Applications of cyclovirobuxine D and pharmaceutically acceptable salts thereof in preparation of antibacterial drugs

Abstract

The invention belongs to the technical field of natural antibacterial drugs, and discloses application of cyclovirobuxine D and pharmaceutically acceptable salts thereof in preparation of antibacterial drugs. The invention discloses the effect of cyclovirobuxine D in preparation of antibacterial drugs, and tests prove that cyclovirobuxine D has remarkable antibacterial activity in inhibition of gram-positive bacteria such as staphylococcus aureus, gram-negative bacteria such as escherichia coli and super drug-resistant bacteria such as MRSA (Methicillin Resistant Staphylococcus Aureus).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of natural antibacterial drugs, and particularly relates to application of cyclodaphniphylline D and pharmaceutically acceptable salts thereof in preparation of antibacterial drugs. BACKGROUND

[0002] In the past few decades, people have been committed to seeking new antibacterial agents and new alternative therapies to make up for the deficiency of antibiotics in the treatment of bacterial infections. Mainly because microorganisms have incredible innate adaptability, and their strong adaptive capacity leads to the easy development of bacterial drug resistance, and there is a limitation in the current effective antibiotic species, and there is a shortage of new antibacterial alternatives. The current situation is pushing the start of the "post-antibiotic era", which is threatening the achievements of modern medicine in antibiotics.

[0003] The current research finds that the use of some antibiotics sometimes causes antagonistic / allergic side effects and infections. Therefore, in this rapidly developing technical era, it is urgent to develop new and effective antibacterial drugs. Plants are undoubtedly the most important source of therapeutic drugs.

[0004] Previous studies have shown that the water extract of Buxus and the alcohol extract of Buxus have antibacterial activity, but the specific antibacterial compound monomer has not been determined. There are many kinds of Buxus alkaloids that can be extracted from Buxus, and there are still undiscovered species, so it is not clear which Buxus alkaloid monomer can be used to play an antibacterial role. At present, only the crude extract of Buxus alkaloids is studied for antibacterial activity, but because the components contained in these crude extracts are complex, they also include other non-alkaloid components such as terpenes, phenols, and flavonoids. Therefore, the specific active ingredients have not been determined. Therefore, it is necessary to develop new and effective antibacterial drugs to alleviate the problem of bacterial drug resistance.

[0005] Cyclodaphniphylline D is a natural compound monomer extracted from Buxus microphylla and its homologous plants. It is a colorless needle-like crystal, easily soluble in chloroform, slightly soluble in water, and has a melting point of 219-222℃. The main functions are to promote blood circulation and relieve pain, and it is suitable for treating chest pain, pulse knot, and other symptoms caused by blood stasis, such as angina pectoris, arrhythmia, etc. The oral dose is 1-2 mg at a time, 2-3 times a day, and it needs to be stored in a light-proof and airtight container. Its preparation form is Buxin tablets, which is included in the Chinese Pharmacopoeia.

[0006] There is no related technical content of cyclodaphniphylline D in inhibiting bacterial growth in the prior art. SUMMARY

[0007] In order to develop new effective antibacterial drugs to alleviate the problem of bacterial drug resistance, the application takes the active monomer ingredient, namely cyclovirobuxin D, separated and purified from a buxus alkaloid as the basis, discusses the antibacterial activity of cyclovirobuxin D on staphylococcus aureus, escherichia coli and MRSA, verifies the antibacterial effect through experiments, and preliminarily explores the antibacterial mechanism, and provides the application of cyclovirobuxin D and its pharmaceutically acceptable salt in the preparation of antibacterial drugs.

[0008] The purpose of the application is achieved by the following technical solutions.

[0009] In a first aspect, the application provides the application of cyclovirobuxin D and its pharmaceutically acceptable salt in the preparation of antibacterial drugs, wherein the molecular formula of the cyclovirobuxin D (CVB-D) is C 26 H 46 N2O, and the chemical structural formula is as follows:

[0010] In order to improve the water solubility of cyclovirobuxin D, the cyclovirobuxin D monomer is applied in the form of a pharmaceutically acceptable salt thereof. The pharmaceutically acceptable salt includes hydrochloride, phosphate, oxalate, sulfate or citrate, and preferably hydrochloride.

[0011] The administration mode is oral or injection administration, and preferably intravenous injection administration.

[0012] The application dose is 1.0 mg of cyclovirobuxin D per kilogram of body weight.

[0013] The antibacterial drug is any one of an antibacterial drug against gram-positive bacteria, an antibacterial drug against gram-negative bacteria or an antibacterial drug against drug-resistant bacteria. Further, the gram-positive bacteria are staphylococcus aureus, the gram-negative bacteria are escherichia coli, and the super drug-resistant bacteria are methicillin-resistant staphylococcus aureus.

