Multi-target-point drug-resistance-free efficient antibacterial compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibiosis, and discloses a multi-target drug-resistance-free efficient antibacterial compound as well as a preparation method and application thereof. According to the invention, organic phenolic acid is used as a skeleton and coordinated with different rare earth ions in an aqueous solution, and then the organic phenolic acid-rare earth supramolecular compound is formed through further self-assembly. The compound can be combined with targets of micromolecular organic phenolic acid and rare earth at the same time, and the efficient antibacterial effect on sensitive and drug-resistant pathogenic bacteria is achieved by destroying bacterial cell walls, cell membranes and DNA. And the multi-target antibacterial mode of the compound endows the compound with the characteristic of no drug resistance or low drug resistance. Therefore, the bacteriostatic agent provided by the invention has the advantages of low cost, easiness in preparation, high activity, multiple targets and no drug resistance, and has a good application prospect in the aspect of drug-resistant pathogenic bacterium infection.

Description

Technical Field

[0001] The present invention relates to the field of antibacterial technology, and specifically to a multi-target, drug-resistant, and highly efficient antibacterial compound, a preparation method, and an application thereof. Background Art

[0002] Antibiotics are one of the most important discoveries of the 20th century. Over the past 80 years, they have greatly reduced the risk of infection caused by routine surgery and brought cures to infectious diseases. However, the overuse or misuse of antibiotics in the medical and animal health fields has caused these indispensable antibiotics to rapidly lose their effectiveness, and complex and refractory bacterial infections have posed a huge threat to clinical treatment. Antibiotic resistance has become one of the most serious challenges facing society today, and the international community has paid close attention to the problem of microbial resistance. In recent years, 700,000 people have died from drug-resistant bacterial infections each year worldwide. If timely measures are not taken to curb the further deterioration of resistance or develop new antibiotic drugs, this number may rise to 10 million by 2050. It can be seen that the problem of antibiotic resistance is becoming more and more serious, and the development of new antibacterial drugs is imminent.

[0003] At present, there are mainly the following solutions to the problem of antibiotic resistance: (1) Develop new antibiotics targeting a specific antibacterial target or mechanism. However, the development cost of new antibiotics is high and the time is long. In addition, antibiotics with a single target or antibacterial mechanism are prone to drug resistance after repeated use. (2) Structural modification of existing antibiotics. For traditional drugs, the modification process of antibiotics is complicated, and it is also difficult to separate and purify the modified antibiotics. (3) New antibacterial therapies: phage therapy, gene editing, and nanoparticles. Phages can usually only infect specific types of bacteria, and some patients may have immune responses, resulting in reduced efficacy or increased treatment risks. Nanoparticles may also cause uncertainty in biodistribution and metabolism in the body, and may also affect long-term safety and efficacy evaluation. (4) Combination therapy. Although it can expand the antibacterial spectrum of antibiotics and slow down the development of bacterial resistance, unreasonable combination therapy will not only increase the frequency of adverse reactions, but also waste drugs and delay the correct treatment time. Therefore, it is very important to develop an antibacterial drug that is easy to prepare, has multiple targets, is highly active, and has no drug resistance.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the problems in the background technology, the present invention provides a multi-target non-resistance high-efficiency antibacterial compound and a preparation method and application thereof. The present invention utilizes the polyphenolic hydroxyl and carboxyl groups of small molecule organic phenolic acid to form coordination bonds with different rare earths to obtain an antibacterial compound, which not only enhances the antibacterial effect, but also more effectively combines the action mechanisms of both parties to reduce the generation of drug resistance.

[0006] To achieve the above object, the first technical solution adopted by the present invention is: A multi-target, non-resistance, and highly effective antibacterial compound comprises small molecule organic phenolic acid and rare earth, wherein the small molecule organic phenolic acid is gallic acid.

[0007] Preferably, the rare earth includes any one of rare earth oxides, rare earth nano-ions, rare earth nitrates and other compounds containing rare earth elements.

[0008] Preferably, the rare earth element is a lanthanide element with an atomic number in the range of 57-71 and capable of interacting with small molecule organic phenolic acid. Preferably, the rare earth element comprises at least one of cerium ion, gadolinium ion and ytterbium ion.

[0009] Preferably, the molar concentration ratio of gallic acid to rare earth is 1: (0.25-8).

[0010] The second technical solution adopted by the present invention is: A method for preparing a multi-target, non-resistant, and highly efficient antibacterial complex comprises: firstly dissolving gallic acid in water, performing ultrasonic dispersion treatment at 15-45°C, then adding a pre-dissolved rare earth solution, and reacting at 10-55°C for 1-6 hours.

