Application of usnic acid in preparation of anti-vibrio parahaemolyticus medicine, pharmaceutical composition and application of pharmaceutical composition

Abstract

The invention provides application of usnic acid in preparation of anti-vibrio parahaemolyticus drugs, a pharmaceutical composition and application of the pharmaceutical composition, and belongs to the technical field of biological medicines. The pharmaceutical composition containing usnic acid and colistin is simple in function addition, can achieve a synergistic antibacterial effect, has an obvious synergistic antibacterial effect, and can synergistically resist vibrio parahaemolyticus. A chessboard method minimum inhibitory concentration test and a minimum bactericidal concentration test prove that the combination of usnic acid and colistin has an obvious synergistic antibacterial activity effect, and the problems of drug resistance of multi-drug-resistant gram-negative bacteria and lack of effective drugs are solved. The invention relates to new application of usnic acid and usnic acid combined with colistin in treatment of vibrio parahaemolyticus infection, and the usnic acid and the colistin have important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of usnic acid in the preparation of an anti-Vibrio parahaemolyticus drug, a drug composition and an application thereof. Background Art

[0002] Vibrio parahemolyticus is an important foodborne pathogen, a Gram-negative bacillus with a strong ability to survive in the environment. Clinically, the main symptoms are acute onset, abdominal pain, vomiting, diarrhea and watery stools. The most effective treatment for Vibrio parahemolyticus infection is generally the use of antibiotics, but the irrational use of antibiotics in aquaculture and clinical practice has also led to an increasingly serious phenomenon of Vibrio parahemolyticus resistance. The development of new antimicrobial drugs has become one of the important ways to curb bacterial resistance in clinical practice. However, the development of new antibiotics lags far behind the rapid evolution and spread of antibiotic resistance. Compared with the discovery of new antimicrobial drugs, the combined strategy brings hope for clinical treatment by restoring the sensitivity of resistant strains to existing antibiotics.

[0003] Colistin (polymyxin E, colistin) exerts its antibacterial effect by binding to the lipopolysaccharide of the outer membrane of Gram-negative bacteria, mediating cell membrane rupture and leakage of intracellular substances, and ultimately leading to bacterial lysis. When treating "super-resistant bacteria" that produce carbapenemase, colistin is considered the last resort and is also the drug of choice for treating severe infections caused by multidrug-resistant Gram-negative bacteria. Although colistin can quickly eliminate bacteria, its in vivo efficacy has always been poor, and as many as 70% of patients do not respond well to colistin treatment. Due to the nephrotoxicity and neurotoxicity of colistin, colistin should not be used excessively. Therefore, the problem of drug resistance of multidrug-resistant Gram-negative bacteria and the lack of effective drugs needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide an application of usnic acid in the preparation of anti-Vibrio parahaemolyticus drugs, a pharmaceutical composition and its application. Usnic acid has a significant inhibitory effect on Vibrio parahaemolyticus. Usnic acid has a significant synergistic effect on Vibrio parahaemolyticus after being used in combination with colistin, which greatly reduces the amount of colistin used in the treatment of Vibrio parahaemolyticus, and shows a strong application value in the process of preparing drugs for the treatment of Vibrio parahaemolyticus infection.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides an application of usnic acid in preparing an anti-Vibrio parahaemolyticus medicament.

[0007] The invention provides a pharmaceutical composition for preparing an anti-Vibrio parahaemolyticus drug, comprising usnic acid and colistin.

[0008] Preferably, the colistin is polymyxin E or a pharmaceutically acceptable salt thereof.

[0009] Preferably, the colistin is colistin sulfate.

[0010] Preferably, the mass ratio of usnic acid to colistin is 2.5 to 160:1.

[0011] Preferably, the mass ratio of usnic acid to colistin is 5 to 20:1.

[0012] Preferably, the dosage form of the pharmaceutical composition is one of tablets, capsules, powders, sustained-release tablets, controlled-release tablets, oral liquids, syrups, injection liquid dosage forms, and lyophilized powder injection dosage forms.

[0013] The present invention provides application of the pharmaceutical composition described in the above technical solution in preparing an anti-Vibrio parahaemolyticus drug.

[0014] The present invention provides an application of usnic acid in the preparation of an anti-Vibrio parahaemolyticus drug and a pharmaceutical composition, wherein the pharmaceutical composition containing usnic acid and colistin is not only a simple addition of functions, but also can achieve a synergistic antibacterial effect, has an obvious synergistic antibacterial synergistic effect, and can synergistically resist Vibrio parahaemolyticus.

