Antibacterial composition containing SU3327 and tannic acid as well as preparation method and application of antibacterial composition

Abstract

The invention provides an antibacterial composition containing SU3327 and tannic acid as well as a preparation method and application of the antibacterial composition, and belongs to the technical field of medicines. The invention provides an antibacterial composition. The antibacterial composition is prepared from SU3327 and tannic acid, the mass ratio of the SU3327 to the tannic acid is (1.25 to 40): (31.25 to 500). The combined use of the SU3327 and the tannic acid not only is simple function addition, but also synergistically achieves an antibacterial effect, the tannic acid can remarkably improve the antibacterial activity of the SU3327 and greatly reduce the use amount of the SU3327 in the medicament for treating bacterial infection, and the combination of the two can effectively kill and / or inhibit drug-resistant bacteria such as escherichia coli and staphylococcus aureus, so that the antibacterial effect of the SU3327 and the tannic acid is greatly improved. The compound shows extremely high application value in the process of treating bacterial infection.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an antibacterial composition containing SU3327 and tannic acid, and a preparation method and application thereof. Background Art

[0002] Bacterial diseases are one of the main causes of illness in humans and animals. The most effective treatment for bacterial infections is generally antibiotics, but due to the long-term use of antibiotics, the problem of bacterial resistance is becoming increasingly serious, and even no drugs are available. The problem of bacterial resistance has become a global public health issue. Researchers found that between 1990 and 2021, more than 1 million deaths worldwide were directly related to antibiotic resistance each year, and the number of deaths indirectly related to antibiotic resistance reached an average of 4.71 million per year. If no measures are taken, according to the current trend, the number of deaths related to antibiotic resistance will steadily increase in the next few decades. It is estimated that between 2025 and 2050, a total of more than 39 million deaths will be directly attributed to antibiotic resistance, and 169 million deaths will be indirectly related. The development of new antibacterial drugs has become one of the important ways to curb bacterial resistance in the clinic. However, since the development of new antibiotics lags far behind the rapid evolution and spread of antibiotic resistance, compared with the discovery of new antimicrobial drugs, combined strategies can restore the sensitivity of resistant strains to existing antibiotics, thus bringing hope for clinical treatment.

[0003] SU3327, CAS No. 40045-50-9, molecular formula C5H3N5O2S3, molecular weight 261.3, is a C-JUN N-terminal kinase (JNK) selective inhibitor, also known as halicin or SU3327, is a new potential antibiotic developed by a research team led by James Collins of the Massachusetts Institute of Technology (MIT) using deep learning artificial intelligence (AI). It has a certain inhibitory effect on a variety of bacteria such as Acinetobacter baumannii, Escherichia coli, Mycobacterium tuberculosis, and Clostridium difficile (Stokes JM, Yang K, Swanson K, Jin W, Cubillos-Ruiz A, Donghia NM, MacNairCR, French S, Carfrae LA, Bloom-Ackermann Z, Tran VM, Chiappino-Pepe A, Badran AH, Andrews IW, Chory EJ, Church GM, Brown ED, Jaakkola TS, BarzilayR, Collins JJ. A Deep Learning Approach to Antibiotic Discovery. Cell. 2020,180(4): 688-702); In Chinese patent CN113491692A (Use of C-JUN N-terminal kinase inhibitor SU3327), the inhibitory effect of SU3327 on Actinobacillus pleuropneumoniae was further reported; subsequently, in Chinese patent CN116236479A (Use of SU3327 in the preparation of drugs for enhancing the efficacy of polymyxin against bacterial infections) and Chinese patent CN116637109A (Use of SU3327 for restoring the sensitivity of vancomycin-resistant bacteria), the effects of SU3327 on synergistic enhancement of colistin against Escherichia coli, Klebsiella pneumoniae and Staphylococcus pseudintermedius containing colistin resistance gene 1 (MCR1), and synergistic enhancement of vancomycin against enterococci were reported. In Chinese patent CN117860741A (Application of SU3327 in the preparation of anti-Vibrio parahaemolyticus drugs), it was found that SU3327 combined with the antibacterial drug colistin has a good synergistic effect. In addition, long-term use of SU3327 may produce certain toxic effects.