[0014] Compared with chloramphenicol and ofloxacin, the cyclovirobuxin D monomer hydrochloride has good resistance to escherichia coli. The cyclovirobuxin D monomer hydrochloride and six antibiotics including chloramphenicol, ofloxacin, cefmetazole sodium, cefpodoxime proxetil, minocycline and penicillin V potassium are subjected to a combined drug sensitivity test, and the results include the following contents:

[0015] (1) When the cyclovirobuxin D hydrochloride and cefmetazole sodium are combined, a synergistic effect is shown;

[0016] (2) When the cyclovirobuxin D hydrochloride is combined with chloramphenicol and ofloxacin, an additive effect is shown;

[0017] (3) When the cyclovirobuxin D hydrochloride is combined with cefpodoxime proxetil, no correlation effect is shown;

[0018] (4) CVB-D hydrochloride shows antagonistic effect when combined with minocycline and penicillin V potassium.

[0019] In a second aspect, the present application further provides an antibacterial drug composition, which is composed of cyclovirobuxin D hydrochloride and cefmetazole sodium; preferably, the mass ratio of the two is (1:1)~(1:4).

[0020] In a third aspect, the present application further provides an antibacterial drug composition, which is composed of cyclovirobuxin D hydrochloride and chloramphenicol; preferably, the mass ratio of the two is (1:2)~(2:1).

[0021] In a fourth aspect, the present application further provides an antibacterial drug composition, which is composed of cyclovirobuxin D hydrochloride and ofloxacin; preferably, the mass ratio of the two is (1:2)~(2:1).

[0022] Beneficial effects:

[0023] In a first aspect, the present application discloses the application value of cyclovirobuxin D in preparing antibacterial drugs. The antibacterial activity, drug resistance and the effect of combination of cyclovirobuxin D are verified by methods such as agar punching, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), half inhibitory concentration (IC 50 ), 24h bactericidal curve determination, drug resistance determination, chessboard method combined antibacterial test, etc. The results show that cyclovirobuxin D has significant antibacterial activity in inhibiting gram-positive bacteria such as Staphylococcus aureus, gram-negative bacteria such as Escherichia coli, and super drug-resistant bacteria such as MRSA. Among them, the MIC of CVB-D hydrochloride on Escherichia coli, Staphylococcus aureus and MRSA is 0.75 mg / mL, 0.75 mg / mL and 1.50 mg / mL respectively; the MBC of CVB-D hydrochloride on Escherichia coli, Staphylococcus aureus and MRSA is 3.12 mg / mL, 1.50 mg / mL and 3.12 mg / mL respectively. The IC 50 of CVB-D hydrochloride on Escherichia coli, Staphylococcus aureus and MRSA is 0.40 mg / mL, 0.35 mg / mL and 0.34 mg / mL respectively. The present application further verifies that the cyclovirobuxin D monomer inhibits bacterial drug resistance by inhibiting the formation of biofilm. The application of cyclovirobuxin D in preparing antibacterial drugs not only expands the application field of buxus alkaloids, but also provides a new natural antibacterial candidate drug for dealing with the increasingly serious problem of bacterial drug resistance.

[0024] The antibacterial drug composition provided in the second to fourth aspects has the effect of reducing the dosage of the original antibiotic while maintaining the original antibiotic drug efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Purification chromatogram of CVB-D in Example 1.

[0026] Figure 2 Infrared spectrum of CVB-D in Example 1.

[0027] Figure 3 Primary mass spectrum of CVB-D in Example 1.

[0028] Figure 4 Identification chromatogram of CVB-D hydrochloride in Example 2.

[0029] Figure 5 Bacteriostatic circle experiment diagram of CVB-D hydrochloride on bacteria in Example 3.

[0030] Figure 6 MIC experiment diagram of CVB-D hydrochloride on E. coli in Example 4.

[0031] Figure 7 MIC experiment diagram of CVB-D hydrochloride on S. aureus in Example 4.

[0032] Figure 8 MIC experiment diagram of CVB-D hydrochloride on MRSA in Example 4.

[0033] Figure 9A 24 h growth curve of CVB-D hydrochloride on E. coli in Example 7.

[0034] Figure 9B 24 h growth curve of CVB-D hydrochloride on S. aureus in Example 7.

[0035] Figure 9C 24 h growth curve of CVB-D hydrochloride on MRSA in Example 7.

[0036] Figure 10 Drug resistance test experiment diagram of CVB-D hydrochloride in Example 8.

[0037] Figure 11 Concentration gradient diagram of combined bacteriostatic test in Example 9.

[0038] Figure 12 CVB-D hydrochloride antibiofilm activity result diagram in Example 10.

[0039] Figure 13A Conductivity diagram of CVB-D hydrochloride on S. aureus in Example 10;

[0040] Figure 13B Conductivity plot of CVB-D hydrochloride against E. coli in Example 10;

[0041] Figure 13C Conductivity plot of CVB-D hydrochloride against MRSA in Example 10;

[0042] Figure 14 Microprotein standard curve plot in Example 10.