[0015] The present invention proves that the combination of usnic acid and colistin has obvious synergistic antibacterial activity (the test bacteria is Vibrio parahaemolyticus) through the chessboard method minimum inhibitory concentration test and the minimum bactericidal concentration test, and solves the problem of drug resistance of multi-drug resistant Gram-negative bacteria and lack of effective drugs. The present invention is a new use of usnic acid and usnic acid combined with colistin in treating Vibrio parahaemolyticus infection, which has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the chessboard method for the combination of usnic acid and colistin against Vibrio parahaemolyticus ATCC17802;

[0017] Figure 2 The chessboard method results of usnic acid combined with colistin against Vibrio parahaemolyticus A20231098;

[0018] Figure 3 The results of the chessboard method for the combination of usnic acid and colistin against Vibrio parahaemolyticus 23b1412;

[0019] Figure 4 The results of the bactericidal curve of usnic acid combined with colistin against Vibrio parahaemolyticus ATCC17802. DETAILED DESCRIPTION

[0020] In the present invention, unless otherwise specified, the required reagents are commercially available products well known to those skilled in the art.

[0021] The invention provides an application of usnic acid in preparing an anti-Vibrio parahaemolyticus medicament.

[0022] The present invention is to use the usnic acid (CAS No. 125-46-2, molecular formula C 18 H 16 O7, molecular weight 344.32) is not particularly limited to its source, and any commercially available product known in the art may be used, and its structural formula is:

[0023]

[0024] The invention provides a pharmaceutical composition for preparing an anti-Vibrio parahaemolyticus drug, comprising usnic acid and colistin.

[0025] In the present invention, the colistin is preferably polymyxin E or a pharmaceutically acceptable salt thereof. The present invention has no particular limitation on the pharmaceutically acceptable salt, and any pharmaceutically acceptable salt of colistin known in the art may be used.

[0026] In the present invention, the colistin is preferably colistin sulfate. The present invention has no particular limitation on the source of the colistin sulfate, and any commercially available product known in the art may be used.

[0027] In the present invention, the mass ratio of usnic acid to colistin is preferably 2.5 to 160:1, more preferably 5 to 100:1, and further preferably 5 to 20:1.

[0028] In the present invention, the dosage form of the pharmaceutical composition is preferably one of tablets, capsules, powders, sustained-release tablets, controlled-release tablets, oral liquids, syrups, injection liquid dosage forms, and freeze-dried powder injection dosage forms. The present invention has no particular limitation on the preparation methods of different dosage forms, and the corresponding dosage forms can be obtained by methods well known in the art to meet the above dosage requirements.

[0029] The present invention provides application of the pharmaceutical composition described in the above technical solution in preparing an anti-Vibrio parahaemolyticus drug.

[0030] The active ingredients of the anti-Vibrio parahaemolyticus drug of the present invention may be only usnic acid and colistin or pharmaceutically acceptable salts thereof, such as colistin sulfate; other antibacterial active ingredients may also be included. The present invention has no particular limitation on the specific types of the other active ingredients, and any active ingredients well known in the art may be used. The present invention preferably determines the amount of the other antibacterial ingredients added based on the antibacterial effect.

[0031] The anti-Vibrio parahaemolyticus drug of the present invention has a killing and / or inhibitory effect on bacteria.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product specifications. The raw materials and equipment used in the examples are well known to those skilled in the art, and can be purchased or easily obtained or prepared on the market.

[0034] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0035] The usnic acid used in the present invention has a CAS number of 125-46-2 and a molecular formula of C 18 H 16 O7, molecular weight 344.32, purchased from Aladdin Reagent Company, catalog number U275043, purity >98%.

[0036] Usnic acid was prepared into a stock solution with a concentration of 40 mg / mL using DMSO and stored in a -20°C refrigerator.

[0037] Colistin sulfate was purchased from Aladdin Reagent Company, catalog number C114323, activity>19000U / mg. Colistin sulfate was prepared with distilled water to a stock solution concentration of 2mg / mL and stored at -20°C for future use.

[0038] The standard strain of Vibrio parahaemolyticus ATCC17802 was purchased from Guangdong Microbiological Culture Collection Center. Vibrio parahaemolyticus A20231098 and Vibrio parahaemolyticus 23b1412 were provided by Sanya People's Hospital.

