A baicalin-Mn complex that is not likely to cause bacterial resistance and a preparation method thereof

Abstract

The invention discloses a baicalin-Mn complex that is not easy to cause bacterial resistance and a preparation method thereof, a preparation method of a baicalin-Mn complex that is not easy to cause bacterial resistance, and the steps are as follows: accurately weigh a certain amount of baicalin pure product and MnCl2 compound and place them in a flask, add distilled water and stir evenly, react at 100°C for 2-3h, and concentrate under reduced pressure to dryness. The baicalin-Mn complex provided by the invention is not easy to induce bacterial resistance, and may also have a certain reversal effect on bacteria resistant to certain antibiotics; it has a significant killing effect on a variety of bacteria, including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa; damage bacteria through a variety of ways, the minimum bactericidal concentration is small, and the bactericidal effect is good; the preparation method does not require high temperature and high pressure and complex instruments and equipment, the method is simple, easy to operate, does not use toxic and harmful chemical reagents, and has no safety hazards.

Description

Technical Field

[0001] The invention relates to the technical field of antibacterial drugs, and in particular to a baicalin-Mn complex which is not likely to cause bacterial resistance and a preparation method thereof. Background Art

[0002] Under normal circumstances, different bacteria are sensitive to different antibiotics. For example, most Gram-positive bacteria are sensitive to broad-spectrum antibiotics. After patients use different antibiotics for different bacteria, the bacteria in the body are highly sensitive to the drugs, and the drugs can kill the bacteria more quickly. However, as the dosage of antibiotics increases and the duration of use extends, the biofilm of the bacteria in the body will have a certain resistance to the drugs. In addition, as the drugs continue to stimulate the bacteria, the extreme environment outside the membrane causes the bacteria inside the membrane to become heterogeneous, which will promote the formation of bacterial resistance / resistance.

[0003] Bacterial resistance to drug, also known as drug resistance, refers to the phenomenon that bacteria are insensitive to antibiotics. It is a special form of bacterial survival. Bacterial resistance can be divided into acquired resistance and natural resistance according to its cause. In clinical practice, acquired resistance is the most common. The use of antibiotics will cause most sensitive strains to be continuously killed, while resistant strains will continue to multiply. Over time, the effect of antibiotics will be significantly reduced or even lose its effect.

[0004] The hazards of bacterial resistance are mainly the following:

[0005] 1. The recovery time of the disease is prolonged. After the bacteria develop drug resistance, the infection of the human body will cause conventional antibiotics to have a poor therapeutic effect. It is necessary to adjust the medication under the guidance of a doctor, improve the drug sensitivity test, and screen for antibiotics that are sensitive to drug-resistant bacteria for treatment, which will lead to a prolonged recovery period of the disease. If sensitive antibiotics cannot be screened, it may pose a serious threat to life and health;

[0006] 2. Increase the dosage and types of drugs. For infections caused by drug-resistant bacteria, the use of a single antibiotic often does not have a good therapeutic effect. It is often necessary to use two to three or even more antibiotics in combination for treatment. The increase in the dosage will also increase the toxicity of the drugs, and the probability of adverse reactions is often higher, which will cause certain adverse effects on life and health. Super-resistant bacteria that are resistant to multiple antibiotics may also appear;

[0007] 3. Concurrent infections: Since resistant bacteria are difficult to be eliminated by antibiotics, these resistant bacteria may invade other parts of the body and cause concurrent infections of other tissues or serious infections, such as sepsis and intracranial infections, endangering life and health, and even leading to a tragic situation where there is no cure.

[0008] Therefore, developing new antibiotics that have good antibacterial and bactericidal effects and are less likely to cause bacterial resistance is an eternal goal for medical researchers. Summary of the invention

[0009] The purpose of the present invention is to provide a baicalin-Mn complex that is not likely to cause bacterial resistance and a preparation method thereof, so as to increase the types of antibacterial drugs, provide new drug options for patients to fight bacteria, improve the sensitivity of bacteria to antibacterial drugs, and reduce the occurrence of bacterial resistance.

[0010] To achieve the above object, the present invention provides a baicalin-Mn complex that is not likely to cause bacterial resistance and a preparation method thereof. The preparation method of the baicalin-Mn complex that is not likely to cause bacterial resistance comprises the following steps: accurately weighing a certain amount of pure baicalin compound and MnCl 2 Place the compound in a flask, add distilled water and stir evenly, react at 100°C for 2-3h, and concentrate to dryness under reduced pressure.

