CN118420694B - Baicalin-Mn complex not easy to cause bacterial drug resistance and preparation method thereof - Google Patents
Baicalin-Mn complex not easy to cause bacterial drug resistance and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a baicalin-Mn complex which is not easy to cause bacterial drug resistance and a preparation method thereof, and the preparation method of the baicalin-Mn complex which is not easy to cause bacterial drug resistance comprises the following steps: accurately weighing a certain amount of baicalin pure product and MnCl 2 compound, placing the baicalin pure product and the MnCl 2 compound into a flask, adding distilled water, uniformly stirring, reacting for 2-3 h at 100 ℃, and concentrating under reduced pressure until the mixture is dried. The baicalin-Mn complex provided by the invention is not easy to induce bacteria to generate drug resistance, and also has a certain reversing effect on bacteria resistant to certain antibiotics, has obvious killing effect on various bacteria including staphylococcus aureus, escherichia coli and pseudomonas aeruginosa, is small in minimum sterilization concentration and good in sterilization effect by various ways, does not need high temperature, high pressure and complex instrument and equipment, is simple in method, easy to operate, does not use toxic and harmful chemical reagents, and has no potential safety hazard.
Description
Technical Field
The invention relates to the technical field of antibacterial medicines, in particular to a baicalin-Mn complex which is not easy to cause bacterial drug resistance and a preparation method thereof.
Background
Under normal conditions, different bacteria have sensitivity to different antibacterial drugs, for example, most gram positive bacteria are sensitive to broad-spectrum antibiotic drugs, after different antibacterial drugs are used for different bacteria, the bacteria in the body have high sensitivity to the drugs, the drugs can kill the bacteria more quickly, but the biological film of the bacteria in the body has certain drug resistance effect along with the increase of the drug dosage and the extension of the use time when the antibiotics are used, and the extreme environment outside the film causes heterogeneity of the bacteria in the film along with the continuous stimulation of the drugs to the bacteria, so that the formation of bacterial resistance/drug tolerance can be promoted.
Bacterial resistance (RESISTANCE TO DRUG), also known as drug resistance, refers to the phenomenon that bacteria are insensitive to antibacterial agents, and is a special manifestation of bacteria in the survival process. Bacterial resistance can be classified into acquired resistance and natural resistance according to the cause of occurrence. Most of the antibiotics are used to kill most of sensitive strains, and the strains with the drug resistance are continuously propagated, so that the effect of the antibacterial agent is obviously reduced or even lost over time.
The harm of bacteria to develop drug resistance mainly comprises the following aspects:
1. After the recovery time of the illness is prolonged and bacteria generate drug resistance, the infected human body can cause the conventional antibiotics to have poor treatment effect, the medicine is required to be regulated under the guidance of doctors, the drug sensitivity test is perfected, the antibiotics sensitive to drug resistant bacteria are screened for treatment, the situation of prolonged recovery period of the illness is further caused, and if the sensitive antibiotics are not screened, serious threat can be brought to life health;
2. The dosage and the type of the drug are increased, for the infection of drug-resistant bacteria, a certain antibiotic is singly adopted, so that a good treatment effect is often not achieved, two to three or more antibiotics are often required to be used for treatment in combination, the toxicity caused by the drug is increased due to the increase of the dosage, the probability of adverse reaction is often higher, a certain adverse effect is caused on life health, and super drug-resistant bacteria resistant to various antibiotics can also be generated;
3. Other complicated infections are caused, and since the drug-resistant bacteria are difficult to be removed by antibiotics, the drug-resistant bacteria may invade other parts of the body, cause complicated infections and serious infections of other tissues, such as sepsis, intracranial infections and the like, endanger life health, and even appear in a non-drug treatable tragic state.
Therefore, the development of new antibiotic drugs with good antibacterial and bactericidal effects and difficult bacterial resistance is a permanent struggle goal for medical researchers.
Disclosure of Invention
The invention aims to provide a baicalin-Mn complex which is not easy to cause bacterial drug resistance and a preparation method thereof, so as to increase the types of antibacterial drugs, provide new drug selection for patient antibacterial drugs, improve the sensitivity of bacteria to antibacterial drugs and reduce the occurrence of bacterial drug resistance.