[0039] Bacterial culture medium was used in the following experiments:

[0040] CAMHB broth culture medium was purchased from BD Biosciences (Shanghai) Co., Ltd. and the preparation method was as follows: weigh 22.0 g into 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C under high pressure for 15 min, and set aside.

[0041] MHA medium was purchased from Beijing Luqiao Technology Co., Ltd. and prepared as follows: weigh 38.0 g into 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121°C under high pressure for 15 min, cool to 55°C and pour into a plate for later use.

[0042] Brain heart infusion medium (BHI) was purchased from Beijing Luqiao Technology Co., Ltd. The preparation method is as follows: weigh 38.5 g of this product, dissolve it in 1000 mL of distilled water with heating and stirring, adjust the pH to 7.3, and sterilize it under high pressure at 121°C for 15 minutes for use.

[0043] BHI solid culture medium was purchased from Beijing Luqiao Technology Co., Ltd. and was prepared as follows: weigh 50.0 g of the product in 1000 mL of distilled water, heat and boil until completely dissolved, sterilize at 121°C under high pressure for 20 min, cool to 55°C and pour into a plate for later use.

[0044] Example 1

[0045] Evaluation of usnic acid and colistin alone and in combination against Vibrio parahaemolyticus:

[0046] Determination of the combined use index (FICI) of usnic acid and colistin against pathogens: The checkerboard method was used to determine the combined antibacterial effect of usnic acid and colistin against Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus A20231098, Vibrio parahaemolyticus 23b1412 and other strains.

[0047] Specifically, the MIC of usnic acid and colistin sulfate alone was detected using the multiple dilution method. CAMHB broth medium was used for multiple dilution, and 50 μL of CAMHB broth medium containing different concentrations of the two drugs was added along the horizontal and vertical axes of the 96-well culture plate, and then 50 μL of various pathogenic bacteria were added respectively, so that the final bacterial count in each well was 2×10 5 CFU (OD value at 600nm absorbance is 0.08), the final concentrations of usnic acid are 160μg / mL, 80μg / mL, 40μg / mL, 20μg / mL, 10μg / mL, 5μg / mL, 2.5μg / mL, and the concentrations of colistin sulfate are 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL. Culture at 37℃ for 24h, and observe the results. When the OD value at 600nm absorbance is less than 0.1, it is judged as antibacterial.

[0048] Specifically, Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus A20231098, and Vibrio parahaemolyticus 23b1412 strains were cultured normally using CAMHB medium, and the checkerboard method was used to test the combined antibacterial effect of the two. The specific operation of the checkerboard method is as follows: usnic acid (as drug A) and colistin (as drug B) were diluted in multiple ratios using CAMHB broth medium at the highest concentrations of 80 μg / mL and 16 μg / mL, respectively. 50 μL of CAMHB broth medium containing different concentrations of the two drugs was added along the horizontal and vertical axes of the 96-well culture plate, and then 50 μL of various pathogenic bacteria were added, so that the final number of bacteria in each well was 2×10 5 CFU, culture at 37℃ for 24h, and observe the results. Record the minimum inhibitory concentration (MIC) of the two drugs when used alone and in combination, and calculate the FICI value (fractional inhibitory concentration index) according to the following formula.

[0049] FICI = MIC of drug A combined / MIC of drug A alone + MIC of drug B combined / MIC of drug B alone, judgment criteria: FICI ≤ 0.5, synergistic effect; 0.5 < FICI ≤ 1, additive effect; 1 < FICI ≤ 2, irrelevant effect; FICI > 2, antagonistic effect. That is: FICI of usnic acid and colistin = MIC (usnic acid combined) / MIC (usnic acid alone) + MIC (colistin combined) / MIC (colistin alone).

[0050] The results are shown in Table 1. The MICs of usnic acid against Vibrio parahaemolyticus ATCC17802, A20231098 and 23b1412 strains were 80 μg / mL, 80 μg / mL and 160 μg / mL, respectively.