[0011] Preferably, the baicalin and Mn 2+ The amount of compound was to ensure that baicalin and Mn 2+ The molar mass ratio is 4:1 to 4:3; the flask is a round-bottom flask, and the total amount of liquid added does not exceed half of the volume of the flask.

[0012] Preferably, the amount of distilled water added is such that the final concentration of baicalin is 2 mmol / L, and the stirring speed is 200-400 rpm / min.

[0013] Preferably, the reduced pressure spin drying is performed by a rotary evaporator, the initial temperature of the reduced pressure spin drying is 60° C., the rotation speed is 60 r / min, the pressure is 150 mbar, the subsequent pressure and rotation speed remain unchanged, and the pressure decreases by 20 mbar every 5 minutes.

[0014] A baicalin-Mn complex which is not likely to cause bacterial resistance and is prepared by the preparation method as described above.

[0015] An application of the baicalin-Mn complex as described above which is not likely to cause bacterial resistance in inhibiting and killing bacteria.

[0016] Therefore, the present invention provides a baicalin-Mn complex that is not likely to cause bacterial resistance and a preparation method thereof, and its specific technical effects are as follows:

[0017] (1) The baicalin-Mn complex provided by the present invention is not likely to cause bacterial resistance and may also have a certain reversal effect on bacteria resistant to certain antibiotics;

[0018] (2) The baicalin-Mn complex provided by the present invention has a significant killing effect on a variety of bacteria, including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa;

[0019] (3) The baicalin-Mn complex provided by the present invention damages bacteria through multiple pathways such as damaging cell membranes, destroying the integrity of bacterial cell walls and bacterial morphological structures, and inducing bacteria to produce a large amount of ROS. The minimum bactericidal concentration is small and the bactericidal effect is good. The minimum bactericidal concentration for Staphylococcus aureus is 0.4 mmol / L.

[0020] (4) The preparation method of the baicalin-Mn complex provided by the present invention does not require high temperature, high pressure and complex instruments and equipment. The method is simple and easy to operate, does not use toxic and harmful chemical reagents, and does not have any safety hazards.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0023] Figure 1 is the UV-visible absorption spectra of BC and BCM aqueous solutions;

[0024] Figure 2 It is the result of the comparison between the precise molecular weight and isotopic distribution of BCM;

[0025] Figure 3 These are the experimental results of the inhibitory effect of BCM on three types of bacteria;

[0026] Figure 4 is the effect of BCM on the growth curve of Staphylococcus aureus;

[0027] Figure 5 is the bactericidal kinetic curve of BCM against Staphylococcus aureus;

[0028] Figure 6 is the scanning electron microscopic observation result of the morphological structure of Staphylococcus aureus before and after BCM treatment;

[0029] Figure 7 is a fluorescence microscopy image of Staphylococcus aureus after BCM treatment;

[0030] Figure 8 is the effect of BC and BCM on the release of AKP activity in bacteria;

[0031] Fig. 9 is the effect of BCM on the ROS content in Staphylococcus aureus;

[0032] Fig.10 It is the resistance of Staphylococcus aureus to BCM and three antibiotics and the reversal effect of BCM. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0034] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0035] The baicalin used in the examples was produced by Chengdu Aifa Biotechnology Co., Ltd., HPLC purity ≥ 98%; MnCl 2 Produced by Aladdin Corporation of the United States; scanning electron microscope produced by Hitachi High-Tech Corporation, model SU8010; fluorescent cell imager produced by BIO-RAD Corporation, model ZOE TM ; AKP test kit was produced by Nanjing Jiancheng Bioengineering Institute; fluorescence spectrophotometer was produced by Gangdong Technology Company, model F-320; constant temperature heating magnetic stirrer was produced by Gongyi Yuhua Instrument Co., Ltd., model DF-101S; rotary evaporator was produced by German Heidolph Company, model Hei-VAP.

[0036] The final concentrations of BCM described in the examples are all based on the molar mass of baicalin added during synthesis.

[0037] Embodiment 1

[0038] A baicalin-Mn complex (BCM) that is not likely to cause bacterial resistance is prepared, and the steps are as follows:

[0039] (1) Accurately weigh 125.84 mg MnCl 2 Dissolve it with 10 mL of distilled water, pipette 300 μL into a round-bottom flask, then add 17.86 mg of pure baicalin (BC) and 20 mL of distilled water into the round-bottom flask in sequence, and stir evenly at a speed of 200 rpm / min.