In order to achieve the above purpose, the invention provides a baicalin-Mn complex which is not easy to cause bacterial drug resistance and a preparation method thereof, and the preparation method of the baicalin-Mn complex which is not easy to cause bacterial drug resistance comprises the following steps: accurately weighing a certain amount of baicalin compound pure product and MnCl 2 compound, placing the pure product and the MnCl 2 compound into a flask, adding distilled water, uniformly stirring, reacting for 2-3 h at 100 ℃, and concentrating under reduced pressure until the pure product and the MnCl 2 compound are dried.
Preferably, the amounts of baicalin and Mn 2+ compound are weighed to ensure that the molar mass ratio of baicalin to Mn 2+ is 4:1-4:3, the flask is a round-bottom flask, and the total liquid amount added is not more than half of the volume of the flask.
Preferably, distilled water is added in an amount such that the final concentration of baicalin is 2mmol/L, the stirring speed is 200-400rpm/min,
Preferably, the decompression spin drying is completed by a rotary evaporator, the initial temperature of the decompression spin drying is 60 ℃, the rotating speed is 60r/min, the pressure is 150mbar, the subsequent pressure and the rotating speed are kept unchanged, and the pressure is reduced by 20mbar every 5 min.
A baicalin-Mn complex which is not easy to cause bacterial drug resistance and prepared by the preparation method.
The application of baicalin-Mn complex which is not easy to cause bacterial drug resistance in inhibiting and killing bacteria is provided.
Therefore, the baicalin-Mn complex which is not easy to cause bacterial drug resistance and the preparation method thereof provided by the invention have the following specific technical effects:
(1) The baicalin-Mn complex provided by the invention is not easy to cause bacterial drug resistance, and can also have certain reversal effect on bacteria resistant to certain antibiotics;
(2) The baicalin-Mn complex provided by the invention has obvious killing effect on various bacteria, including staphylococcus aureus, escherichia coli and pseudomonas aeruginosa;
(3) The baicalin-Mn complex provided by the invention damages bacteria by various ways such as damaging cell membranes, destroying the integrity and the thallus form structure of bacterial cell walls, inducing bacteria to generate a large amount of ROS, and the like, and has small minimum sterilization concentration and good sterilization effect, and the minimum sterilization concentration on staphylococcus aureus is 0.4mmol/L;
(4) The preparation method of the baicalin-Mn complex provided by the invention does not need high-temperature high-pressure and complex instruments and equipment, is simple and easy to operate, does not use toxic and harmful chemical reagents, and has no potential safety hazard.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments of the present invention will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of the ultraviolet-visible absorption spectra of aqueous BC and BCM solutions;
FIG. 2 is a graph showing the exact molecular weight and isotope distribution comparison of BCM;
FIG. 3 is the experimental results of BCM inhibition against 3 bacteria;
FIG. 4 is the effect of BCM on Staphylococcus aureus growth curve;
FIG. 5 is a graph of the bactericidal kinetics of BCM against Staphylococcus aureus;
FIG. 6 is a scanning electron microscope observation of the morphology of Staphylococcus aureus before and after BCM treatment;
FIG. 7 is a fluorescence microscope image of Staphylococcus aureus after BCM treatment;
FIG. 8 is the effect of BC and BCM on the release of AKP activity in bacteria;
FIG. 9 is the effect of BCM on the ROS content in Staphylococcus aureus bacteria;
FIG. 10 is a graph showing resistance and reverse resistance of Staphylococcus aureus to BCM and 3 antibiotics.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
In order to make the objects, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application will be clearly and completely described below through the accompanying drawings and examples. The following detailed description is of embodiments, and is intended to provide further details of the application. Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The baicalin used in the examples is produced by Chengdu Biotechnology Co., ltd, HPLC purity is not less than 98%, mnCl 2 is produced by Aladin Co., ltd. In the United states, a scanning electron microscope is produced by Riligaku corporation, model SU8010, a fluorescence cell imager is produced by BIO-RAD Co., model ZOE TM, an AKP kit is produced by Nanjing Biotechnology institute, a fluorescence spectrophotometer is produced by Gangdong technology Co., model F-320, a constant temperature heating magnetic stirrer is produced by Heidolph Co., ltd. In the Gongyi city, model DF-101S, and a rotary evaporator is produced by Heidolph Co., germany, model Hei-VAP.