[0051] Table 1 MIC values ​​of usnic acid and colistin sulfate against Vibrio parahaemolyticus

[0052]

[0053] The results of combined antimicrobial Figures 1 to 3 As shown:

[0054] From Table 1 and Figure 1It can be seen that for Vibrio parahaemolyticus ATCC17802 strain, the MICs of usnic acid and colistin sulfate alone were 80 μg / mL and 2 μg / mL, respectively; when the two were used in combination, when the usnic acid concentration was in the range of 1.25-20 μg / mL and the colistin sulfate concentration was in the range of 0.25-0.5 μg / mL, there was an obvious synergistic effect, and the FICI was less than or equal to 0.5, that is, when the ratio of usnic acid to colistin sulfate was 2.5-80:1, they could synergistically resist bacteria; further, when the concentration of colistin sulfate was 0.25 μg / mL and the concentration of usnic acid was 1.25 μg / mL, the FICI was the smallest, which was 0.14, that is, when the optimal ratio of usnic acid to colistin sulfate was 5:1, the best synergistic effect was achieved.

[0055] From Table 1 and Figure 2 It can be seen that for Vibrio parahaemolyticus A20231098 strain, the MICs of usnic acid and colistin sulfate alone were 80 μg / mL and 8 μg / mL, respectively. When the two were used in combination, when the concentration of usnic acid was 20 μg / mL and the concentration of colistin sulfate was in the range of 1-2 μg / mL, there was an obvious synergistic effect, and the FICI was less than or equal to 0.5, that is, when the ratio of usnic acid to colistin sulfate was 10-20:1, they could synergistically resist bacteria; further, when the concentration of colistin sulfate was 1 μg / mL and the concentration of usnic acid was 20 μg / mL, the FICI was the smallest, which was 0.375, that is, when the optimal ratio of usnic acid to colistin sulfate was 20:1, the best synergistic effect was achieved.

[0056] From Table 1 and Figure 3 It can be seen that for Vibrio parahaemolyticus 23b1412 strain, the MICs of usnic acid and colistin sulfate alone were 160 μg / mL and 8 μg / mL, respectively. When the two were used in combination, when the usnic acid concentration was in the range of 20-40 μg / mL and the colistin sulfate concentration was in the range of 0.25-2 μg / mL, there was an obvious synergistic effect, and the FICI was less than or equal to 0.5, that is, when the ratio of usnic acid to colistin sulfate was 10-160:1, they could synergistically resist bacteria; further, when the concentration of colistin sulfate was 1 μg / mL and the concentration of usnic acid was 20 μg / mL, the FICI was the smallest, which was 0.25, that is, when the optimal ratio of usnic acid to colistin sulfate was 20:1, the best synergistic effect was achieved.

[0057] The above test results show that usnic acid has a good antibacterial effect on Vibrio parahaemolyticus. At the same time, the combined use of usnic acid and colistin has an obvious synergistic antibacterial effect, which can significantly reduce the minimum inhibitory concentration (MIC) of Vibrio parahaemolyticus to colistin (from 2-8μg / mL to 0.25-2μg / mL), and the FICI is in the range of 0.14 to 0.375, corresponding to a concentration ratio of 5 to 160:1.

[0058] Example 2

[0059] Determination of the minimum bactericidal concentration (MBC) of usnic acid and colistin sulfate used alone and in combination:

[0060] The minimum bactericidal combination of usnic acid and colistin sulfate against Vibrio parahaemolyticus ATCC17802, A20231098 and 23b1412 strains was determined by the checkerboard method.

[0061] Specifically, after the Vibrio parahaemolyticus ATCC17802, A20231098 and 23b1412 strains were tested by the chessboard method, 10 μL of the culture solution from each well without growth in the 96 wells was aspirated and spotted on the MHA agar culture plate, and cultured at 37°C for 24 hours. The lowest concentration in the well with no growth was the MBC. The results are shown in Table 2:

[0062] Table 2 MBC values ​​of usnic acid and colistin sulfate alone or in combination against Vibrio parahaemolyticus

[0063]

[0064] As shown in Table 2, for Vibrio parahaemolyticus ATCC17802 strain, the MBCs of usnic acid and colistin used alone were 160 μg / mL and 4 μg / mL, respectively, and the minimum bactericidal combination concentration MBC of the two were 10 μg / mL and 0.25 μg / mL, respectively, and the synergistic bactericidal index was 0.125.

[0065] For Vibrio parahaemolyticus A20231098, the MBCs of usnic acid and colistin used alone were 160μg / mL and 8μg / mL, respectively, and the minimum bactericidal combination concentration MBC of the two were 20μg / mL and 2μg / mL, respectively, with a synergistic bactericidal index of 0.375.

