Antibacterial compound, preparation method and application thereof, and pesticide agent

Abstract

The invention relates to the technical field of natural antibacterial compounds, in particular to an antibacterial compound, a preparation method and application thereof and a pesticide agent. The antibacterial compound is selected from compounds shown in the following structural formula: the compound has an inhibition effect on methicillin-resistant staphylococcus aureus and other drug-resistant bacteria, staphylococcus aureus, escherichia coli, salmonella, pseudomonas aeruginosa and other bacteria, and alternaria, fusarium, colletotrichum and other fungi; the compound can be applied as broad-spectrum antibacterial, broad-spectrum antifungal and drug-resistant bacterium resistant drugs.

Description

Technical Field

[0001] This invention relates to the field of natural antibacterial compounds, and more specifically, to antibacterial compounds, their preparation methods, their applications, and pesticide formulations. Background Technology

[0002] With the continued rise in multidrug-resistant bacterial and invasive fungal infections, global public health systems face severe challenges. Existing antibiotics and antifungal drugs face two major challenges: first, the rapid development of drug resistance leads to a significant decline in drug efficacy; second, single compounds possessing both broad-spectrum antibacterial and broad-spectrum antifungal activity are extremely scarce. Natural products are a treasure trove for discovering novel antibacterial lead compounds and are an indispensable resource for human survival. Fungi, second only to insects in nature's biological resources, inhabit almost all known ecosystems on Earth and are an important source for clinical drug development.

[0003] Trichiaptum podocarpi is a fungus belonging to the genus Trichiaptum in the family Polyporaceae. There is limited research on its applications, and even less research on its fermentation products.

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

[0005] The purpose of this invention is to provide antibacterial compounds, their preparation methods, their applications, and pesticide formulations. This invention provides a novel compound that exhibits inhibitory effects against methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant bacteria, including Staphylococcus aureus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa, as well as fungi such as Alternaria, Fusarium, and Anthrax. It can be used as a broad-spectrum antibacterial, broad-spectrum antifungal, and anti-drug-resistant drug.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides an antibacterial compound selected from compounds shown in the following structural formula:

[0008]

[0009] Secondly, the present invention provides a method for preparing the antibacterial compound described in the foregoing embodiments, comprising: fermenting Podocarpus macrophyllus to form a fermentation product; and then extracting and separating the fermentation product by column chromatography.

[0010] In an optional embodiment, the fermentation product is extracted using an ester solvent to form an ester extract.

[0011] Then, an alcohol-water system is used for elution.

[0012] In an optional embodiment, the fermentation product is extracted with ethyl acetate to form an ethyl acetate extract.

[0013] The ethyl acetate extract was eluted isocratically with a first mixed solvent, followed by isocratically eluted with a second mixed solvent.

[0014] Wherein, the first mixed solvent is a mixed solvent formed by mixing methanol and water in a volume ratio of (60-78):(22-40);

[0015] The second mixed solvent is a mixed solvent formed by mixing methanol and water in a volume ratio of (80-87):(13-20).

[0016] Thirdly, the present invention provides the application of the antibacterial compound described in the foregoing embodiments in the preparation of a medicament against drug-resistant bacteria.

[0017] In an optional embodiment, the drug-resistant bacteria include multidrug-resistant bacteria; preferably methicillin-resistant Staphylococcus aureus.

[0018] Fourthly, the present invention provides the application of the antibacterial compound described in the foregoing embodiments in the preparation of antifungal agents.

[0019] In an optional embodiment, the fungi include Alternaria, Fusarium, and Anthrax fungi.

[0020] Fifthly, the present invention provides a pesticide agent comprising the antibacterial compound described in the foregoing embodiments.

[0021] Sixthly, the present invention provides the use of the antibacterial compound described in the foregoing embodiments in the preparation of antibacterial agents.

[0022] Preferably, the bacteria include Staphylococcus aureus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa.