[0004] Tannic acid (TA) is a natural polyphenol compound that can complex with ions. Tannic acid has certain antibacterial, antiviral and antifungal effects, can inhibit bacterial proliferation and pathogenic microbial activity, and indirectly reduce tissue swelling caused by inflammation or infection. The U.S. Food and Drug Administration (FDA) has approved tannic acid as a food additive. In addition, TA has been shown to reduce bacterial fatty acid synthesis by irreversibly inhibiting β-ketoacyl-ACP reductase (Wu D, Wu XD, You XF, Ma XF, Tian WX. Inhibitory effects on bacterial growthand beta-ketoacyl-ACP reductase by different species of maple leaf extractsand tannic acid. Phytother Res. 2010 Jan;24 Suppl 1:S35-41. doi: 10.1002 / ptr.2873. Erratum in: Phytother Res. 2010 Apr;24(4):632. PMID: 19444866.).

[0005] So far, there is no literature report on tannic acid as an antibacterial adjuvant to enhance the efficacy of SU3327. Summary of the invention

[0006] In view of this, the object of the present invention is to provide an antibacterial composition containing SU3327 and tannic acid, and a preparation method and application thereof. SU3327 and tannic acid have a synergistic effect when used in combination, and can significantly improve the antibacterial effect on bacteria (including drug-resistant bacteria).

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The invention provides application of tannic acid in preparing SU3327 synergist.

[0008] The invention also provides an antibacterial composition, comprising SU3327 and tannic acid.

[0009] Preferably, the mass ratio of SU3327 to tannic acid is 1.25-40:31.25-500.

[0010] Preferably, the bacteria include Gram-negative bacteria and Gram-positive bacteria, the Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus.

[0011] The present invention also provides a method for preparing the antibacterial composition, comprising the following steps: SU3327 and tannic acid are mixed to obtain an antibacterial composition.

[0012] The present invention also provides the use of the antibacterial composition or the antibacterial composition prepared by the preparation method in preparing drugs for resisting bacterial infection.

[0013] Preferably, the dosage form of the drug includes powder, tablet, pill, capsule, oral solution, injection, ointment or aerosol.

[0014] The invention also provides application of tannic acid and SU3327 in preparing medicine for resisting bacterial infection.

[0015] Preferably, the bacteria include Gram-negative bacteria and Gram-positive bacteria, the Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus.

[0016] Preferably, the dosage form of the drug includes powder, tablet, pill, capsule, oral solution, injection, ointment or aerosol.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The invention provides an antibacterial composition, comprising SU3327 and tannic acid; the combined use of SU3327 and tannic acid is not only a simple addition of functions, but a synergistic antibacterial effect, tannic acid can significantly enhance the antibacterial activity of SU3327, and greatly reduce the amount of SU3327 used in the drug for treating bacterial infection, and the combination of the two can effectively kill and / or inhibit drug-resistant bacteria such as Escherichia coli and Staphylococcus aureus, and shows extremely strong application value in the process of treating bacterial infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the bactericidal curve of SU3327 combined with tannic acid against Escherichia coli 25922; Figure 2 This is the bactericidal curve of SU3327 combined with tannic acid against Escherichia coli B2; Figure 3 This is the bactericidal curve of SU3327 combined with tannic acid against Staphylococcus aureus 33591. DETAILED DESCRIPTION

[0019] The invention provides application of tannic acid in preparing SU3327 synergist.

[0020] The invention also provides an antibacterial composition, comprising SU3327 and tannic acid.

[0021] In the present invention, the mass ratio of SU3327 to tannic acid is preferably 1.25-40:31.25-500, and more preferably 5-20:62.5-250; the bacteria include Gram-negative bacteria and Gram-positive bacteria, the Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus.

[0022] The present invention also provides a method for preparing the antibacterial composition, comprising the following steps: SU3327 and tannic acid are mixed to obtain an antibacterial composition.

[0023] The present invention also provides the use of the antibacterial composition or the antibacterial composition prepared by the preparation method in preparing drugs for resisting bacterial infection.

[0024] In the present invention, the dosage form of the drug includes powder, tablet, pill, capsule, oral solution, injection, ointment or aerosol.

[0025] The invention also provides application of tannic acid and SU3327 in preparing medicine for resisting bacterial infection.

[0026] In the present invention, the bacteria include Gram-negative bacteria and Gram-positive bacteria, the Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus; the dosage form of the drug includes powder, tablet, pill, capsule, oral solution, injection, ointment or aerosol.

[0027] 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.