[0011] In the above preparation method, gallic acid is used as a skeleton structure to form a complex with rare earth, and a high-efficiency antibacterial complex is obtained by self-assembly into a supramolecular structure.

[0012] Preferably, the ultrasonic dispersion treatment time is 4-30 min.

[0013] Preferably, the reaction pH is 2-7 and the rotation speed is 100-500 rpm.

[0014] The third technical solution adopted by the present invention is: Application of a multi-target, drug-resistant and highly effective antibacterial compound in bacterial inhibition.

[0015] Preferably, the bacteria include Gram-positive bacteria.

[0016] Preferably, the Gram-positive bacteria include sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0017] The multi-target, drug-resistant and highly effective antibacterial compound provided by the present invention has an inhibition zone of 25-30 mm for sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0018] The multi-target, drug-resistant and highly effective antibacterial compound provided by the present invention has a minimum inhibitory concentration of 0.25-1 mmol / L and a minimum bactericidal concentration of 1-2 mmol / L for sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0019] The antibacterial targets of the multi-target, drug-resistant and highly effective antibacterial compound provided by the present invention for methicillin-resistant Staphylococcus aureus mainly include cell wall, cell membrane and DNA.

[0020] The multi-target, non-resistance, and highly effective antibacterial compound provided by the present invention does not produce drug resistance even after continuous use for 60 days.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a new design and preparation method of a multi-target, non-resistant, and highly efficient antibacterial complex, which is to mix different rare earth elements and small molecular organic phenolic acids to form a complex, and then form a supramolecular compound through self-assembly. Then, the antibacterial advantages of both parties are brought into play, thereby achieving the characteristics of synergistic antibacterial.

[0022] 2. The present invention provides a new design concept for a multi-target non-resistance antibacterial complex. According to the drug resistance mechanism produced by bacteria, small molecule organic phenolic acid is combined with rare earth. The antibacterial complex can simultaneously combine the targets of small molecule organic phenolic acid and rare earth, and achieve efficient antibacterial effect on sensitive and resistant pathogens by destroying bacterial cell walls, cell membranes and DNA, thereby achieving multi-target inhibition. Traditional antibacterial agents are generally designed for a certain target, for example, inhibiting the synthesis of cell membranes and cell walls, enhancing the permeability of bacterial cell membranes, interfering with the synthesis of bacterial proteins, and inhibiting DNA and RNA replication and transcription. In comparison, it is more difficult for bacteria to mutate antibiotics at multiple targets than at single targets, so it is more difficult for multi-targets to produce drug resistance.

[0023] 3. The method provided by the present invention selectively combines small molecule organic phenolic acid with different rare earths. The method is simple and flexible and has strong antibacterial activity against drug-resistant bacteria.

[0024] 4. The antibacterial compound prepared by the present invention did not produce drug resistance after continuous induction for 60 days, and compared with penicillin, its MIC on day 28 was 2096 times that on day 1. Therefore, the antibacterial compound prepared by the present invention has the advantage of no drug resistance or low drug resistance.

[0025] 5. The antibacterial compound of the present invention can achieve multi-target antibacterial effect by inhibiting the integrity of bacterial cell wall membrane, destroying bacterial organelles and DNA, and thus inhibiting the growth of MRSA bacteria.

[0026] 6. The preparation process of the present invention is simple, low in cost, has low requirements on equipment, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Fourier transform infrared visible spectroscopy (FTIR) characterization of the antibacterial composite and monomer prepared in Example 2; Figure 2 Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antibacterial complex prepared in the experimental group and the control group in Example 3 against sensitive Staphylococcus aureus; Figure 3 Determination of MIC and MBC of the antibacterial complex prepared for the experimental group and the control group in Example 4 against methicillin-resistant Staphylococcus aureus; Figure 4 The scanning electron microscope (SEM) images of the antibacterial composites prepared in the experimental group and the control group in Example 5 against MRSA bacteria; Figure 5 Transmission electron microscopy (TEM) images of the antibacterial composites prepared in the experimental group and the control group in Example 5 against MRSA bacteria; Figure 6 The agarose gel electrophoresis diagram of the antibacterial complex and DNA prepared in the experimental group and the control group in Example 6; Figure 7 The sustained drug resistance of the antibacterial complex prepared in Example 7 to ASSA. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.

[0029] In order to make the technical solution of the present invention clearer, the preparation method of the multi-target non-resistant and efficient antibacterial complex and its performance are described in detail through a plurality of specific embodiments below.

