New use of curcumin in enhancing antibacterial effect of tigecycline

Abstract

The present application relates to a new use of curcumin in synergistic tigecycline antibacterial effect. The use amount of curcumin is 32 μg / mL-128 μg / mL, and the use amount of tigecycline is 0.015-4 μg / mL. In the preparation of composition or compound preparation, when curcumin is used to synergize the antibacterial effect of tetracycline antibiotic tigecycline, the ratio of the use amount of curcumin to tigecycline is between 8:1-8533:1. The present application can restore the sensitivity of drug-resistant bacteria to tigecycline, reduce the dosage of tigecycline, improve the antibacterial treatment effect, reduce the toxic side effects, and has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to a new use of curcumin as a tigecycline synergist. Background Art

[0002] Tigecycline belongs to the new generation of tetracycline antibiotics and is a glycylcycline antibiotic. It was approved for marketing in the United States in 2005 and in my country in early 2012. Its primary mechanism of action is similar to that of other tetracyclines: reversible binding to the helical region (H34) of the bacterial ribosome 30S subunit, preventing tRNA entry and inhibiting bacterial protein translation (i.e., peptide chain elongation), ultimately blocking bacterial protein synthesis and limiting bacterial growth. However, the addition of an N-alkyl-glycylamino side chain at the C9 position of the D ring enhances ribosome binding and broadens its antibacterial spectrum. Its ability to inhibit protein synthesis is 3-20 times greater than that of other tetracyclines. Tigecycline can effectively overcome the main mechanisms of tetracycline resistance, such as the tetracycline-specific efflux pump Tet(A) and the ribosomal protection protein Tet(M).

[0003] Curcumin is a natural active ingredient extracted from the dried rhizome of Curcuma longa, a plant of the genus Curcuma in the Zingiberaceae family. It has low toxicity and is well tolerated. Curcumin has a molecular formula of C21H206, a molecular weight of 368.37 g / mol, and a melting point of 183°C. It is crystalline and is one of the few diketone pigments in the plant kingdom. The curcumin molecule contains multiple double bonds. The structural formula of curcumin is shown below:

[0004]

[0005] In recent years, with the increasing use of tigecycline and the abuse of other antibiotics, the therapeutic effect of tigecycline has declined, leading to the emergence of tigecycline resistance. According to the EUCAST European Union Antimicrobial Susceptibility Testing Standard, bacteria are considered resistant when the MIC value of tigecycline against Enterobacteriaceae is 4μg / mL. Currently, tigecycline resistance is showing a significant upward trend. Therefore, reducing its occurrence through potentiators has important clinical application significance. To date, there have been no reports of curcumin as a potentiator for tigecycline. Summary of the Invention

[0006] The present invention provides a novel use of a curcumin compound for enhancing the antibacterial effect of tigecycline. When the curcumin is used in an amount of 32 μg / mL to 128 μg / mL, the tigecycline is used in an amount of 0.015 to 4 μg / mL. When preparing a composition or compound preparation, the curcumin is used to enhance the antibacterial effect of tigecycline, and the ratio of curcumin to tigecycline is between 8:1 and 8533:1.

[0007] Preferably, the dosage of tigecycline is 0.5-2 μg / mL and the dosage of curcumin is 32 μg / mL-128 μg / mL.

[0008] Preferably, the dosage of tigecycline is 0.015-0.06 μg / mL and the dosage of curcumin is 32 μg / mL-128 μg / mL.

[0009] Preferably, the dosage of tigecycline is 1-4 μg / mL and the dosage of curcumin is 32 μg / mL-128 μg / mL.

[0010] Another object of the present invention is to provide a novel use of curcumin for enhancing the antibacterial activity of tigecycline against Escherichia coli. When the curcumin dosage is 32 μg / mL to 128 μg / mL, the tigecycline dosage is 0.015 to 4 μg / mL. When preparing a composition or compound preparation, the curcumin dosage ratio for enhancing the antibacterial activity of tigecycline is between 8:1 and 8533:1.