[0028] The tannic acid used in this application was purchased from aladdin, with a purity of ≥90% and a CAS number of 1401-55-4. Before use, dimethyl sulfoxide (DMSO) was used to adjust the concentration of tannic acid to 20 mg / mL and placed in a -20 °C refrigerator for later use. The SU3327 used in this application was purchased from MedChemExpress with a purity of 98.03%. Before use, the concentration of SU3327 needs to be adjusted to 10 mg / mL using dimethyl sulfoxide (DMSO) and placed in a -20 °C refrigerator for later use. Staphylococcus aureus 33591, Escherichia coli 25922, and Escherichia coli B2 used in this application were from the National Center for Veterinary Drug Safety (Beijing); The Mueller Hinton Ⅱ Cation Adjusted Broth (CAMHB Broth) medium used in this application was purchased from BD Biosciences (Shanghai) Co., Ltd. The preparation method is as follows: weigh 22.0 g of this product in 1 L of distilled water, heat and boil until completely dissolved, sterilize at 121 ° C for 15 min, and set aside; The MH agar medium used in this application was purchased from Beijing Luqiao Technology Co., Ltd. and was prepared as follows: weigh 38.0 g of the product in 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; The brain heart infusion broth medium (BHI broth medium) used in this application 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 min for use.

[0029] Example 1 Effects of SU3327 and tannic acid alone and in combination on Escherichia coli 25922, Escherichia coli B2, and Staphylococcus aureus 33591 The chessboard method was used to investigate the effects of SU3327 and tannic acid alone and in combination on various bacteria. The specific steps were as follows: SU3327 and tannic acid were taken separately, and dimethyl sulfoxide (DMSO) was used to prepare 40 µg / mL SU3327 and 500 µg / mL tannic acid solutions, which were then diluted in series using CAMHB broth to obtain tannic acid concentrations of 500 µg / mL, 250 µg / mL, 125 µg / mL, 62.5 µg / mL, 31.25 µg / mL, 15.625 µg / mL, 7.8125 µg / mL, 3.9063 µg / mL, 1.9531 µg / mL, 0.9766 µg / mL, and 0.4883 µg / mL, respectively; the concentrations of SU3327 were 40 µg / mL, 20 µg / mL, 10 µg / mL, 5 µg / mL, 2.5 50 μL of CAMHB broth containing different concentrations of SU3327 and tannic acid were added along the horizontal and vertical axes of the 96-well culture plate, and then 50 μL of various pathogenic bacteria were added to make the final bacterial count per well 2×10 5 CFU, incubate in a 37 ℃ constant temperature incubator for 24 h, 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: FICI of SU3327 and tannic acid = MIC (combination of SU3327) / MIC (SU3327 alone) + MIC (combination of tannic acid) / MIC (tannic acid alone); judgment criteria: FICI≤0.5, synergistic effect; 0.5<FICI≤1, additive effect; 1﹤FICI≤2, no effect; FICI>2, antagonistic effect.

[0030] Experimental results: as shown in Table 1.

[0031] Table 1 MIC and FICI values ​​of SU3327 and tannic acid alone and in combination against various bacteria

[0032] As can be seen from Table 1, against E. coli 25922, the MICs of SU3327 and tannic acid alone were 20 µg / mL and greater than 1000 µg / mL, respectively. When the two were used in combination, SU3327 was at 5 µg / mL and tannic acid was at 31.25 µg / mL, and the two achieved the best synergistic effect, with a FICI index of 0.266.

[0033] As can be seen from Table 1, for E. coli B2, the MICs of SU3327 and tannic acid alone were 20 µg / mL and greater than 1000 µg / mL, respectively. When the two were used in combination, SU3327 was at 5 µg / mL and tannic acid was at 31.25 µg / mL, and the two achieved the best synergistic effect, with a FICI index of 0.266.

[0034] As can be seen from Table 1, for Staphylococcus aureus 33591, the MICs of SU3327 and tannic acid alone were 10 µg / mL and 125 µg / mL, respectively. When the two were used in combination, SU3327 was at 5 µg / mL and tannic acid was at 31.25 µg / mL, and the two achieved the best additive effect, with a FICI index of 0.75.

[0035] In conclusion, SU3327 combined with tannic acid had a significant synergistic antibacterial effect against Escherichia coli 25922 (ATCC25922) and Escherichia coli B2, and a significant additive antibacterial effect against Staphylococcus aureus 33591, with FICI ranging from 0.266 to 0.75.

[0036] Example 2 Determination of the minimum bactericidal concentration (MBC) of SU3327 and tannic acid used alone and in combination 10 μL of the solution from each well of the 96-well culture plate in Example 1 where no bacteria were observed to grow was spotted on MH agar medium and placed in a 37°C constant temperature incubator for 18 h. The lowest concentration in the wells where no bacteria were observed to grow was the MBC.

[0037] Experimental results: as shown in Table 2.