[0030] ASSA, MRSA, GA, Ce, Gd, Yb, RE and PEN used in the following examples are sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, gallic acid, cerium nitrate hexahydrate, gadolinium nitrate hexahydrate, ytterbium nitrate pentahydrate, rare earth and penicillin G, respectively.

[0031] Example 1 The size of the inhibition zone of ASSA with different molar ratios of antibacterial compounds First, GA of different concentrations was ultrasonically dissolved and dispersed in water, and then Ce, Gd and Yb solutions dissolved in advance were added at 35°C, so that the molar concentration ratio of GA to RE was 1:4, 1:2, 1:1 and 2:1. After mixing evenly, the mixture was reacted in the dark for 4 h to obtain GA-Ce, GA-Gd and GA-Yb complex solutions, i.e., antibacterial complexes. The inhibition zone of the antibacterial complex and its monomer against ASSA bacteria was then determined by the Oxford cup horizontal diffusion method.

[0032] The inhibition zones of ASSA with different concentration ratios of antibacterial complex solutions are shown in Table 1.

[0033] Table 1 .

[0034] Note: “-”: no antibacterial activity As shown in Table 1, the inhibition zone of the GA-RE group was significantly larger than that of GA and RE used alone, and the size of the inhibition zone increased with the increase of GA concentration. It can be seen that GA and RE have a synergistic antibacterial effect.

[0035] Example 2 FTIR Characterization of Antibacterial Composites GA-Ce, GA-Gd and GA-Yb First, GA was ultrasonically dissolved and dispersed in water, and then Ce, Gd and Yb were added at 35°C to make the molar concentration ratio of GA and RE 2:1. Then the reaction was continued for 4 h to obtain GA-Ce, GA-Gd and GA-Yb complexes, i.e., antibacterial complexes. Figure 1 The FTIR spectra of the prepared antibacterial complex GA-RE and its ligand are shown in Figure 1. Compared with GA, the FTIR of the complex has a significant difference. As can be seen from the figure, GA has a peak at 3494.9 cm -1 There is a broad absorption peak at 1667-1423 cm, which is the OH bond stretching vibration absorption peak in the phenolic hydroxyl group. When GA and RE form a complex, the absorption peak here shifts, indicating that RE and the hydroxyl oxygen of GA are coordinated. -1 Related to the vibration of the aromatic ring skeleton of GA, 1322.8 cm -1 and 1222.2 cm -1represents the bending vibration of OH and CO. Compared with GA, in the spectrum of the complex GA-RE, the peaks of these three locations have changed and shifted, indicating that RE and GA have formed a complex.

[0036] Example 3 MIC and MBC determination of GA-RE against ASSA The GA-RE antibacterial complex was prepared by the same operation as in Example 2. The MIC and MBC tests were performed using the test tube method. The GA-RE antibacterial complex was first diluted by a two-fold gradient. Then 1 mL of the GA-RE complex and 1 mL of the bacterial suspension were mixed in each test tube and cultured at 37°C for 24 h. The MIC was defined as the highest dilution concentration that completely inhibited bacterial growth. The MBC value was defined as the minimum drug concentration required to kill 99.9% of the bacteria. The results are shown in Figure 2. Figure 2 As shown in the figure, the MICs of GA-Ce, GA-Gd and GA-Yb against ASSA are 0.25, 0.5 and 0.5 mmol / L, respectively. The MBCs are all 2 mmol / L. It can be seen that GA-RE has a strong antibacterial activity against ASSA.

[0037] Example 4 MIC and MBC determination of GA-RE against MRSA The GA-RE antibacterial compound was prepared and the MIC and MBC determination experiments were performed according to the same operation as in Example 3, except that the bacteria were replaced by MRSA from ASSA. Figure 3 As shown in the figure, the MIC of GA-Ce, GA-Gd and GA-Yb against MRSA is 0.5 mmol / L. The MBCs are 4.0, 2.0 and 2.0 mmol / L, respectively. It can be seen that GA-RE also has strong antibacterial activity against MRSA.

[0038] Example 5 Effect of GA-Gd on the microstructure of MRSA MRSA was used as the experimental strain, and GA-Gd was prepared according to the same operation as in Example 3. Then, 2 mmol / L GA, 1 mmol / L Gd and 1 mmol / L GA-Gd solutions were added to the MRSA suspension, and cultured at 37 °C for 3 h. SEM results (such as Figure 4 ) showed that the MRSA bacteria treated in the control group were spherical or elliptical, with a smooth surface and well-preserved cell morphology. After Gd treatment, the surface of MRSA bacteria was concave. After GA treatment, the surface of MRSA became rougher and concave. The bacteria treated with GA-Gd were severely concave or even damaged, and the integrity of the cell wall and cell membrane was also significantly damaged. TEM results (such as Figure 5) show that the MRSA bacteria structure morphology and organelles of the control group are complete. After Gd treatment, the bacterial cell membrane dissolves but the organelles remain relatively intact. After GA treatment, the MRSA bacteria bacterial cell membrane structure is preserved intact, but the cells produce slightly cavitation. And the bacteria after GA-Gd treatment, the cell wall cell membrane is dissolved, the overall structure of the bacteria is destroyed, and the cells produce serious cavitation. SEM and TEM results further prove that GA-Gd has a synergistic antibacterial effect, and can cause serious damage to the organelles by affecting the integrity of the bacterial cell wall and cell membrane, thereby killing bacteria.