[0040] (2) The round-bottom flask was placed on a constant temperature heating magnetic stirrer and heated to 100°C and condensed under reflux. During this period, it was still placed on the constant temperature magnetic stirrer and continued to heat and stir for 2 hours. After the reaction was completed, it was placed on a rotary evaporator and concentrated to dryness under reduced pressure to obtain BCM. The initial temperature of the reduced pressure rotary dryer was 60°C, the speed was 60r / min, and the pressure was 150mbar. The subsequent pressure and speed remained unchanged, and the pressure dropped by 20mbar every 5 minutes.

[0041] Embodiment 2

[0042] A baicalin-Mn complex that is not likely to cause bacterial resistance was prepared by the same method as in Example 1, except that 100 μL MnCl was absorbed in step (1). 2 Aqueous solution, stirring speed is 300 rpm / min.

[0043] Embodiment 3

[0044] A baicalin-Mn complex that is not likely to cause bacterial resistance was prepared by the same method as in Example 1, except that 200 μL MnCl was absorbed in step (1). 2 The aqueous solution was stirred at a speed of 400 rpm / min and the reaction time in step (2) was 3 h.

[0045] Effect Example 1

[0046] Structural characterization of BCMs

[0047] Take 1 mL of the BCM reaction solution from Example 1 that has not been dried by spin drying and dilute it to 10 mL of distilled water to make a BCM dilution solution with a concentration of 2 mmol / L; then weigh 17.86 mg of BC in a round-bottom flask, add 20 mL of distilled water, heat at 100°C for 2 hours, and take 1 mL of the reaction solution while hot and add it to 10 mL of distilled water to make the concentration 2 mmol / L. Take 2 mL of the BCM dilution solution and BC solution respectively and scan at a wavelength of 250 nm-500 nm to obtain a UV-visible absorption spectrum, as shown in FIG. Figure 1 .

[0048] Another 3 mL of BCM dilution was centrifuged at 8000 r / min for 5 min, and the supernatant was detected by ESI-TOF MS using infusion injection, positive ion mode detection, mass detection range of 100-1500, drying air flow rate of 800 L / h, temperature of 500 °C. The test results are shown in Table 1, and the precise molecular weight and isotope distribution comparison results are shown in Figure 2 ,in Figure 2 The A part is C 21 H 17 O 11 Theoretical isotope distribution diagram of Mn, part B is C 21 H17 O 11 The measured isotope distribution of Mn, the C part is C 42 H 35 O 22 Theoretical isotope distribution diagram of Mn, with D part being C 42 H 35 O 22 Measured Mn isotope distribution diagram.

[0049] Effect Example 2

[0050] Determination of Minimum Bactericidal Concentration (MBC) of BCM

[0051] Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were inoculated on solid culture medium and cultured at 37°C for 24 h. Then, a single colony was picked and placed in TSB liquid culture medium. The culture was shaken until the logarithmic growth phase and diluted to 10 with fresh TSB liquid culture medium. 5 CFU / mL.

[0052] According to the method of Example 1, 17.86 mg BC and 125.84 mg MnCl were weighed respectively. 2 , BCM was prepared, dissolved in 2.5 mL of distilled water containing 20% ​​DMSO as a mother solution for later use, and then distilled water was used to make BCM solutions with concentrations of 16.00, 8.00, 4.00, 2.00, 1.00, and 0.50 mmol / L, and then the BCM of the above different concentrations was added to the 1st to 3rd rows (4 rows in total) of a sterile 24-well plate, 0.4 mL per well, and then 1.6 mL of Staphylococcus aureus was added to each well of the 1st row, 1.6 mL of Escherichia coli was added to each well of the 2nd row, and 1.6 mL of Pseudomonas aeruginosa was added to each well of the 3rd row, so that the final concentrations of BCM were 3.20, 1.60, 0.80, 0.40, 0.20, and 0.10 mmol / L, respectively.

[0053] After culturing at 37℃ for 18h, the samples were spread on TSB solid medium and cultured at 37℃ for another 18h. The samples were taken out and photographed. The lowest concentration without bacterial growth was recorded as the minimum bactericidal concentration (MBC). The final concentrations of BC and MnCl were 3.20, 1.60, 0.80, 0.40, 0.20, 0.10mmol / L and 2.40mmol / L, respectively. 2 As a control, each group of experiments were repeated 3 times. Figure 3 ,From left to right in the figure, the final concentrations of BCM were 3.20, 1.60, 0.80, 0.40, 0.20, and 0.10 mmol / L, and the final concentrations of BC were 3.20, 1.60, 0.80, 0.40, 0.20, and 0.10 mmol / L, respectively.