The final concentrations of BCM in the examples are based on the molar mass of baicalin added during synthesis.
Example 1
The preparation method of the baicalin-Mn complex (BCM) which is not easy to cause bacterial drug resistance comprises the following steps:
(1) 125.84mg of MnCl 2 is accurately weighed and dissolved in 10mL of distilled water, 300 mu L of the solution is sucked and added into a round-bottom flask, 17.86mg of Baicalin (BC) pure product and 20mL of distilled water are sequentially added into the round-bottom flask, and the solution is uniformly stirred at a speed of 200 rpm/min.
(2) Heating the round bottom flask to 100 ℃ on a constant temperature heating magnetic stirrer, condensing and refluxing, continuously heating and stirring the round bottom flask on the constant temperature magnetic stirrer for 2 hours during the period, concentrating the round bottom flask on a rotary evaporator under reduced pressure until the round bottom flask is dry after the reaction is finished, obtaining BCM, and rotating the round bottom flask at the initial temperature of 60 ℃ under reduced pressure, wherein the rotating speed of 60r/min is 150mbar, the subsequent pressure and the rotating speed are kept unchanged, and the pressure is reduced by 20mbar every 5min.
Example two
The preparation method of the baicalin-Mn complex which is not easy to cause bacterial drug resistance is exactly the same as that of the first embodiment, except that 100 mu LMnCl 2 of aqueous solution is sucked in the step (1), and the stirring speed is 300rpm/min.
Example III
The preparation method of the baicalin-Mn complex which is not easy to cause bacterial drug resistance is exactly the same as that of the first embodiment, except that 200 mu LMnCl 2 of aqueous solution is sucked in the step (1), the stirring speed is 400rpm/min, and the reaction time in the step (2) is 3h.
Effect example 1
Structure characterization of BCM
1ML of the reaction solution is taken out of the non-spin-dried BCM reaction solution in the first embodiment to 10mL of distilled water while the reaction solution is hot, the reaction solution is diluted to obtain BCM diluent with the concentration of 2mmol/L, 17.86mg of BC is weighed into a round-bottom flask, 20mL of distilled water is added, the temperature is 100 ℃ and the reaction solution is heated for 2 hours, and 1mL of the reaction solution is taken out to 10mL of distilled water while the reaction solution is hot, and the concentration of 2mmol/L. The ultraviolet-visible absorption spectrum is obtained by scanning the 2mLBCM dilution and BC solution at the wavelength of 250nm-500nm, respectively, as shown in figure 1.
In addition, 3mL BCM diluent is taken and centrifuged at 8000r/min for 5min, supernatant is detected by ESI-TOF MS, an infusion sample injection mode is adopted, the mass detection range is 100-1500, the drying air flow speed is 800L/h, the temperature is 500 ℃, the detection result is shown in Table 1, the accurate molecular weight and isotope distribution comparison result is shown in FIG. 2, wherein part A in FIG. 2 is a theoretical isotope distribution diagram of C 21H17O11 Mn, part B is a measured isotope distribution diagram of C 21H17O11 Mn, part C is a theoretical isotope distribution diagram of C 42H35O22 Mn, and part D is a measured isotope distribution diagram of C 42H35O22 Mn.
Effect example two
Determination of Minimum Bactericidal Concentration (MBC) of BCM
Staphylococcus aureus, escherichia coli and pseudomonas aeruginosa are inoculated on a solid culture medium, after the culture is carried out for 24 hours at 37 ℃, single bacterial colony is selected to be put into TSB liquid culture solution, shake culture is carried out until logarithmic phase, and fresh TSB liquid culture solution is used for dilution to 10 5 CFU/mL.
17.86Mg BC and 125.84mg MnCl 2 are weighed respectively according to the method of example one, BCM is prepared, 2.5mL distilled water containing 20% DMSO is used as mother solution for standby, then distilled water is used for preparing BCM solutions with the concentration of 16.00, 8.00, 4.00, 2.00, 1.00 and 0.50mmol/L, BCM with different concentrations is added into lines 1-3 (total 4 lines) of a sterile 24-hole plate, each hole is 0.4mL, 1.6mL staphylococcus aureus is added into each hole of line 1, 1.6mL escherichia coli is added into each hole of line 2, and 1.6mL pseudomonas aeruginosa is added into each hole of line 3, so that the final concentration of BCM is 3.20, 1.60, 0.80, 0.40, 0.20 and 0.10mmol/L respectively.