[0043] Figure 15 Bacterial extracellular protein content plot in Example 10.

[0044] Figure 16 Scanning electron microscope (SEM) determination of morphological changes of S. aureus in Example 10.

[0045] Figure 17 Scanning electron microscope (SEM) determination of morphological changes of MRSA in Example 10.

[0046] Figure 18 Scanning electron microscope (SEM) determination of morphological changes of E. coli in Example 10. DETAILED DESCRIPTION

[0047] The technical solutions of the present application are described in detail below through specific examples, but the protection scope of the present application is not limited to the examples. If the specific techniques or conditions are not specified in the examples, the techniques or conditions are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, and if the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased through regular channels.

[0048] Ofloxacin was purchased from Shanghai Macklin Biochemical Science and Technology Co., Ltd.; Minocycline was purchased from Shanghai Macklin Biochemical Science and Technology Co., Ltd.; Cefmetazole sodium was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Cefpodoxime proxetil was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Penicillin V potassium was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Chloramphenicol was purchased from Shanghai Macklin Biochemical Science and Technology Co., Ltd.; S. aureus was purchased from Beijing Beisan Biological Company; MRSA was purchased from Ningbo Mingzhou Biological Company; E. coli was purchased from Nanjing Lezhen Biological Company; Agar was purchased from China National Pharmaceutical Group; Trypsinase and yeast extract were both purchased from OXOID Company; 0.1% TTC was purchased from China National Pharmaceutical Group;

[0049] Luria-Bertani liquid medium: 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride were weighed respectively and dissolved in 1000 mL of ultrapure water, 121 ℃ high-pressure sterilization for 15 min, and stored at 4 ℃ for standby;

[0050] Luria-Bertani solid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar were weighed into 1000 mL of ultrapure water, respectively, and sterilized at 121 ℃ for 15 min. The prepared solid medium was aliquoted into sterile culture dishes and stored at 4 ℃ for standby use.

[0051] Example 1: Isolation, purification, and characterization of cyclovirobuxin D

[0052] (1) Purification of cyclovirobuxin D by multiple recrystallization in methanol: 7.5 g of Buxus chinensis extract was added with an appropriate amount of methanol, and stirred and heated in a 65 ℃ oil bath. After the solution became clear, it was filtered while hot, and the filtrate was cooled in a 4 ℃ refrigerator to crystallize. The main component of the crystals was CVB-D. The above steps were repeated, and CVB-D was crystallized twice based on the obtained crystals. In this way, high-purity CVB-D was obtained after three recrystallizations.

[0053] (2) Purity analysis of cyclovirobuxin D: 3.0 mg of cyclovirobuxin D was precisely measured and prepared into a 0.5 mg / mL test solution. The mobile phase was prepared and dissolved according to the method in Table 1. 20 μL of the prepared test solution was precisely measured and injected into the high-performance liquid chromatograph, and the chromatogram was recorded. The purity of CVB-D was calculated by the peak area normalization method, as shown in Table 2. Figure 1

[0054] Table 1 HPLC purity detection chromatographic conditions

[0055]

[0056] Table 2 Purity of CVB-D extracted by methanol

[0057]

[0058] (3) IR analysis of cyclovirobuxin D: a small amount of dried CVB-D crystal was added with an appropriate amount of potassium bromide in a dry mortar and ground evenly. The sample was pressed into a nearly transparent sheet using a tablet press, and the infrared absorption of the sample was measured in the wave number range of 4000-400 cm -1 Figure 2

[0059] Table 3 Determination results of CVB-D infrared absorption spectrum

[0060]

[0061] ​​​(4) QTOF-MS analysis of cyclovirobuxin D: Mass spectrometry conditions: ion source: electrospray ion source (ESI); ion mode: positive ion mode (positive); capillary voltage: 3000 V; fragment voltage: 120 V; scan range: m / z: 300-500; drying gas temperature: 350 ℃; drying gas flow rate: 10 L / min; sheath gas temperature: 350 ℃; sheath gas flow rate: 12 L / min, and the results are as follows Figure 3 .

[0062] From the primary mass spectrum, the m / z of [M+H]+ is 403.36, so the relative molecular mass of the sample is 402.36, which is consistent with the relative molecular mass of C 26 H 46 N2O, so it can be inferred that the sample is CVB-D.

[0063] Example 2: Preparation and identification of cyclovirobuxin D hydrochloride

[0064] (1) Preparation of cyclovirobuxin D hydrochloride: Take 7.5 g of CVB-D in a round-bottom flask, add 90 mL of water, and place it in a 65 ℃ oil bath for heating. During the heating process, add 10% HCl dropwise until the pH value of the solution is about 6. Filter while hot, and place the filtrate in a 4 ℃ environment for crystallization. The crystals that precipitate are placed in a vacuum drying oven for drying. The dried crystals at this time are CVB-D hydrochloride.