[0054] Effect Example 3

[0055] Effects on bacterial growth curve

[0056] According to the results of Example 2, Staphylococcus aureus was selected for this experiment. The BCM prepared in Example 1 was dissolved in 2.5 mL of distilled water containing 20% ​​DMSO, and then diluted with distilled water to concentrations of 0.2, 0.4, 0.8, and 1.6 mmol / L, respectively, and added to 1.6 mL of 10 5 CFU / mL of Staphylococcus aureus suspension, the final concentration of BCM was 0.05, 0.10, 0.20, and 0.40 mmol / L, with BC and Mn 2+ As a control, the final concentrations were 1.60mmol / L and 1.20mmol / L, respectively, by adding 0.4mL distilled water + 1.6mL 10 5 CFU / mL of Staphylococcus aureus was used as the blank control group. Incubate at 37°C and measure bacterial growth using an ELISA reader (OD600) at 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, and 20 hours of incubation for 20 hours. Figure 4 .

[0057] Effect Example 4

[0058] Bactericidal kinetics curve of BCM

[0059] According to the results of Example 2, Staphylococcus aureus was selected for this experiment. The BCM prepared in Example 1 was dissolved in 2.5 mL of distilled water containing 20% ​​DMSO, and then diluted with distilled water to concentrations of 0.2, 0.4, 0.8, and 1.6 mmol / L, respectively. 1.6 mL of bacterial solution with a concentration of 10 5 CFU / mL of Staphylococcus aureus suspension, the final concentrations of BCM were 0.05, 0.10, 0.20, and 0.40 mmol / L, BC and Mn 2+ The final concentrations were 1.60mmol / L and 1.20mmol / L, respectively, by adding 0.4mL distilled water + 1.6mL 10 5 CFU / mL of Staphylococcus aureus was used as the blank control group. Incubate at 37°C, remove the bacterial solution at 0, 1, 2, 3, 4, 5, and 6 hours of incubation, dilute with sterile PBS, spread, and culture at 37°C for 18 hours, and record the number of colonies. Figure 5 .

[0060] Effect Example 5

[0061] Effects on bacterial morphology

[0062] Take 50 mL of logarithmic phase Staphylococcus aureus, centrifuge at 5000 r / min for 5 min, collect the bacteria, and resuspend the bacteria in PBS to an OD of 600 0.3, add BCM (final concentration is 2MBC, 0.80mmol / L) to the bacterial suspension, add 0.4mL distilled water + 1.6mL 10 5 CFU / mL of Staphylococcus aureus was used as the blank control group.

[0063] Incubate at 37°C for 2 hours, centrifuge, collect the precipitated bacteria, wash with PBS, centrifuge again, collect the bacteria, resuspend in 4 mL of 2.5% glutaraldehyde, and fix overnight at 4°C. Wash off the glutaraldehyde solution on the bacteria with PBS, fix the bacteria with 1% osmium acid solution for 1-2 hours, wash off the osmium acid solution on the bacteria with PBS; and dehydrate the bacteria with gradient concentrations of ethanol solution (including five concentrations of 30%, 50%, 70%, 80%, 90% and 95%), treat each concentration for 15 minutes, and then treat twice with 100% ethanol, each time for 20 minutes. Treat the sample with a mixture of ethanol and isoamyl acetate (V / V=1 / 1) for 30 minutes, and then treat the sample with pure isoamyl acetate for 1 hour or leave overnight. Critical point drying. Plate, and observe the ultrastructure of bacteria after BCM treatment under a scanning electron microscope. The morphology and external changes of the cell surface of Staphylococcus aureus treated with BCM were observed by scanning electron microscopy. The results are shown in Figure 6 ,in Figure 6 Part A is the control group, and part B is Staphylococcus aureus treated with 2MBC's BCM.