After incubation at 37 ℃ for 18 hours, spread on TSB solid medium, removed after further incubation at 37 ℃ for 18 hours, recorded by photographing, and recorded as the lowest bactericidal concentration (MBC) at the lowest concentration without bacterial growth. As a result of 3 times of experiments per group, with BC having 3.20, 1.60, 0.80, 0.40, 0.20, 0.10mmol/L and MnCl 2 having 2.40mmol/L as controls, respectively, the results are shown in FIG. 3, in which BCM has 3.20, 1.60, 0.80, 0.40, 0.20, 0.10mmol/L and BC has 3.20, 1.60, 0.80, 0.40, 0.20, 0.10mmol/L, respectively, from left to right.
Effect example three
Effects on bacterial growth curve
According to the results of effect example II, staphylococcus aureus was selected for the experiment. The BCM prepared in example I was dissolved in 2.5mL of distilled water containing 20% DMSO, diluted with distilled water to a concentration of 0.2, 0.4, 0.8 and 1.6mmol/L, respectively, and added to 1.6mL of a suspension of Staphylococcus aureus having a concentration of 10 5 CFU/mL, respectively, the final concentration of BCM was 0.05, 0.10, 0.20 and 0.40mmol/L, BC and Mn 2+ were used as controls, the final concentrations were 1.60mmol/L and 1.20mmol/L, respectively, and Staphylococcus aureus having a concentration of 10 5 CFU/mL, respectively, was added with 0.4mL of distilled water+1.6 mL as a blank. Incubation at 37 ℃, bacterial growth was measured using an enzyme-labeled instrument (OD 600) at 1, 2,3, 4,5, 6, 7, 8, 10, 12, 14, 16, 18, 20h of incubation for 20h. The results are shown in FIG. 4.
Effect example four
Sterilization kinetics curve for BCM
According to the results of effect example II, staphylococcus aureus was selected for the experiment. The BCM prepared in example I was dissolved in 2.5mL of distilled water containing 20% DMSO, diluted with distilled water to a concentration of 0.2, 0.4, 0.8 and 1.6mmol/L, respectively, and added to 1.6mL of a suspension of Staphylococcus aureus having a bacterial liquid concentration of 10 5 CFU/mL, so that the final concentration of BCM was 0.05, 0.10, 0.20, 0.40mmol/L, and the final concentrations of BC and Mn 2+ were 1.60mmol/L and 1.20mmol/L, respectively, and a blank control group was Staphylococcus aureus having a bacterial liquid concentration of 10 5 CFU/mL, to which 0.4mL of distilled water+1.6 mL was added. Incubation at 37 ℃, removing bacterial solutions at 0, 1,2, 3, 4, 5 and 6h of incubation, diluting with sterile PBS, spreading, culturing at 37 ℃ for 18h, and recording colony numbers. The results are shown in FIG. 5.
Effect example five
Effects on bacterial morphology
And 50mL of staphylococcus aureus in logarithmic phase is centrifuged for 5min at 5000r/min, thalli are collected, the thalli are resuspended in PBS to make OD 600 of the bacterial liquid be 0.3, BCM (the final concentration is 2MBC,0.80 mmol/L) is added into the bacterial suspension, and 0.4mL of distilled water and 1.6mL of staphylococcus aureus with the concentration of 10 5 CFU/mL are used as a blank control group.
Incubation at 37 ℃ for 2h, centrifugation, collection of precipitated cells, washing with PBS, re-centrifugation, collection of cells, 4mL of 2.5% glutaraldehyde resuspension, fixation at 4 ℃ overnight. Washing the cells with PBS to remove glutaraldehyde solution, fixing the cells with 1% osmium acid solution for 1-2h, washing the cells with PBS to remove osmium acid solution, and dehydrating the cells with gradient ethanol solutions (including 30%,50%,70%,80%,90% and 95% ethanol) for 15min each, and 100% ethanol twice for 20min each. Samples were treated with a mixture of ethanol and isoamyl acetate (V/v=1/1) for 30min, and then treated with pure isoamyl acetate for 1h or left overnight. And (5) drying the critical point. And (3) coating and observing the ultrafine morphology of bacteria after BCM treatment under a scanning electron microscope. The morphology and external change results of the BCM treated staphylococcus aureus cell surface were observed by scanning electron microscopy and are shown in fig. 6, wherein part a in fig. 6 is a control group and part B is 2MBC of BCM treated staphylococcus aureus.