[0065] (2) Identification of cyclovirobuxin D hydrochloride: HPLC is used to detect hydrochloric acid CVB-D, and the retention time and purity of the main component before and after salting are compared to explore whether the main component is affected during the salting process. The results are shown in Table 4.

[0066] Table 4 Change in cyclovirobuxin D component and purity before and after salting

[0067]

[0068] From Table 4 and Figure 4 , it can be seen that the retention time of cyclovirobuxin D before and after salting is consistent, the main component does not change during the preparation of cyclovirobuxin D hydrochloride, and no other impurities are introduced. The purity after salting is improved compared to the cyclovirobuxin D monomer before salting.

[0069] Example 3: Determination of the antibacterial activity of CVB-D hydrochloride by agar punching method

[0070] The test strain is diluted to OD 600 The corresponding value is 0.1, which corresponds to 10 8CFU / mL. Then it was diluted twice by 10 times, and then adjusted to 10 7 CFU / mL, 100 μL of the bacterial solution was taken to Luria-Bertani solid medium plate, and after waiting for 5 min, it was punched using a sterilized 6 mm puncher. The sample solution was prepared in a gradient, i.e. 5 mg of CVB-D hydrochloride was dissolved in 25 μL of CHCl3 and 4975 μL of PBS to prepare a sample solution of 1 mg / mL, and then the sample solution was gradient diluted so that its final concentration was 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL, and directly as a liquid medicine, an equal volume of liquid medicine was added to the hole so that the final content of the liquid medicine was 1600 μg, 800 μg, and 400 μg. The culture medium with the added liquid medicine was placed in a 4°C refrigerator to allow the liquid medicine to penetrate, and then placed in a 37°C CO2 incubator for 12 h, and after multiple experiments, the diameter of the inhibition zone was measured by cross method, and part of the test sample results are shown in Figure 5 A, B, and C in the drawings, Figure 5 A, B, and C in the drawings, Figure 5 A, B, and C in the drawings,

[0071] All sample statistical results are shown in Table 5.

[0072] Table 5: Inhibition zone range (mm) of CVB-D hydrochloride

[0073]

[0074] According to the pharmacological method, the diameter (d) of the inhibition zone <10 mm represents drug resistance and no inhibition effect, d = 10 mm is slightly sensitive, 11-15 mm is moderately sensitive, and d≥16 mm is highly sensitive. From Table 5, it can be seen that when the content of CVB-D hydrochloride is 1600 μg, it is highly sensitive to E. coli, and moderately sensitive to S. aureus and MRSA; when the content of the drug is 400 μg, it does not show inhibition effect on MRSA. At the same time, the experimental results show that the inhibition activity of CVB-D hydrochloride on the three test strains is: E. coli > S. aureus > MRSA.

[0075] Example 4: Determination of minimum inhibitory concentration (MIC)

[0076] Prepare the CVB-D hydrochloride sample solution with an initial concentration of 12.5 mg / mL in advance; sterilize the test tubes, number them, dilute the sample solution by the double dilution method, prepare 12 concentration gradients, with a total volume of 1 mL, each test tube containing 10 uL of 0.1% TTC, add 50 uL of the strain with an initial bacterial concentration of 5 x 10 5 CFU / mL, so that the final concentration of the sample is 12.5, 6.25, 3.125, 1.5, 0.75, 0.375, 0.1875, 0.090, 0.047, 0.024, 0.012, 0.006 mg / mL; place in a 37°C shaking incubator for 12 h, observe the color change of the solution in the test tube, the TTC added can react with the bacteria to change color, if the bacteria grow, the solution color will change to red or pink, the lowest concentration without color change of the solution is the minimum inhibitory concentration (MIC), the MIC experimental results of CVB-D hydrochloride on E. coli, S. aureus and MRSA are shown in Figure 6 、 Figure 7 、 Figure 8

[0077] Example 5: Determination of Minimum Bactericidal Concentration (MBC)

[0078] Take 100 uL of the culture in each test tube with the minimum inhibitory concentration of the above drug and above which no bacteria grow, i.e. the test tube does not change color, spread it evenly on a sterilized ordinary agar medium, and incubate in a 37°C incubator for 24 h, observe the bacterial growth, the minimum drug concentration corresponding to 5 or fewer colonies is the minimum bactericidal concentration (MBC), and the results are shown in Table 6.