[0023] The present invention has the following beneficial effects: (1) The embodiments of the present invention provide a new compound that has anti-methicillin-resistant Staphylococcus aureus (MRSA) activity; it can be used in the preparation of drugs against drug-resistant bacteria. At the same time, its ability to prevent the development of drug resistance expands its application range.

[0024] (2) This compound also has antibacterial activity against bacteria such as Staphylococcus aureus, Escherichia coli, Salmonella and Pseudomonas aeruginosa, and can be used as a broad-spectrum antibacterial drug.

[0025] (3) This compound also has inhibitory activity against fungi such as Alternaria, Fusarium and Anthracnose, and can be used as a broad-spectrum antifungal drug. At the same time, it has potential effects on plant diseases caused by the above fungi and can be used as a pesticide.

[0026] (4) This compound is derived from Podocarpus macrophyllus, a natural antibacterial compound that expands the application range of Podocarpus macrophyllus. At the same time, the preparation process of this compound is simple, can be prepared efficiently and stably, and the compound has high safety. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Morphological diagram of Podocarpus macrophyllus provided in an embodiment of the present invention;

[0029] Figure 2 The structural formula and crystal structure diagram of the antibacterial compound provided in the embodiments of the present invention;

[0030] Figure 3 The 1H NMR spectrum of the antibacterial compound provided in the embodiments of the present invention;

[0031] Figure 4 The carbon NMR spectrum of the antibacterial compound provided in the embodiments of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0033] This invention provides an antibacterial compound selected from compounds with the following structural formulas:

[0034] It exhibits inhibitory effects against methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant bacteria, as well as bacteria such as Staphylococcus aureus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa, and fungi such as Alternaria, Fusarium, and Anthracnose. It can be used alone as a broad-spectrum antibacterial, broad-spectrum antifungal, and anti-drug-resistant agent, or as a simultaneous antifungal, antibacterial, and anti-drug-resistant agent. It can also be used as a pesticide to improve vegetation survival rates.

[0035] This antibacterial compound was prepared using *Polygonum aviculare*, and the specific preparation process is as follows:

[0036] Liquid fermentation products were obtained by fermenting Podocarpus macrophyllus with a culture medium. The culture medium was selected from existing technologies, such as, but not limited to, potato dextrose broth (PDB) and rice culture medium.

[0037] The PDB medium consists of potatoes, glucose, yeast extract, peptone, potassium dihydrogen phosphate, and anhydrous magnesium sulfate, but is not limited to changes in fermentation conditions based on the PDB medium. The rice medium consists of rice and water, but is not limited to changes in fermentation conditions based on the rice medium.

[0038] The fermentation conditions are conventional, such as a fermentation temperature of 26-28℃, a fermentation time of 20-25 days, and a normal amount of oxygen.

[0039] The liquid fermentation products are then centrifuged to form mycelia and bacterial broth. Next, the fermentation products are extracted using ester solvents to form ester extracts; specifically, the bacterial broth is concentrated and evaporated, then extracted using ester solvents, and concentrated under reduced pressure to form ester extracts; wherein, the ester solvents include, but are not limited to, ethyl acetate.

[0040] The ester extracts are then separated using column chromatography, for example, by medium-pressure liquid chromatography to obtain the desired compounds. The elution process utilizes an alcohol-water system, where the alcohol solvent includes, but is not limited to, C1-C3 solvents, such as methanol.

[0041] Specifically, the elution process involves isocratic elution of the ethyl acetate extract with a first mixed solvent for 180-250 min to remove impurities, followed by isocratic elution with a second mixed solvent.

[0042] The first mixed solvent is a mixed solvent formed by mixing methanol and water in a volume ratio of (60-78):(22-40); for example, the volume ratio of methanol to water is any value between 60:40, 72:28, 73:27, 74:26, 75:25, 76:24, 77:23, 78:22 or (60-78):(22-40).