Effect example six
Effects on bacterial cell membranes
Taking 50mL of staphylococcus aureus in logarithmic phase, centrifuging for 5min at 5000r/min, collecting thalli, washing for 3 times by PBS, re-suspending the thalli by PBS to enable the OD 600 of the thalli to be 0.3, respectively adding BCM (with the final concentration of 2MBC,0.80 mmol/L) and BC (with the final concentration of 0.80 mmol/L) into the bacterial suspension, incubating for 2h at 37 ℃, centrifuging, collecting thalli, washing for 3 times by PBS, and re-suspending by PBS. Taking 500 mu L of bacteria after being resuspended, adding 20 mu L of dye DAPI (10 mu g/mL), incubating for 15min in a dark place, adding 20 mu L of dye PI (15 mu g/mL), incubating for 15min in a dark place, tabletting, taking another 500 mu L of bacteria after being resuspended, adding 30 mu L of dye DiSC (5) (30 mu M), incubating for 60min in a dark place, tabletting, observing under a fluorescent cell imager, photographing and recording.
DAPI is a fluorescent dye capable of penetrating intact cell membranes and binding to DNA, emits blue light, can be used for staining living and dead cells, PI is a fluorescent dye incapable of passing through living cell membranes but capable of penetrating broken cell membranes and binding to DNA, emits red light, is commonly used for staining dead cells, diSC (5) is a membrane potential sensitive probe capable of accumulating in phospholipid bilayer, and DiSC (5) is released outside cells when depolarization of cell membranes occurs, and emits green light. The survival of the bacteria was observed by simultaneous staining of DAPI and PI, and the results are shown in fig. 7.
Effect example seven
Effects on bacterial cell walls
50ML of logarithmic phase staphylococcus aureus is taken and centrifuged for 5min at 5000r/min, bacterial cells are collected, PBS is used for washing for 3 times, the bacterial cells are resuspended to make OD 600 of bacterial liquid be 0.3, BCM and BC pure products prepared in the first embodiment are respectively added into bacterial suspension, the BCM final concentration is respectively 1/4×, 1/2×,1×,2×,4×MBC, the BC final concentration is respectively 1.60, 0.80, 0.40, 0.20 and 0.10mmol/L, the blank group is staphylococcus aureus which is not treated by any reagent, and each experiment is carried out for 3 times.
Incubation at 37 ℃ for 2h, centrifugation at 5000r/min for 10min, taking supernatant, and measuring the activity of bacterial AKP in the supernatant according to the AKP kit instructions.
Alkaline phosphatase (AKP) is present between the cell wall and the cell membrane, and bacteria are not normally secreted into the bacterial culture, and alkaline phosphatase leaks into the bacterial culture only when the cell wall of the bacteria is destroyed, so that the integrity of the bacterial cell wall can be observed by detecting the activity of alkaline phosphatase in the bacterial culture. The results are shown in FIG. 8.
Effect example eight
Effect on ROS content in cells
50ML of logarithmic phase staphylococcus aureus is taken and centrifuged for 5min at 5000r/min, thalli are collected, PBS is used for washing for 3 times, the thalli are resuspended by PBS to enable the OD 600 of the thalli to be 0.3, the thalli are incubated with H 2 DCFDA (the final concentration is 10 mu mol/L) for 20min at 37 ℃, then the thalli are collected by centrifugation, PBS is used for washing for 3 times, and PBS is used for resuspension of the thalli. BCM (final concentration of 2MBC, concentration of 0.80 mmol/L) and BC (final concentration of 0.80 mmol/L) are respectively added into the bacterial suspension, the bacterial suspension is incubated at 37 ℃, samples are respectively taken at 30, 60, 90, 120, 150 and 180min of incubation, fluorescence intensity is measured by a fluorescence spectrophotometer, excitation wavelength is 488nm, and emission wavelength is 525nm. The blank control group was staphylococcus aureus after being re-suspended in PBS and incubated with H 2 DCFDA, without reagents and culture medium, and each group was run in parallel 3 times.