[0079] Table 6: Colony growth of CVB-D hydrochloride on test strains

[0080]

[0081] Note: + for the number of colonies ≥5: - for the number of colonies ≤5

[0082] From Figure 6 、 Figure 7 、 Figure 8 ​As can be seen from Table 6, the hydrochloride salt of cyclovirobuxin D monomer, referred to as hydrochloric acid CVB-D, has certain inhibitory effect on gram-negative bacteria Escherichia coli and gram-positive bacteria Staphylococcus aureus. The MIC of hydrochloric acid CVB-D on Staphylococcus aureus and Escherichia coli is 0.75 mg / mL, and the MIC on MRSA is 1.5 mg / mL. The MBC of hydrochloric acid CVB-D on MRSA and Escherichia coli is 3.12 mg / mL, and the MBC on Staphylococcus aureus is 1.50 mg / mL. Therefore, hydrochloric acid CVB-D is a broad-spectrum antibacterial drug, which has certain inhibitory effect on gram-positive bacteria, gram-negative bacteria and drug-resistant bacteria.

[0083] Example 6: Determination of half inhibitory concentration (IC 50 )

[0084] Escherichia coli, Staphylococcus aureus and MRSA were respectively inoculated in 2 mL of Luria-Bertani liquid medium, and placed in a 37°C, 200 rpm incubator for shaking culture. After 16 h of activation, the bacteria were in the late exponential growth phase. An appropriate amount of the above bacterial solution was adjusted to have an OD 600 of 0.1, i.e. about 1×10 8 CFU / mL. The bacterial solution was diluted to have a concentration of 1×10 4 CFU / mL. CVB-D hydrochloride was dissolved in Luria-Bertani liquid medium to prepare a drug solution with an initial concentration of 3 mg / mL. In the experimental group, 100 uL of the drug solution and 100 uL of the bacterial strain were added, and then a two-fold gradient dilution method was used for dilution to have a final drug concentration of 1.50, 0.75, 0.375, 0.1875, 0.09, 0.047, 0.024, 0.012 mg / mL. In the blank control well, 200 uL of Luria-Bertani liquid medium was added, and in the bacterial growth well, 100 uL of Luria-Bertani liquid medium and 100 uL of the bacterial strain were added. Then, the 96-well plate was placed in a 37°C shaking incubator for 24 h. The OD value was measured at 600 nm by using an enzyme-labeled instrument. The bacteriostatic rate Y was calculated according to the following formula. The IC 50 value of each group was calculated by using GraphPad Prism.

[0085] Bacteriostatic rate Y = 1-[(OD 实验组 - OD 空白控制孔 ) / (OD 细菌生长控制孔 - OD 空白控制孔 )]

[0086] The calculated IC50 of CVB-D hydrochloride for E. coli, S. aureus, and MRSA were 0.40 mg / mL, 0.35 mg / mL, and 0.34 mg / mL, respectively. The IC50 of CVB-D hydrochloride for S. aureus and MRSA were close, indicating that CVB-D hydrochloride still had a good inhibitory effect on drug-resistant S. aureus. 50 The calculated IC50 of CVB-D hydrochloride for E. coli, S. aureus, and MRSA were 0.40 mg / mL, 0.35 mg / mL, and 0.34 mg / mL, respectively. The IC50 of CVB-D hydrochloride for S. aureus and MRSA were close, indicating that CVB-D hydrochloride still had a good inhibitory effect on drug-resistant S. aureus.

[0087] Example 7: Determination of 24 h time-kill curve

[0088] According to the preparation method in Example 3, drug solutions with concentrations of 0.75 mg / mL and 1.5 mg / mL were prepared, and the bacterial solution group was used as a blank control group. 500 uL of activated bacterial solution was taken into a test tube containing 2 mL of Luria-Bertani medium for overnight subculture. 20 uL of the subcultured bacterial solution was placed in 3.5 mL of drug-containing culture solution, and the OD value at 600 nm was measured every hour to draw the 24 h growth curve of the bacteria. A group without drug addition and a bacterial growth control group with equal amount of PBS were set for each experiment.

[0089] The 24 h growth curves of E. coli, S. aureus, and MRSA are shown in Figures Figure 9A , Figure 9B and Figure 9C respectively. It can be seen that CVB-D hydrochloride has a concentration-dependent effect on E. coli, S. aureus, and MRSA. CVB-D hydrochloride at a concentration of 1.5 mg / mL is a flat line, indicating that it can completely inhibit the growth and proliferation of E. coli, S. aureus, and MRSA. Compared with the bacterial solution control group, it can be seen that drugs with concentrations of 0.75 and 1.50 mg / mL can inhibit the above three bacteria, showing a concentration-dependent relationship.

[0090] Example 8: Determination of drug resistance of CVB-D hydrochloride to E. coli

[0091] In this example, antibiotics ofloxacin and chloramphenicol were used as positive controls, and E. coli was used as the test strain to observe the drug resistance of CVB-D hydrochloride.