[0011] Preferably, the dosage of tigecycline is 0.5-2 μg / mL and the dosage of curcumin is 32 μg / mL-128 μg / mL.

[0012] Preferably, the dosage of tigecycline is 0.015-0.06 μg / mL and the dosage of curcumin is 32 μg / mL-128 μg / mL.

[0013] Preferably, the dosage of tigecycline is 1-4 μg / mL and the dosage of curcumin is 32 μg / mL-128 μg / mL.

[0014] Another object of the present invention is to provide an antibacterial test method or a target test method for the use of curcumin in enhancing the antibacterial activity of tigecycline against Escherichia coli, the method comprising the following steps:

[0015] S1: dissolving curcumin and tigecycline in a solvent;

[0016] S2: curcumin and tigecycline are mixed in proportion to obtain a composition;

[0017] S3: Using the composition of step S2 for MIC test, FICI test, time-kill curve test, or target test.

[0018] The solvent in step S1 includes dimethyl sulfoxide or deionized water.

[0019] In step S2, the ratio of curcumin to tigecycline is 8:1-8533:1. Preferably, the ratio of curcumin to tigecycline is 8:1-2133:1.

[0020] When the amount of curcumin in step S3 is 32μg / mL-128μg / mL, the amount of tigecycline is 0.015-4μg / mL. Preferably, the amount of tigecycline is 0.5-2μg / mL in combination with the amount of curcumin of 32μg / mL-128μg / mL. Preferably, the amount of tigecycline is 0.015-0.06μg / mL in combination with the amount of curcumin of 32μg / mL-128μg / mL. Preferably, the amount of tigecycline is 1-4μg / mL in combination with the amount of curcumin of 32μg / mL-128μg / mL;

[0021] The term "synergistic effect" as used herein means that the effect of two drugs taken together is greater than the algebraic sum of the effects of the individual drugs.

[0022] As used herein, the term "additive effect" means that the combined effect of two drugs is equal to the algebraic sum of their individual effects.

[0023] As used herein, the term "antagonism" means that the effect of two drugs taken together is less than the sum of their individual effects.

[0024] The present invention has the following beneficial effects:

[0025] (1) When the dosage of curcumin was 32-128 μg / mL, the dosage of tigecycline was 0.015-4 μg / mL, which could significantly enhance the antibacterial activity of tigecycline against the large intestine; (2) The combination of curcumin and tigecycline showed significant synergistic effect, with the FIC index ranging from 0.09375 to 0.375 (all less than 0.5), that is, the combination of the two showed significant synergistic effect; (3) The dosage of tigecycline can be effectively reduced. The dosage of tigecycline used alone is 267-8 times that of the combined use of curcumin and tigecycline; (4) The combined use of curcumin and tigecycline can play a synergistic role in reversing drug resistance, with the FIC index ranging from 0.09375 to 0.375. The combined use showed significant bactericidal effect after 12 hours and killed all bacteria within 24 hours. At 24 h, the colony counts in both combination groups were reduced by more than 2 log10 CFU / mL compared with TIG alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the time-killing curve of curcumin and tigecycline against strain CTR2. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0028] Example 1: MIC test

[0029] Materials and methods

[0030] 1. Drug testing

[0031] Curcumin (CUR): Beijing Solaibao Technology Co., Ltd., purity: 95%

[0032] Tigecycline (TIG): Beijing Solaibao Technology Co., Ltd., purity: 98%

[0033] Curcumin was prepared in dimethyl sulfoxide to a concentration of 5120 μg / mL. Tigecycline was prepared in deionized water to a concentration of 1280 μg / mL. The test drugs were stored at -20°C. Prior to testing, the drug stock solution was removed and thawed at room temperature, thoroughly mixed, and then pharmacodynamic testing was performed.