[0064] Effect Example 6

[0065] Effects on bacterial cell membranes

[0066] Take 50 mL of logarithmic phase Staphylococcus aureus, centrifuge at 5000 r / min for 5 min, collect the cells, wash 3 times with PBS, and resuspend the cells in PBS to make the OD of the bacterial solution 600 The concentration of 0.3 was used. BCM (final concentration was 2MBC, 0.80mmol / L) and BC (final concentration was 0.80mmol / L) were added to the bacterial suspension, incubated at 37℃ for 2h, centrifuged, and the bacteria were collected, washed 3 times with PBS, and resuspended with PBS. Take 500μL of the resuspended bacteria, add 20μL of the dye DAPI (10μg / mL), incubate in the dark for 15min, then add 20μL of the dye PI (15μg / mL), incubate in the dark for 15min, and then prepare the slide. Take another 500μL of the resuspended bacteria, add 30μL of the dye DiSC3 (5) (30μM), incubate in the dark for 60min, and then prepare the slide. Observe under a fluorescent cell imager, take pictures and record.

[0067] DAPI is a fluorescent dye that can pass through intact cell membranes and bind to DNA. It emits blue light and can be used to stain live and dead cells. PI is a fluorescent dye that cannot pass through live cell membranes but can pass through damaged cell membranes and bind to DNA. It emits red light and is often used to stain dead cells. DiSC3(5) is a membrane potential sensitive probe that can accumulate in the phospholipid bilayer. When the cell membrane is depolarized, DiSC3(5) is released outside the cell and emits green light. By staining bacteria with DAPI and PI simultaneously, the survival status of the bacteria can be observed. The results are shown in Figure 7 .

[0068] Effect Example 7

[0069] Effects on bacterial cell wall

[0070] Take 50mL of logarithmic phase Staphylococcus aureus, centrifuge at 5000r / min for 5min, collect the cells, wash 3 times with PBS, and resuspend the cells in PBS to make the OD of the bacterial solution 600 The concentration of BCM was 0.3, and the pure BCM and BC prepared in Example 1 were added to the bacterial suspension to make the final concentrations of BCM 1 / 4×, 1 / 2×, 1×, 2×, and 4×MBC, and the final concentrations of BC were 1.60, 0.80, 0.40, 0.20, and 0.10 mmol / L, respectively. The blank control group was Staphylococcus aureus not treated with any reagents. Each group of experiments was conducted in parallel 3 times.

[0071] Incubate at 37°C for 2 h, centrifuge at 5000 r / min for 10 min, take the supernatant, and determine the activity of bacterial AKP in the supernatant according to the instructions of the AKP kit.

[0072] Alkaline phosphatase (AKP) exists between the cell wall and the cell membrane. Bacteria will not secrete it into the bacterial culture medium under normal circumstances. Only when the bacterial cell wall is damaged will alkaline phosphatase leak into the bacterial culture medium. Therefore, the integrity of the bacterial cell wall can be observed by detecting the activity of alkaline phosphatase in the bacterial culture medium. Figure 8 .

[0073] Effect Example 8

[0074] Effects on ROS content in bacteria

[0075] Take 50mL of logarithmic phase Staphylococcus aureus, centrifuge at 5000r / min for 5min, collect the cells, wash 3 times with PBS, and resuspend the cells in PBS to make the OD of the bacterial solution 600 is 0.3, bacterial solution and H 2After incubation with DCFDA (final concentration of 10 μmol / L) at 37°C for 20 min, the cells were collected by centrifugation, washed 3 times with PBS, and resuspended in PBS. BCM (final concentration of 2MBC, concentration of 0.80mmol / L) and BC (final concentration of 0.80mmol / L) were added to the bacterial suspension, incubated at 37°C, and samples were taken at 30, 60, 90, 120, 150, and 180 min of incubation. The fluorescence intensity was measured by a fluorescence spectrophotometer with an excitation wavelength of 488nm and an emission wavelength of 525nm. The blank control group was resuspended in PBS with H 2 After co-incubation with DCFDA, no reagents or culture medium were added to Staphylococcus aureus. Each group of experiments was repeated three times in parallel.

[0076] ROS is a product of aerobic metabolism. Excessive ROS can damage nucleic acids, proteins, lipids, etc., thus affecting the growth of bacteria. The H2DCFDA fluorescent probe can pass through the cell membrane and be oxidized and hydrolyzed by intracellular esterases and ROS to generate DCF that emits green fluorescence. Therefore, the ROS content in the bacteria is proportional to the fluorescence intensity. Fig. 9 .