[0092] Drug-containing MIC determination: Prepare a CVB-D hydrochloride sample solution with an initial concentration of 12.5 mg / mL in advance. Sterilize the test tubes and dilute the sample solution by the double dilution method to prepare eight concentration gradients, with a total volume of 1 mL in each test tube, containing 10 uL of 0.1% TTC, 50 uL of the strain, so that the final concentration of the sample is 12.5, 6.25, 3.125, 1.5, 0.75, 0.375, 0.1875, 0.09, 0.047, 0.024, 0.012, 0.006 mg / mL, and incubate at 37°C on a shaker for 12 h.

[0093] Antibiotic MIC determination: Prepare sample solutions of ofloxacin and chloramphenicol with an initial concentration of 16 mg / mL in advance. Sterilize the test tubes and dilute the CVB-D hydrochloride sample solution by the double dilution method to prepare eight concentration gradients, with a total volume of 1 mL in each test tube, containing 10 uL of 0.1% TTC, 50 uL of the strain, so that the final concentration of the sample is 16, 8, 4, 2, 1, 0.5, 0.25, 0.175 mg / mL, and incubate at 37°C on a shaker for 12 h.

[0094] Record the MIC values of the strain after 12 h, and adjust the OD value at 600 nm of the bacterial solution in the concentration lower than the MIC test tube to 5 x 10 5 CFU / mL. Repeat the above MIC determination test steps, and observe the changes in the MIC values after continuous culture for 7 days. The results are shown in Table 7 and Figure 10

[0095] Table 7 MIC determination values of E. coli

[0096]

[0097] It should be noted that in Table 7, MIC (7th day) / MIC (1st day) > 1 represents easy drug resistance. As can be seen from Table 7 and Figure 10 Table 8, ofloxacin is the most likely to produce drug resistance when acting on E. coli, followed by chloramphenicol. The MIC of CVB-D does not change in seven days, indicating that the drug resistance of the CVB-D monomer is good.

[0098] Example 9: Checkerboard microdilution method combined drug sensitivity test

[0099] The selected combined drugs in this example are: ofloxacin, minocycline, cefmetazole sodium, cefpodoxime proxetil, penicillin V potassium, and chloramphenicol. The test strain is E. coli.

[0100] ​Dissolve A drug, i.e. CVB-D hydrochloride, with sterilized liquid medium, dissolve B drug, i.e. the above-mentioned various existing antibiotics, respectively, and the concentrations of the two drugs are diluted to 8 MIC, 4 MIC, 2 MIC, MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC in turn, respectively, and CVB-D hydrochloride and the corresponding antibiotics are combined in vertical and horizontal columns, as follows:

[0101] A plate: in the first row of the 96-well plate, the first 8 wells are first added with 100 µL of 8 MIC concentration of A drug, and then 50 µL of bacterial solution is added to rows 2-7, and then 50 µL of 8 MIC, 4 MIC, 2 MIC, 1 MIC, 1 / 2 MIC, 1 / 4 MIC concentration of A drug is added in turn, so that the final concentrations of rows 2-7 are 4 MIC, 2 MIC, MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, respectively;

[0102] B plate: in the first row of the 96-well plate, the first 8 wells are first added with 100 µL of 8 MIC concentration of B drug, and then 50 µL of bacterial solution is added to rows 2-7, and then 50 µL of 8 MIC, 4 MIC, 2 MIC, 1 MIC, 1 / 2 MIC, 1 / 4 MIC concentration of B drug is added in turn, so that the final concentrations of rows 2-7 are 4 MIC, 2 MIC, MIC, 1 / 2 MIC, 1 / 4 MIC, 1 / 8 MIC, respectively;

[0103] A plate and B plate combination: the first column of the B plate is added to the first row of the A plate, the second column of the B plate is added to the second row of the A plate, and so on until the seventh row, at this time the eighth column of the combined A plate and B plate is used to determine the MIC of CVB-D hydrochloride alone, and the eighth row of the combined A plate and B plate is used to determine the MIC of the antibiotic alone. The intersection of the 8th row and the 8th column is finally 100 µL of bacterial solution broth, which is used as a positive control. Add 10 µL of 0.1% TTC solution to all the wells with bacterial solution and liquid medium. The combined 96-well plate is incubated at 37 ℃ for 18-24 h, each test is repeated three times, and the color change of each well is observed, and the concentration corresponding to the well without color change is the combined antibacterial concentration of the two drugs.

[0104] After the combination of CVB-D hydrochloride and antibiotics, the B drug (antibiotic) is used as the horizontal coordinate concentration, and the A drug (CVB-D hydrochloride) is used as the vertical coordinate concentration, and the concentration distribution of each drug in each well after combination is as follows Figure 11 .