[0039] Example 6 Reaction of GA-Gd with MRSA DNA DNA is the basis of cell genetic material. Agarose gel electrophoresis was used to analyze the interaction between GA-Gd and DNA at different concentrations. GA-Gd was prepared according to the same operation as in Example 3, and the concentration was diluted to 8.0, 4.0, 2.0, 1.0, 0.5 and 0.1 mmol / L with ultrapure water. Then, according to the method provided by the kit (OMEGA Bacterial Genomic DNA Extraction Kit), the DNA of MRSA bacteria was extracted, and then 0.3 μg of DNA extracted from MRSA was mixed with different concentrations of GA-Gd, and after reacting at 4°C for 60 min, agarose gel electrophoresis analysis was performed. Figure 6 It can be seen that the DNA electrophoresis bands in the blank group are neat and clear, without tailing. As the concentration of GA-Gd increases, the brightness of the DNA electrophoresis bands decreases, resulting in diffusion and tailing. Therefore, GA-Gd can affect the structure of DNA, causing it to lose its own electrophoretic properties.

[0040] Example 7 Evaluation of the sustained resistance of GA-Gd to ASSA GA-Gd was prepared according to the same operation as in Example 3. ASSA was cultured in MH medium at 37°C for 10 h and then diluted to 1 × 10 5 CFU / mL was used as the working suspension. GA-Gd and PEN were added to the suspension at a concentration of 0.5 × MIC and incubated at 37°C for 24 h. The suspension was diluted 100 times with MH medium and mixed with 0.5 × MIC antimicrobial complexes to perform a new cell-drug incubation cycle. The MIC values ​​of GA-Gd and PEN against Staphylococcus aureus were tested every 2 days. In the drug resistance test, the concentrations of GA-Gd and PEN were adjusted to maintain at 0.5 × MIC. Antibiotic resistance is manifested as an increase in MIC. The results are shown in Figure 7As shown in the figure, after repeated use for 60 days, the MIC of GA-Gd was still the same as the initial MIC, indicating that ASSA did not develop resistance to GA-Gd. However, after 28 days of use, the MIC of PEN increased by 1024 times compared with the initial MIC. Therefore, GA-Gd has the characteristics of low or no resistance, and its long-term use can still effectively exert antibacterial effects.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-target, non-resistant and highly effective antibacterial compound, characterized in that: It includes small molecule organic phenolic acid and rare earth, among which the small molecule organic phenolic acid is gallic acid.

2. The multi-target, non-resistance, and highly effective antibacterial compound according to claim 1, characterized in that: The rare earth includes any one of rare earth oxides, rare earth nanoparticles, and rare earth nitrates.

3. According to the multi-target, non-resistance, and highly effective antibacterial complex of claim 2, the rare earth element is a lanthanide element with an atomic number in the range of 57-71 and capable of interacting with small molecule organic phenolic acids.

4. According to the multi-target, non-resistance, and highly effective antibacterial composite of claim 3, the rare earth element comprises at least one of cerium ions, gadolinium ions, and ytterbium ions.

5. The multi-target, non-resistance, and highly effective antibacterial compound according to claim 1, characterized in that: The molar ratio of gallic acid to rare earth is 1:(0.25-8).

6. The method for preparing the multi-target, non-resistance, and highly effective antibacterial compound according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: firstly dissolving gallic acid in water, performing ultrasonic dispersion treatment at 15-45°C, then adding the rare earth solution dissolved in advance, and reacting at 10-55°C for 1-6 hours.

7. The preparation method according to claim 6, characterized in that: The reaction pH is 2-7 and the rotation speed is 100-500 rpm.

8. Use of the multi-target, drug-resistant, and highly effective antibacterial complex according to any one of claims 1 to 5 in bacterial inhibition.

9. The use according to claim 8, characterized in that: The bacteria are Gram-positive bacteria.

10. The use according to claim 9, characterized in that: The Gram-positive bacteria include sensitive Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

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