[0038] Table 2 MBC values ​​of SU3327 and tannic acid alone and in combination against various bacteria

[0039] As can be seen from Table 2, against Escherichia coli 25922 (ATCC25922), the MBCs of SU3327 and tannic acid alone were 40 µg / mL and greater than 1000 µg / mL, respectively, and the minimum bactericidal combination concentrations of the two combined were 10 µg / mL and 500 µg / mL, respectively; As can be seen from Table 2, against E. coli B2, the MBCs of SU3327 and tannic acid alone were 40 µg / mL and greater than 1000 µg / mL, respectively, and the minimum bactericidal combination concentrations of the two combined were 10 µg / mL and 250 µg / mL and 20 µg / mL and 31.25 µg / mL, respectively; As can be seen from Table 2 , against S. aureus 33591, the MBCs of SU3327 and tannic acid alone were 20 µg / mL and 500 µg / mL, respectively, and the minimum bactericidal combination concentrations of the two combined were 1.25 µg / mL and 250 µg / mL, respectively; In summary, the combined treatment of SU3327 and tannic acid had a significant bactericidal effect on Escherichia coli 25922 (ATCC25922), Escherichia coli B2, and Staphylococcus aureus 33591, and significantly reduced the dosage of SU3327 and tannic acid.

[0040] Example 3 Evaluation of the bactericidal effects of SU3327 and tannic acid alone and in combination against Escherichia coli B2, Escherichia coli 25922 and Staphylococcus aureus 33591 1. Escherichia coli 25922 Escherichia coli 25922 (ATCC25922) was inoculated into BHI broth medium for bacterial enrichment culture at 37 °C, 200 rpm, and cultured for 6 h. The bacterial solution was adjusted to a turbidity of 0.5, i.e., 1×10 8 CFU / mL, and then the bacterial solution was diluted to 1×10 6CFU / mL; the bacterial solution was divided into 4 groups: 40 µg / mL SU3327 group, 250 µg / mL tannic acid group, 250 µg / mL tannic acid + 40 µg / mL SU3327 group and control group; SU3327 was added to the bacterial solution of the 40 µg / mL SU3327 group to prepare a mixed solution, wherein the final concentration of SU3327 was 40 µg / mL; tannic acid was added to the bacterial solution of the 250 µg / mL tannic acid group to prepare a mixed solution, wherein the final concentration of tannic acid was 250 µg / mL; SU3327 and tannic acid were added to the bacterial solution of the 250 µg / mL tannic acid + 40 µg / mL SU3327 group to prepare a mixed solution, wherein the final concentration of SU3327 was 40 µg / mL, and the final concentration of tannic acid was 250 µg / mL; The mixed solution was placed in a 37 ℃ constant temperature incubator for culture. After 1 h, 3 h, 6 h, 12 h, and 24 h of culture, 100 μL of each mixed solution was spread on MH agar medium with a diameter of 10 cm, and the colonies were counted after overnight culture.

[0041] Experimental results: Figure 1 As shown. Figure 1 It can be seen that for Escherichia coli 25922 strain, at the 24th hour, SU3327 and tannic acid alone had no significant effect on the bacterial colony count compared with the control group; when SU3327 and tannic acid were treated together, at the 6th hour, the colony count was significantly lower than that of the control group and the single-drug treatment group, until 24 hours, the colony count of the combined treatment group was significantly reduced, lower than that of the control group and the single-drug group; therefore, the combined treatment of SU3327 and tannic acid has an obvious synergistic bactericidal effect on Escherichia coli 25922.

[0042] 2. Escherichia coli B2 Escherichia coli B2 was inoculated into BHI broth medium for bacterial enrichment culture at 37 °C, 200 rpm, and cultured for 6 h. The bacterial solution was adjusted to a turbidity of 0.5 using a McFarland turbidimeter, i.e., 1×10 8 CFU / mL, and then the bacterial solution was diluted to 1×10 6 CFU / mL; the bacterial solution was divided into 4 groups: 20 µg / mL SU3327 group, 62.5 µg / mL tannic acid group, 62.5 µg / mL tannic acid + 20 µg / mL SU3327 group and control group; SU3327 was added to the bacterial solution of the 20 µg / mL SU3327 group to prepare a mixed solution, wherein the final concentration of SU3327 was 20 µg / mL; tannic acid was added to the bacterial solution of the 62.5 µg / mL tannic acid group to prepare a mixed solution, wherein the final concentration of tannic acid was 62.5 µg / mL; SU3327 and tannic acid were added to the bacterial solution of the 62.5 µg / mL tannic acid + 20 µg / mL SU3327 group to prepare a mixed solution, wherein the final concentration of SU3327 was 20 µg / mL, and the final concentration of tannic acid was 62.5 µg / mL; The mixed solution was placed in a 37 ℃ constant temperature incubator for culture. After 1 h, 3 h, 6 h, 12 h, and 24 h of culture, 100 μL of each mixed solution was spread on MH agar medium with a diameter of 10 cm, and the colonies were counted after overnight culture.