[0105] The fractional inhibitory concentration index (FICI) is used as the basis for judging the combined antibacterial experiment: FICI = (MICA药联用 / MIC A药单用 )+ (MIC B药联用 / MIC B药单用 )wherein FICI≤0.5 represents synergistic effect; 0.5

[0106] Table 8 Inhibition of E. coli by combination of drugs

[0107]

[0108] From the above-mentioned combination drug sensitivity test, it can be seen that CVB-D hydrochloride combined with cefmetazole sodium shows synergistic effect, combined with ofloxacin and chloramphenicol shows additive effect, shows no correlation with cefpodoxime proxetil, and shows antagonistic effect with minocycline and penicillin V potassium. It shows that when CVB-D hydrochloride is used with cefmetazole sodium, ofloxacin and chloramphenicol, the original efficacy can be maintained, and the amount of antibiotics can be reduced.

[0109] Example 10: Study on the antibacterial mechanism of CVB-D hydrochloride

[0110] In this example, the effects of CVB-D hydrochloride on bacterial cells were observed by conductivity meter, determination of in vitro antibiofilm activity by crystal violet method, determination of extracellular protein content, and SEM observation. The antibacterial mechanism of CVB-D hydrochloride on the test bacteria was preliminarily explored by observing the leakage of charged substances, the change trend of extracellular protein content, and the change process of cell morphology.

[0111] (1) Determination of in vitro antibiofilm activity by crystal violet method

[0112] Bacterial monoclonal was inoculated into sterile solid medium and placed at 37°C with 200 rpm shaking for 24 h, then centrifuged to collect the bacterial strain. The bacteria were washed twice with PBS and centrifuged (5000 rpm x 5 min). The washed bacterial cells were suspended in liquid medium and the bacterial concentration was adjusted to 1 x 10 8 CFU / mL for biofilm formation.

[0113] The adjusted bacterial solution was evenly divided into 4 parts, 1.5 mL of each was taken into a test tube, and reacted at 37°C for 3 h, then centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with PBS (10000 rpm x 10 min).

[0114] The drug solution was prepared as 3.0, 1.5, 0.75 mg / mL, respectively, 1.5 mL of the above different concentrations of drug solution was added to the centrifuged bacteria, and 1.5 mL of liquid medium without drug was added to the centrifuged bacteria as a control group. Four tubes of bacteria solution were placed in a 37°C shaking bed for 16 h, the supernatant was discarded (10000 rpm x 10 min), the bacteria were washed twice with PBS (10000 rpm x 10 min), 1.5 mL of 10 μg / mL crystal violet was added, and it was placed at 37°C for 1 h, centrifuged (10000 rpm x 15 min), the supernatant was discarded, the bacteria were washed with UP water for 3 times, 1.5 mL of 95% ethanol was added, and it was placed at room temperature for 45 min, the OD value was measured at 570 nm, and the results are shown in Table 9. Figure 12 Table 9.

[0115] Table 9: Inhibition rate of CVB-D hydrochloride on bacterial biofilm

[0116]

[0117] In Table 9, NI represents no obvious inhibition. From Figure 12 and Table 9, when the concentration of CVB-D hydrochloride is 0.75 mg / mL, there is no obvious inhibition on MRSA. When the concentration of the drug is 1.5, 3.0 mg / mL, it has an inhibitory effect on the three test bacteria. The formation ability of biofilm has a concentration-dependent relationship with the drug, and the higher the concentration of the drug, the stronger the inhibitory ability of the biofilm.

[0118] (2) Determination of bacterial content leakage by conductivity

[0119] CVB-D drug with a concentration of 2 mg / mL was prepared with PBS, 15 mL of the drug was placed in a centrifuge tube, and 15 mL of liquid medium without drug was placed in a centrifuge tube, 5 mL of test strain (106 CFU / mL) was added to the above two tubes, respectively, and the culture solution was centrifuged (6000 rpm x 10 min) at 0, 4, 8, 12, 16 h, the supernatant was taken, and the conductivity was measured.

[0120] Figure 13A , Figure 13B , Figure 13CThe test results of the three test strains of S. aureus, E. coli and MRSA in turn showed that the conductivity of S. aureus and E. coli increased first and then stabilized, and the conductivity of S. aureus tended to be stable after 4 h, indicating that the damage of CVB-D hydrochloride to S. aureus mainly occurred in the early stage of the test; and the conductivity of MRSA showed a growing trend within 16 h. The conductivity of the three test bacteria was higher than that of the bacterial liquid control group, indicating that CVB-D hydrochloride can damage the cell membrane of the three test bacteria, cause the leakage of charged substances in the bacterial cells, and play its antibacterial activity.

[0121] (3) Bacterial extracellular protein content determination

[0122] In this test, the BCA method was used to detect the protein content.

[0123] Working solution (WR) preparation: Mix BCA reagent and Cu reagent at a ratio of 50:1 to obtain a light green solution as the working solution.

[0124] Standard protein solution preparation: Dilute the BSA standard solution with PBS to 80, 40, 20, 10, 5, 2.5 and 1.25 µg / mL.

