with a certain concentration, and drying and storing the domiphen bromide solution in a dark place for later use;
(3) and (3) mixing the nano silver solution obtained in the step (1) and the domiphen bromide solution obtained in the step (2) in proportion to obtain the nano silver/domiphen bromide composite antibacterial agent.
2. The preparation method of the nano-silver/domiphen bromide composite antibacterial agent according to claim 1, wherein in the step (1), the concentration of silver nitrate is 1.5-1.8 mg/mL, and the concentration of the folium artemisiae argyi extract is 1-3 mg/mL.
3. The method for preparing a nano-silver/domiphen bromide composite antibacterial agent according to claim 1, wherein the concentration of the nano-silver solution in step (3) is not lower than 0.5ppm, and the concentration of the domiphen bromide solution is not lower than 1 ppm.
4. The composite antibacterial agent prepared by the preparation method according to claim 1.
5. Use of the composite antimicrobial agent of claim 4 in the manufacture of a sanitary antimicrobial article.
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| CN111149816A (en) * | 2020-01-19 | 2020-05-15 | 中南民族大学 | Preparation method and application of nano-silver from Chinese mugwort water extract |
| CN111184757A (en) * | 2020-03-20 | 2020-05-22 | 蒋成聚 | Plant folium artemisiae argyi extract and silver ion compound bactericide and preparation method thereof |
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| CN111184757A (en) * | 2020-03-20 | 2020-05-22 | 蒋成聚 | Plant folium artemisiae argyi extract and silver ion compound bactericide and preparation method thereof |
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| MARÍA DEL PILAR RODRÍGUEZ-TORRES 等，: "Artemisia absinthium-based silver nanoparticles antifungal evaluation against three Candida species", 《MATERIALS RESEARCH EXPRESS》 * |
| N KHATOON 等: "Mode of action and anti-Candida activity of Artemisia annua mediated-synthesized silver nanoparticles", 《JOURNAL DE MYCOLOGIE MEDICALE》 * |
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