ROS are products of aerobic metabolism, and excessive ROS can damage nucleic acids, proteins, lipids, etc., thereby affecting bacterial growth. The H2DCFDA fluorescent probe can penetrate through a cell membrane and be oxidized and hydrolyzed by intracellular esterase and ROS to generate DCF which emits green fluorescence, so that the ROS content in a bacterium body is in direct proportion to the fluorescence intensity. The results are shown in FIG. 9.
Effect example nine
Drug resistance study of BCM
After incubation at 37 ℃ for 18h, the MIC value was recorded as the 1 st generation experiment with the lowest concentration of flocculent turbidity in the macroscopic wells as the lowest inhibitory concentration (MIC) by adding 40 μl of test solution and 160 μl of bacterial solution to each well.
After the bacterial liquid in 1/2MIC holes in the previous generation experiment is taken to be cultivated to the mid-log phase, a test solution is added into bacterial suspension (the bacterial liquid concentration is 10 5 CFU/mL), the bacterial suspension is cultivated for 18 hours at 37 ℃, MIC values are recorded and recorded as the 2 nd generation experiment, and the experimental steps are repeated to the 20 th generation.
The test sample solution comprises BCM with final concentrations of 6.40, 3.20, 1.60, 0.80, 0.40, 0.20, 0.10, 0.05, 0.02 and 0.01mmol/L, and the positive control medicines comprise azithromycin, clindamycin and amikacin with maximum final concentrations of 40 mug/mL, 40 mug/mL and 200 mug/mL.
The antibiotic used for treating staphylococcus aureus infection clinically takes 3 antibiotics including macrolide antibiotics azithromycin and lincomycin antibiotics clindamycin and aminoglycoside antibiotics amikacin as positive control, and whether the staphylococcus aureus is induced to generate drug resistance after the BCM is treated for a long time is studied. The results are shown in FIG. 10.
Effect example ten
Reverse drug resistance study of BCM
Taking drug-resistant bacteria cultured to the 20 th generation in the ninth effect example, continuously culturing for 10 generations by using a culture solution containing BCM (final concentration is 0.20 mmol/L), and respectively measuring MIC values of azithromycin, clindamycin and amikacin for each generation of bacteria.
Analysis of results
As can be seen from FIG. 1, BC has maximum absorbance peaks at 275nm and 317nm, which correspond to absorbance peaks of the benzoyl conjugated system and the cinnamoyl conjugated system, respectively. The ultraviolet-visible absorption spectrum of the BCM aqueous solution prepared in example one was substantially identical to that of baicalin, and the maximum absorption wavelength was also different by only 1nm, but the absorption values at 275nm and 317nm were reduced by 16.55% and 20.89%, respectively, which indicates that the preparation method BC of example one was applied to react with Mn 2+ to generate a new substance.
TABLE 1
As can be seen from table 1, in ESI positive ion mode, compounds with not completely reacted BC (M/z= 447.0925) and M/z 500.0150, 946.1000 respectively could be detected from supernatant of BCM, and by accurate molecular weight versus isotope distribution (fig. 2), these 2 compounds were presumed to be M + or [ m+h ] + excimer ions of complexes formed by baicalin and Mn 2+ in molar mass ratios of 1:1, 2:1, respectively.
As can be seen from FIG. 3, mn 2+ is inactive to 3 bacteria at the experimental dose, BC has bactericidal effect to staphylococcus aureus at the concentration of 3.20mmol/L, but has weak antibacterial effect to escherichia coli and pseudomonas aeruginosa, after baicalin and Mn 2+ form a complex (BCM), the antibacterial activity is obviously enhanced, the bactericidal effect to staphylococcus aureus is 8 times that of baicalin, meanwhile, obvious bactericidal effect to escherichia coli and pseudomonas aeruginosa is also generated, and the minimum bactericidal concentration of MBC is 1.60mmol/L and 0.80mmol/L respectively. Since BCM has the best antibacterial effect on Staphylococcus aureus (minimum bactericidal concentration of MBC is 0.40 mmol/L), staphylococcus aureus was selected for subsequent experiments.