[0125] Measurement method: Take 40 µL of the above prepared standard protein solution of different concentrations in a 96-well plate, add 200 µL of working solution, react at 37 ℃ for 30 min, measure the OD value at 562 nm, and draw the standard curve as Figure 14 . According to the above method, measure the protein content in the supernatant after the bacterial liquid reaction, calculate the protein content according to the standard curve, and the results are as follows Figure 15 .

[0126] As can be seen from Figure 15 , different concentrations of CVB-D hydrochloride caused the absorbance of extracellular protein of the three test bacteria to increase, and was significantly higher than that of the control group. The higher the drug concentration, the greater the absorbance value of extracellular protein, showing a concentration-dependent relationship. Extracellular protein was also measured in the control group, which may be caused by cytoplasm leakage due to natural death of bacteria.

[0127] (4) Scanning electron microscope (SEM) determination of bacterial morphological changes

[0128] Take a large number of bacteria after overnight culture (ensure that the OD value of the bacteria is more than 0.6), centrifuge (4000 rpm, 4 min), remove the supernatant, wash once with 0.1 M PBS with pH 7.2 (5000 rpm x 15 min), remove dead bacteria, give medicine, continue to culture for 12 h, centrifuge and discard the supernatant, wash 3 times with PBS (5000 rpm x 15 min); use 2.5% glutaraldehyde to fix for 8 h (fix under the condition of 4 ℃), after fixing, wash twice with PBS (5000 rpm x 15 min); adjust ethanol to concentrations of 30%, 50%, 70%, 80%, 90% and 100%, respectively, gradient dehydration with the above-mentioned concentrations of ethanol, once for the rest of the concentrations except for 100% ethanol which is dehydrated twice, the washing speed and time are 5000 rpm and 15 min; drop the treated sample on a glass slide (control the sample concentration, the sample concentration should not be too high), pre-freeze in a-10 ℃ refrigerator for 2 h, and then put it into a-80 ℃ freeze dryer for freeze-drying; paste carbon conductive tape on the sample holder of the scanning electron microscope, cut the glass slide with bacteria to a suitable shape, paste the sample to be tested on the carbon conductive tape, after ion sputtering, use the scanning electron microscope to observe, and the results are shown in Figure 16 、 Figure 17 and Figure 18 represent Staphylococcus aureus, MRSA and Escherichia coli groups, respectively, and Figure 16 、 Figure 17 and Figure 18 The left graph of each of them represents the blank control group, and the right graph represents the drug-added group.

[0129] It can be seen that the Staphylococcus aureus cells in the blank control group are complete and similar in shape, and no obvious changes have occurred. After adding the drug, exudates appear around the bacteria, the contents surround the bacterial cells, and the shape is irregular, and the two cells show adhesion phenomenon; MRSA and Escherichia coli both show that the bacterial cells in the blank control group are smooth, the shape is single, and the cell membrane is complete. After adding the drug, the bacterial morphology changes, the cells are concave, adhesion occurs, and the cell morphology is irregular. It is shown that hydrochloric acid CVB-D has a certain destructive effect on the three test bacteria.

[0130] As described above, although the present application has been shown and described with reference to specific preferred embodiments, it is to be understood that such is by way of illustration and not of limitation. Various changes and modifications can be made therein without departing from the spirit and scope of the application as defined in the appended claims.

Claims

1. Use of cyclovirobuxin D and pharmaceutically acceptable salts thereof for the preparation of an antibacterial medicament, characterized in that, The chemical structural formula of cyclovirobuxin D is as follows: ； The monomer molecular formula of cyclovirobuxin D is: C 26 H 46 N2O.

2. Use according to claim 1, characterized in that, Cyclovirobuxin D and any one or a combination of its pharmaceutically acceptable salts are the only drug active substances.

3. Use according to claim 1, characterized in that, The pharmaceutically acceptable salt is a hydrochloride salt; oral or injection administration is adopted, and the administration dose is 1.0 mg / kg.

4. Use according to claim 1, characterized in that, The antibacterial drug is any one of an anti-Gram-positive bacterial drug, an anti-Gram-negative bacterial drug or an anti-drug-resistant bacterial drug.

5. Use according to claim 4, characterized in that, The Gram-positive bacteria are Staphylococcus aureus.

6. Use according to claim 4, characterized in that, The Gram-negative bacteria are Escherichia coli.

7. Use according to claim 4, characterized in that, The super drug-resistant bacteria are methicillin-resistant Staphylococcus aureus.

8. An antibacterial pharmaceutical composition, characterized by, The pharmaceutical composition consists of cyclovirobuxin D hydrochloride and cefmetazole sodium.

9. An antibacterial pharmaceutical composition, characterized by, The pharmaceutical composition consists of cyclovirobuxin D hydrochloride and chloramphenicol.

10. An antibacterial pharmaceutical composition, characterized by, The pharmaceutical composition consists of cyclovirobuxin D hydrochloride and ofloxacin.

Images