[0034] 2. Strains

[0035] Clinically isolated E. coli strains CTR2, CTR4, and CE34 were provided by the Pharmacology Laboratory of Henan Agricultural University. E. coli were activated by streaking onto MacConkey medium, incubated at 37°C for 16–18 h, and stored at 4°C until ready for use.

[0036] 3. Culture medium

[0037] LB broth: Produced by Beijing Aoboxing Biotechnology Co., Ltd., prepare 1000 ml of broth according to the product instructions, autoclave at 121°C for 15 minutes, and store at 4°C until used. SS agar: Produced by Beijing Aoboxing Biotechnology Co., Ltd., prepare 1000 ml of broth according to the product instructions, boil thoroughly to dissolve and sterilize, cool to 60°C, pour into sterilized plates, and store at 4°C until used. MHB broth: Produced by Beijing Aoboxing Biotechnology Co., Ltd., prepare 1000 ml of broth according to the product instructions, autoclave at 121°C for 15 minutes, and store at 4°C until used.

[0038] 4. Instruments

[0039] High-pressure steam sterilization pot (Shanghai Shen'an Medical Instrument Factory); AB204-N electronic analytical balance (Mettler-Toledo Instrument Shanghai Co., Ltd.); HZQ-R air bath oscillator (Beijing Liuyi Instrument Factory); DH-600 electric heating constant temperature incubator (Beijing Keyi Yongxing Instrument Co., Ltd.); DHG-9140A vacuum drying oven (Shanghai YiHeng Experimental Instrument Co., Ltd.); SW-CJ-JC super clean bench (Suzhou Antai Air Technology Co., Ltd.).

[0040] 5. Preparation of bacterial solution

[0041] Single colony of E. coli was picked from the MacConkey medium stored at 4℃ and inoculated into 5 mL LB broth medium, and cultured at 37℃ with 180 rpm shaking to activate the bacteria, so that the bacteria were in the late exponential growth phase. The bacterial solution was taken into MHB broth medium, and the original bacterial solution concentration was diluted to 10 5 CFU / mL. The corresponding concentration of curcumin was added to the bacterial suspension for standby. The final concentration of curcumin in the bacterial suspension was 32 μg / mL, 64 μg / mL and 128 μg / mL, respectively.

[0042] 6. Preparation of drug sensitivity plate

[0043] The bacterial suspension in step 5 was used for drug sensitivity test. A sterile 96-well reaction plate was taken, 126 μL of fresh prepared bacterial suspension containing curcumin and 14 μL of 1280 μg / mL tigecycline were added to the first row, first well; 70 μL of fresh prepared bacterial suspension containing curcumin was added to the 2-12 wells; 70 μL of fresh prepared bacterial suspension containing curcumin was also added to the second row, 1-7 wells, and 70 μL of MHB broth was added to the 8th well as a blank control. The first row, 1-12 wells and the second row, 1-6 wells were serially diluted by 2 times, so that the final concentration of tigecycline in the first row, 1-12 wells and the second row, 1-6 wells was 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, 0.008, 0.004, 0.002, 0.001 μg / mL, respectively; the second row, 7th well was MHB bacterial suspension without drug as negative control; the 8th well was bacterial solution without drug as positive control. The drug sensitivity plate was cultured at 37℃ for 16-20 h.

[0044] 7. Determination of MIC value

[0045] After incubating the bacteria-containing 96-well plates at 37°C for 16-20 hours, the MIC (minimum inhibitory concentration) results were read. The drug MIC value (minimum inhibitory concentration) is the drug concentration in the clear well immediately preceding the turbid well in the 96-well plate. Each experiment was repeated three times in parallel, and only samples with accurately replicated MIC values ​​were accepted. If the MIC values ​​differed by more than one concentration, a new experiment was performed until the results met the requirements. The results are shown in Table 1.