As can be seen from fig. 4, the control staphylococcus aureus enters the logarithmic phase after 3 hours of culture and enters the stationary phase after 7 hours. The growth of staphylococcus aureus after BCM treatment of Mn 2+, 1/8 and 1/4MBC is not inhibited basically, the growth of staphylococcus aureus after BCM treatment of baicalin and 1/2MBC is inhibited to a certain extent, and the growth of staphylococcus aureus after BCM treatment of MBC is inhibited completely.
As can be seen from fig. 5, the sterilization rate of BCM is dependent on concentration and time. When MBC, 2MBC and BCM of 4MBC act for 0-3 hours, the number of living bacteria in the culture solution is not obviously different from that of a control group, but after the action for 3 hours, the number of living bacteria starts to be reduced, and when the action for 6 hours is finished, no bacteria exist in the BCM solution of 4 MBC. In addition, the bactericidal effect of 1/2MBC BCM against staphylococcus aureus was not obvious, indicating that low concentration BCM had no bactericidal activity against staphylococcus aureus in a short time.
In FIG. 6, part A is a non-BCM treated Staphylococcus aureus, with full cells, intact morphology and smooth surface. In FIG. 6, the BCM with part B being 2MBC was treated for 2 hours with Staphylococcus aureus, the surface of the bacterial cells became uneven, and the surface of the cell membrane was seen to be partially collapsed and shrunken. This suggests that BCM can destroy the morphological structure of staphylococcus aureus cells.
As can be seen from fig. 7, the bacteria treated with BCM for 2MBC for 2h were PI stained, and the bacteria treated with BC for 2h at the same concentration and the bacteria in the blank were not PI stained, whereas the bacteria treated with BCM, BC for 2h were incubated with DiSC (5) and showed substantially no fluorescence emission. This suggests that BCM may cause bacterial death by damaging the bacterial cell membrane, but does not destroy the cell membrane by depolarizing the cell membrane.
As can be seen from fig. 8, the activity of AKP in the bacterial culture after 2h of BCM treatment increased with increasing BCM concentration, and the activity of AKP in the bacterial culture after 2h of BC treatment at the same concentration and the blank control did not substantially change. This suggests that BCM can disrupt the integrity of bacterial cell walls, causing AKP to leak into bacterial culture.
As can be seen from fig. 9, the fluorescence intensity of the 2 MBC-treated BCM bacteria was significantly higher than that of BC-treated bacteria, and the fluorescence intensity continued to increase with time, which suggests that the ROS content in the cells also increased rapidly with the prolonged BCM action time. This result suggests that BCM can damage bacteria by inducing them to produce a large amount of ROS.
As can be seen from fig. 10, staphylococcus aureus developed resistance to amikacin, azithromycin and clindamycin at the 2 nd, 7 th and 17 th generations, respectively, and after 20 th generation of culture, the MIC of the three antibiotics increased to 32 times (amikacin), 8 times (azithromycin) and 4 times (clindamycin) of the first generation, respectively, but the MIC values of BCM did not change, indicating that staphylococcus aureus was not prone to develop resistance to BCM.
Furthermore, after 10 passages of continuous incubation of resistant bacteria with BCM at a final concentration of 0.20mmol/L, the MICs of azithromycin and amikacin decreased to 1/2 and 1/4 of the 20 th passage, respectively, but there was no change in the MIC of clindamycin-resistant Staphylococcus aureus, indicating that BCM may also have some reversal effects on bacteria resistant to certain antibiotics.
Therefore, the baicalin-Mn complex which is not easy to cause bacterial drug resistance, and also has a certain reversing effect on bacteria which are resistant to certain antibiotics, has obvious killing effect on various bacteria including staphylococcus aureus, escherichia coli and pseudomonas aeruginosa, damages the bacteria through various ways, has small minimum sterilization concentration and good sterilization effect, does not need high temperature, high pressure and complex instrument and equipment, and has the advantages of simple method, easy operation, no use of toxic and harmful chemical reagents and no potential safety hazard.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted by the same, and the modified or substituted technical solution may not deviate from the spirit and scope of the technical solution of the present invention.
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| 中药中金属配合物的药理探索与指纹图谱研究;武荣兰;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20041215(第04期);B016-382 * |
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