[0077] Effect Example 9

[0078] Study on drug resistance of BCM

[0079] Take a sterile 96-well plate and add 40 μL of the test solution and 160 μL of the bacterial solution to each well. After incubation at 37°C for 18 h, the lowest concentration at which flocculent turbidity is visible to the naked eye in the well is the minimum inhibitory concentration (MIC). Record the MIC value and record it as the first generation experiment.

[0080] Take the bacterial suspension in 1 / 2MIC wells of the previous experiment and culture it to the mid-logarithmic phase, then add the bacterial suspension (bacterial concentration is 10 5 CFU / mL), add the test solution, culture at 37℃ for 18h, record the MIC value, and record it as the second generation experiment. Repeat the above experimental steps to the 20th generation.

[0081] The test solution included: BCM with final concentrations of 6.40, 3.20, 1.60, 0.80, 0.40, 0.20, 0.10, 0.05, 0.02, and 0.01 mmol / L, respectively; the positive control drugs were azithromycin, clindamycin, and amikacin, with maximum final concentrations of 40 μg / mL, 40 μg / mL, and 200 μg / mL, respectively.

[0082] Three antibiotics used clinically to treat Staphylococcus aureus infections: macrolide antibiotic azithromycin, lincomycin antibiotic clindamycin, and aminoglycoside antibiotic amikacin were used as positive controls to study whether long-term treatment of Staphylococcus aureus with BCM would induce drug resistance. Fig.10 .

[0083] Effect Example 10

[0084] Study on reversal of drug resistance by BCM

[0085] The drug-resistant bacteria cultured to the 20th generation in Effect Example 9 were cultured for 10 generations in a culture medium containing BCM (final concentration of 0.20 mmol / L), and the MIC values ​​of azithromycin, clindamycin and amikacin against each generation of bacteria were determined.

[0086] Results Analysis

[0087] Depend on Figure 1 It can be seen that BC has maximum absorption peaks at 275nm and 317nm, corresponding to the absorption peaks of the benzoyl conjugated system and the cinnamoyl conjugated system, respectively. The UV-visible absorption spectrum of the BCM aqueous solution prepared in Example 1 is basically the same as that of baicalin, and the maximum absorption wavelength is only 1nm different, but the absorbance values ​​at 275nm and 317nm are reduced by 16.55% and 20.89%, respectively, which shows that the preparation method of BC in Example 1 is different from that of Mn 2+ The reaction produces new substances.

[0088] Table 1

[0089]

[0090] As can be seen from Table 1, in the ESI positive ion mode, incompletely reacted BC (m / z = 447.0925) and compounds with m / z of 500.0150 and 946.1000, respectively, can be detected from the supernatant of BCM. Figure 2 ), it is speculated that these two compounds are baicalin and Mn 2+ The M of the complex formed with a molar mass ratio of 1:1 and 2:1 + or [M+H] + Quasi-molecular ions.

[0091] from Figure 3 It can be seen that Mn 2+ At the experimental dose, BC had no activity against the three bacteria; BC had a bactericidal effect on Staphylococcus aureus at a concentration of 3.20mmol / L, but had only a weak inhibitory effect on Escherichia coli and Pseudomonas aeruginosa; Baicalin and Mn 2+After the complex (BCM) was formed, the antibacterial activity was significantly enhanced. Its bactericidal effect on Staphylococcus aureus was 8 times that of baicalin. It also had a significant bactericidal effect on Escherichia coli and Pseudomonas aeruginosa, with the minimum bactericidal concentrations of MBC being 1.60mmol / L and 0.80mmol / L, respectively. Since BCM had the best antibacterial effect on Staphylococcus aureus (the minimum bactericidal concentration of MBC was 0.40mmol / L), Staphylococcus aureus was selected for subsequent experiments.

[0092] Depend on Figure 4 It can be seen that the Staphylococcus aureus in the control group entered the logarithmic growth phase after 3 hours of culture and entered the stable phase after 7 hours. 2+ The growth of Staphylococcus aureus treated with 1 / 4, 1 / 8 and 1 / 4MBC BCM was basically not inhibited, the growth of Staphylococcus aureus treated with baicalin and 1 / 2MBC BCM was inhibited to a certain extent, and the growth of Staphylococcus aureus treated with MBC BCM was completely inhibited.