[0046] Table 1 MIC of curcumin combined with tigecycline against the tested strains

[0047]

[0048] Note: 1 / 2MIC curcumin: curcumin added at 128μg / mL; 1 / 4MIC curcumin: curcumin added at 64μg / mL; 1 / 8MIC curcumin: curcumin added at 32μg / mL

[0049] The results showed that the MIC values ​​of tigecycline against strains CTR2, CTR4 and CE34 were 16, 4 and 32 μg / mL, respectively, and the MIC values ​​of curcumin against strains CTR2, CTR4 and CE34 were all 256 μg / mL. When 1 / 2MIC (128 μg / mL), 1 / 4MIC (64 μg / mL) and 1 / 8MIC (32 μg / mL) of curcumin were added, the MICs of tigecycline against CTR2 were 0.5, 1 and 2 μg / mL, respectively; the MICs against CTR4 were 0.015, 0.03 and 0.06 μg / mL, respectively; and the MICs against CE34 were 1, 2 and 4 μg / mL, respectively.

[0050] Compared with the MIC values ​​of tigecycline alone, when 128 μg / mL, 64 μg / mL, and 32 μg / mL of curcumin were added, the MICs of tigecycline against strain CTR2 decreased by 32, 16, and 8 times, respectively; the MICs of tigecycline against strain CTR4 decreased by 267, 133, and 67 times, respectively; and the MICs of tigecycline against strain CE34 decreased by 32, 16, and 8 times, respectively.

[0051] The results showed that curcumin at a concentration of 32-128 μg / mL significantly enhanced the antibacterial activity of tigecycline against Escherichia coli CTR2, CTR4, and CE34. Tigecycline alone was 267-8 times more effective than the combined dose of curcumin and tigecycline.

[0052] Example 2: FICI testing

[0053] Materials and methods

[0054] 1. Drug testing:

[0055] Same as Example 1.

[0056] 2. Strains

[0057] Same as Example 1.

[0058] 3. Culture medium

[0059] Same as Example 1.

[0060] 4. Instruments

[0061] Same as Example 1.

[0062] 5. Bacterial Liquid Preparation

[0063] Same as Example 1.

[0064] 6. Preparation of Drug Sensitivity Plates

[0065] Using the broth microdilution method, we determined the MIC values ​​for CUR and TIG alone. Concentrations of CUR and TIG ranging from 1 to 1 / 1024 times the MIC were selected and placed on a drug susceptibility checkerboard. Specifically, 100 μL of MHB was dispensed into an 8×8 96-well plate. TIG and CUR were then diluted 2-fold along the horizontal and vertical axes, respectively. The MIC values ​​for CUR and TIG alone, as well as negative and positive controls, were also used. (The 96-well plate was placed in a constant temperature incubator at 37°C for 16–18 hours. The results were observed and recorded. The experiment was repeated three times.)

[0066] The Fractional Inhibitory Concentration Index (FICI) was used as the criterion for the chessboard susceptibility test. The FICI of two compounds A and B was defined by the following formula:

[0067] FICI=FIC A +FIC B =MIC AB / MIC A +MIC BA / MIC B

[0068] MIC A is the MIC of compound A alone, MIC AB is the MIC of A when used in combination, MIC B is the MIC of compound B alone, MIC BA is the MIC of B when used in combination. A FIC < 0.5 indicates synergism; 0.5 ≤ FIC ≤ 1 indicates additive effect; 1 < FIC ≤ 2 indicates indifference; and FIC > 2 indicates antagonism. The results are shown in Table 2.

[0069] Table 2 Combined antibacterial effect of curcumin and tigecycline

[0070]

[0071] The results show that the curcumin alone has weak bacteriostatic effect on the tested strains, and the combination of curcumin and TIG shows synergistic effect on TIG, with FIC index of 0.09375-0.375, all less than 0.5, i.e. the combination of the two shows synergistic effect.

[0072] Example 3: Time-kill curve determination

[0073] Materials and methods

[0074] 1. Reagents:

[0075] The same as in Example 1.

[0076] 2. Strains

[0077] The same as in Example 1.