[0043] Experimental results: Figure 2 As shown. Figure 2 It can be seen that for Escherichia coli B2 strain, at the 24th hour, compared with the control group, the treatment with SU3327 and tannic acid alone had no significant effect on the bacterial colony count; when SU3327 and tannic acid were treated together, at the 6th hour, the colony count was significantly lower than that of the control group and the single-drug treatment group, until 24 hours, the colony count of the combined treatment group was significantly reduced, lower than that of the control group and the single-drug group; therefore, the combined treatment of SU3327 and tannic acid has an obvious synergistic bactericidal effect on Escherichia coli B2.

[0044] 3. Staphylococcus aureus 33591 Staphylococcus aureus 33591 was inoculated into BHI broth medium for bacterial enrichment culture at 37 °C, 200 rpm, and cultured for 6 h. The bacterial solution was adjusted to a turbidity of 0.5 (1×10 8 CFU / mL, and then the bacterial solution was diluted to 1×10 6 CFU / mL; the bacterial solution was divided into 4 groups: 20 µg / mL SU3327 group, 125 µg / mL tannic acid group, 125 µg / mL tannic acid + 20 µg / mL SU3327 group and control group; SU3327 was added to the bacterial solution of the 20 µg / mL SU3327 group to prepare a mixed solution, wherein the final concentration of SU3327 was 20 µg / mL; tannic acid was added to the bacterial solution of the 125 µg / mL tannic acid group to prepare a mixed solution, wherein the final concentration of tannic acid was 125 µg / mL; SU3327 and tannic acid were added to the bacterial solution of the 125 µg / mL tannic acid + 20 µg / mL SU3327 group to prepare a mixed solution, wherein the final concentration of SU3327 was 20 µg / mL, and the final concentration of tannic acid was 125 µg / mL; The mixed solution was placed in a 37 ℃ constant temperature incubator for culture. After 1 h, 3 h, 6 h, 12 h, and 24 h of culture, 100 μL of each mixed solution was spread on MH agar medium with a diameter of 10 cm, and the colonies were counted after overnight culture.

[0045] Experimental results: Figure 3 As shown. Figure 3 It can be seen that for Staphylococcus aureus 33591 strain, at the 24th hour, SU3327 and tannic acid alone had no significant effect on the bacterial colony count compared with the control group; when SU3327 and tannic acid were treated together, at the 6th hour of treatment, the colony count was significantly lower than that of the control group and the single-drug treatment group, until 12 hours, there was no bacterial growth and the colony count was 0; therefore, the combined treatment of SU3327 and tannic acid had an obvious synergistic bactericidal effect on Staphylococcus aureus 33591.

[0046] It can be seen from the above examples that the combined use of SU3327 and tannic acid of the present invention can effectively kill and / or inhibit Escherichia coli and Staphylococcus aureus.

[0047] 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. Application of tannic acid in the preparation of SU3327 synergist.

2. An antibacterial composition, characterized in that Includes SU3327 and tannic acid.

3. The antibacterial composition according to claim 2, characterized in that The mass ratio of SU3327 to tannic acid is 1.25-40:31.25-500.

4. The antibacterial composition according to claim 2 or 3, characterized in that The bacteria include Gram-negative bacteria and Gram-positive bacteria. The Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus.

5. The method for preparing the antibacterial composition according to any one of claims 2 to 4, characterized in that: The following steps are involved: SU3327 and tannic acid are mixed to obtain an antibacterial composition.

6. Use of the antibacterial composition according to any one of claims 2 to 4 or the antibacterial composition prepared by the preparation method according to claim 5 in the preparation of drugs for antibacterial infection.

7. The use according to claim 6, characterized in that: The dosage form of the drug includes powder, tablet, pill, capsule, oral solution, injection, ointment or aerosol.

8. Application of tannic acid and SU3327 in the preparation of drugs for resisting bacterial infection.

9. The use according to claim 8, characterized in that: The bacteria include Gram-negative bacteria and Gram-positive bacteria. The Gram-negative bacteria include Escherichia coli, and the Gram-positive bacteria include Staphylococcus aureus.

10. The use according to claim 8, characterized in that: The dosage form of the drug includes powder, tablet, pill, capsule, oral solution, injection, ointment or aerosol.

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