[0093] Depend on Figure 5 It can be seen that the bactericidal rate of BCM is dependent on concentration and time. When the BCM of MBC, 2MBC, and 4MBC was applied for 0 to 3 hours, the number of live bacteria in the culture medium was not significantly different from that of the control group, but after 3 hours, the number of live bacteria began to decrease, and by 6 hours, no bacteria survived in the BCM solution of 4MBC. In addition, the bactericidal effect of 1 / 2MBC BCM on Staphylococcus aureus was not obvious, indicating that low concentrations of BCM had no bactericidal activity against Staphylococcus aureus in a short period of time.

[0094] Figure 6 Part A in the middle is Staphylococcus aureus that has not been treated with BCM. The bacteria are plump, complete in shape, and have a smooth surface. Figure 6 Part B shows that after 2 hours of treatment with 2MBC BCM, the surface of Staphylococcus aureus became uneven, and the cell membrane surface was partially collapsed and shrunken. This shows that BCM can destroy the morphological structure of Staphylococcus aureus.

[0095] Depend on Figure 7 It can be seen that the bacteria treated with 2MBC BCM for 2h were stained with PI, while the bacteria treated with BC at the same concentration for 2h and the bacteria in the blank control were not stained with PI, and the bacteria treated with BCM and BC for 2h showed almost no fluorescence emission after being co-incubated with DiSC3(5). This suggests that BCM may cause bacterial death by damaging the bacterial cell membrane, but not by depolarizing the cell membrane.

[0096] from Figure 8It can be seen that the activity of AKP in the bacterial culture fluid treated with BCM for 2 hours increased with the increase of BCM concentration, while the activity of AKP in the bacterial culture fluid treated with BC for 2 hours and the blank control at the same concentration did not change. This indicates that BCM can destroy the integrity of the bacterial cell wall, causing AKP to leak into the bacterial culture fluid.

[0097] from Fig. 9 It can be seen that the fluorescence intensity of the bacteria treated with 2MBC's BCM is significantly higher than that of the bacteria treated with BC, and the fluorescence intensity continues to increase over time, which indicates that the content of ROS in the bacteria increases rapidly as the BCM action time increases. This result suggests that BCM can damage bacteria by inducing bacteria to produce a large amount of ROS.

[0098] from Fig.10 It can be seen that Staphylococcus aureus developed resistance to amikacin, azithromycin and clindamycin in the 2nd, 7th and 17th generations, respectively. After 20 generations of culture, the MICs of the three antibiotics increased to 32 times (amikacin), 8 times (azithromycin) and 4 times (clindamycin) of the initial generation, respectively, but the MIC value of BCM did not change, which indicates that Staphylococcus aureus is not easy to develop resistance to BCM.

[0099] In addition, after continuous incubation of resistant bacteria with BCM at a final concentration of 0.20 mmol / L for 10 generations, the MICs of azithromycin and amikacin decreased to 1 / 2 and 1 / 4 of that in the 20th generation, respectively, but the MIC of clindamycin-resistant Staphylococcus aureus did not change, indicating that BCM may also have a certain reversal effect on bacteria resistant to certain antibiotics.

[0100] Therefore, the baicalin-Mn complex provided by the present invention is not likely to cause bacterial resistance and may also have a certain reversal effect on bacteria resistant to certain antibiotics; it has an obvious killing effect on a variety of bacteria, including Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa; it damages bacteria through multiple pathways, has a small minimum bactericidal concentration and a good bactericidal effect; the preparation method does not require high temperature and high pressure and complex instruments and equipment, the method is simple and easy to operate, does not use toxic and harmful chemical reagents, and has no safety hazards.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of a baicalin-Mn complex that is not likely to cause bacterial resistance in the preparation of drugs for inhibiting and killing bacteria, characterized in that: The preparation method of the baicalin-Mn complex that is not likely to cause bacterial resistance is as follows: accurately weigh a certain amount of pure baicalin compound and MnCl2 compound and place them in a flask, add distilled water and stir evenly, react at 100° C. for 2 to 3 hours, and concentrate to dryness under reduced pressure; Weigh the baicalin and Mn 2+ The amount of compound was to ensure that baicalin and Mn 2+ The molar ratio is 4:1~4:3; the flask is a round-bottom flask, and the total amount of liquid added does not exceed half of the volume of the flask; The reduced pressure concentration is completed by a rotary evaporator, the initial temperature of the reduced pressure rotary drying is 60°C, the speed is 60r / min, the pressure is 150mbar, the subsequent pressure and speed remain unchanged, and the pressure drops by 20mbar every 5min; The amount of distilled water added was such that the final concentration of baicalin was 2 mmol / L, and the stirring speed was 200-400 rpm / min.

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