[0078] 3. Culture medium

[0079] The same as in Example 1.

[0080] 4. Instruments

[0081] The same as in Example 1.

[0082] 5. Preparation of bacterial solution

[0083] The same as in Example 1.

[0084] 6. Time-kill curve

[0085] Escherichia coli CTR2 was selected for time-kill curve test. A single colony was inoculated into fresh LB broth and placed in a 37°C, 180 r / min shaker for overnight culture; then 1 mL of the bacterial solution was transferred into 10 mL of fresh LB broth and divided into 5 test groups:

[0086] Group A: Control;

[0087] Group B: 1 / 2 MIC TIG;

[0088] Group C: 1 / 2 MIC CUR;

[0089] Group D: 1 / 2 MIC CUR + 1 / 2 MIC TIG;

[0090] Group E: 1 / 4 MIC CUR + 1 / 2 MIC TIG.

[0091] After grouping, place the cells at 37°C and shake culture at 180 rpm. 100 μL of bacterial suspension was taken at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. 100 μL of bacterial suspension was serially diluted 10-fold with PBS buffer, and 100 μL of the dilution was spread on LB agar plates. The cells were cultured at 37°C for 16 to 18 h. Finally, the colonies on the agar plates were counted, with time as the horizontal axis, and log 10 The time-kill curve was drawn with CFU / mL as the vertical axis, and the experiment was repeated three times.

[0092] Result determination: Compared with single drug, the colony count of the combination drug group decreased by ≥2log 10 CFU / mL, it is defined as synergistic effect, and the results are as follows Figure 1 As shown. The experimental results showed that the bactericidal curve of single drug 1 / 2MIC CUR against the strain was almost the same as that of the blank control (control), indicating that the antibacterial effect of CUR was weak; single drug 1 / 2MIC TIG initially showed a slight antibacterial effect on the strain, but then the growth trend was consistent with the blank control and 1 / 2MIC CUR; the bactericidal curve of 1 / 2MIC CUR or 1 / 4MIC CUR combined with 1 / 2MIC TIG was consistent, that is, it showed obvious bactericidal effect after 12h, and killed all bacteria in 24h. At 24h, compared with TIG alone, the colony count of the two combination groups was reduced by more than 2log 10 The time-kill curve results were basically consistent with the combined susceptibility results, indicating that CUR alone had a weak antibacterial effect. The combination of 1 / 2MIC CUR or 1 / 4MIC CUR with TIG significantly reduced the MIC value of TIG against resistant bacteria, demonstrating a good synergistic effect in reversing resistance.

[0093] These experiments demonstrated that curcumin at concentrations of 32 to 128 μg / mL significantly enhanced the antibacterial activity of tigecycline against Escherichia coli CTR2, CTR4, and CE34. The combination of curcumin and tigecycline exhibited significant synergistic effects, with FIC indices ranging from 0.09375 to 0.375, all below 0.5, indicating a significant synergistic effect. Time-kill curves revealed that the combined drug showed a significant bactericidal effect after 12 hours and completely eliminated all bacteria within 24 hours, consistent with the combined drug susceptibility results, indicating that CUR alone had a weak antibacterial effect. The combination of 1 / 2 MIC CUR or 1 / 4 MIC CUR with TIG significantly reduced the MIC value of TIG against resistant bacteria, demonstrating a strong synergistic effect in reversing resistance.

[0094] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Application of curcumin in the preparation of a synergist for the antibacterial effect of tigecycline on Escherichia coli.

2. The use according to claim 1, characterized in that When the dosage of curcumin is 32 μg / mL-128 μg / mL, the dosage of tigecycline is 0.015 μg / mL-4 μg / mL.

3. The use according to claim 1, characterized in that , the dosage ratio of curcumin to tigecycline was 8:1-8533:

1.

4. A tigecycline compound preparation, characterized in that: The invention comprises curcumin, wherein the ratio of the curcumin to tigecycline is 8:1-8533:1.

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