[0066] For Vibrio parahaemolyticus 23b1412, the MBCs of usnic acid and colistin used alone were 160 μg / mL and 8 μg / mL, respectively, and the minimum bactericidal combination concentration (MBC) of the two combined was 40 μg / mL and 1 μg / mL, respectively, with a synergistic bactericidal index of 0.375.

[0067] The above data show that, according to the MBC data, usnic acid and colistin sulfate have obvious synergistic bactericidal effects on Vibrio parahaemolyticus, and the optimal ratio of usnic acid and colistin sulfate is in the range of 10-40:0.25-2.

[0068] Example 3

[0069] In vitro bactericidal curve test of usnic acid and colistin combined against Vibrio parahaemolyticus

[0070] 3.1 Test materials

[0071] The source of Vibrio parahaemolyticus ATCC17802 strain is the same as that in Example 1.

[0072] 3.2 In vitro sterilization curve test

[0073] Vibrio parahaemolyticus ATCC17802 was cultured in BHI broth for 6 hours and then usnic acid, colistin, and a mixture of usnic acid and colistin were added. The groups and dosages were as follows:

[0074] Control group (Control): bacteria were treated with an equal dose of solvent, i.e., 1 wt% DMSO;

[0075] Usnic acid-1: The final concentration of usnic acid is 1.25 μg / mL;

[0076] Colistin group-1 (Colistin): the final concentration of colistin was 0.25 μg / mL;

[0077] Usnic acid and colistin combined group-1: the final concentration of usnic acid was 1.25 μg / mL, and the final concentration of colistin was 0.25 μg / mL;

[0078] Usnic acid-2: The final concentration of usnic acid is 2.5 μg / mL;

[0079] Colistin group-2 (Colistin): the final concentration of colistin was 0.5 μg / mL;

[0080] Usnic acid and colistin combined group-2: the final concentration of usnic acid was 2.5 μg / mL, and the final concentration of colistin was 0.5 μg / mL;

[0081] Usnic acid-3: Usnic acid final concentration 5 μg / mL;

[0082] Colistin group-3 (Colistin): the final concentration of colistin was 1 μg / mL;

[0083] Usnic acid and colistin combined group-3: the final concentration of usnic acid was 5 μg / mL, and the final concentration of colistin was 1 μg / mL.

[0084] Then, 100 μL of bacterial solution was spread on an MHA agar culture plate with a diameter of 10 cm at 1 h, 3 h, 6 h, 12 h and 24 h, and the colonies were counted after overnight culture.

[0085] 3.3 Test results

[0086] The results are as follows Figure 4As shown, for Vibrio parahaemolyticus ATCC17802 strain, at 24h, compared with the control group, 0.25μg / mL colistin, 0.5μg / mL colistin, 1μg / mL colistin, 1.25μg / mL usnic acid, 2.5μg / mL usnic acid, and 5μg / mL usnic acid alone had no significant effect on the number of bacterial colonies. When usnic acid and colistin were treated together, at 12h, the number of colonies was significantly reduced compared with the control group and usnic acid or colistin alone. After 1μg / mL colistin and 5μg / mL usnic acid were used together, the colony count was 0Log10 CFU / mL at 24h.

[0087] The above data indicate that the combined treatment of usnic acid and colistin has a significant synergistic bactericidal effect against Vibrio parahaemolyticus.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of usnic acid in the preparation of an anti-Vibrio parahaemolyticus drug.

2. A pharmaceutical composition for preparing an anti-Vibrio parahaemolyticus drug, characterized in that: Including usnic acid and colistin.

3. The pharmaceutical composition according to claim 2, characterized in that The colistin is polymyxin E or a pharmaceutically acceptable salt thereof.

4. The pharmaceutical composition according to claim 2, characterized in that The colistin is colistin sulfate.

5. The pharmaceutical composition according to claim 4, characterized in that The mass ratio of usnic acid to colistin is 2.5 to 160:

1.

6. The pharmaceutical composition according to claim 5, characterized in that The mass ratio of usnic acid to colistin is 5 to 20:

1.

7. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is one of tablets, capsules, powders, sustained-release tablets, controlled-release tablets, oral liquids, syrups, injection liquid dosage forms, and freeze-dried powder injection dosage forms.

8. Use of the pharmaceutical composition according to any one of claims 2 to 7 in the preparation of an anti-Vibrio parahaemolyticus drug.

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