[0043] The second mixed solvent is a mixed solvent formed by mixing methanol and water in a volume ratio of (80-87):(13-20). For example, the volume ratio of methanol to water is any value between 80:20, 82:18, 83:17, 84:16, 85:15, 86:14, 87:13 or (80-87):(13-20).

[0044] The *Polyporus truncatus* strain used in this invention was collected from the Bawangling Nature Reserve in Changjiang County, Hainan Province. Identification was performed by analyzing its internal transcriptional spacer (ITS), and the accession number is OQ449081.1 (max identity: 98%; querycover: 86%). The strain was stored at 4°C in the Higher Fungal Culture Bank of the School of Pharmacy, South-Central University for Nationalities, with the accession number CGBWSHF-00865. The morphology of the strain is as follows: Figure 1 As shown, (this is an existing species of fungus, and its relevant information has been recorded in the NCBI database). All other plant pathogenic fungi are preserved in the Higher Fungal Culture Bank of the School of Pharmacy, South-Central University for Nationalities.

[0045] Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 strain, Staphylococcus aureus strain, Escherichia coli strain, Salmonella, and Pseudomonas aeruginosa were all purchased from the American Type Culture Collection (ATCC) and stored in 20% glycerol at -80°C.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1

[0048] This invention provides a method for preparing an antibacterial compound, comprising:

[0049] Poria cocos was cultured in PDB medium for 25 days (fermentation conditions also included a temperature of 26-28℃) to obtain liquid fermentation products. The liquid fermentation products were separated into mycelia and bacterial broth using a high-speed centrifuge. The bacterial broth was evaporated and concentrated, then redissolved in a small amount of water, and extracted with ethyl acetate solution. The mixture was thoroughly mixed three times, allowed to stand for layering, and the upper ethyl acetate extract was collected. This portion of the sample was separated using medium-pressure liquid chromatography. Impurities were removed by isocratic elution for 200 min with a methanol-water system (75:25, v / v, 20.0 mL / min), followed by isocratic elution with a methanol-water system (85:15, v / v, 20.0 mL / min) to obtain the target compound. The compound's structural formula and crystal structure are shown below. Figure 2As shown, the 1H NMR spectrum of the compound is shown in [reference needed]. Figure 3 See the carbon NMR spectrum. Figure 4 .

[0050] Experiment Example 1: Antibacterial activity of antibacterial compounds against methicillin-resistant Staphylococcus aureus

[0051] method:

[0052] The MRSA strain, frozen at -80℃, was removed and activated by streak plating onto MH agar plates. The plates were then incubated upside down in a 37℃ incubator until single colonies appeared. A single colony was picked using a sterile inoculation loop and placed in MH broth, incubated overnight at 37℃ with a shaking incubator at 200 rpm. 50 μL of the overnight culture was transferred to a medium containing 5 mL of fresh MH broth and incubated at 37℃ with a shaking incubator at 200 rpm for 3 hours until the logarithmic growth phase was reached.

[0053] Following the performance standards for antimicrobial susceptibility testing established by CLSI, the MIC and MBC of the antimicrobial compound provided in Example 1 (hereinafter referred to as FHTP) and the positive control drug vancomycin against the standard quality control strain ATCC 43300 of methicillin-resistant Staphylococcus aureus (MRSA) were determined using the microbroth dilution method.

[0054] FHTP was dissolved in dimethyl sulfoxide (DMSO) and then serially diluted with phosphate-buffered saline (PBS, pH 7.4) to a stock solution concentration of 20 mg / mL. Vancomycin was prepared with sterile physiological saline, with a stock solution concentration of 10 mg / mL. After aliquoting, the solutions were stored at -20°C for later use. Logarithmic growth phase bacterial cultures were taken and calibrated using an ELISA reader (OD600) and colony count to adjust the concentration to 5 × 10⁻⁶. 5 CFU / mL was used to obtain a bacterial suspension.

[0055] A series of two-fold dilutions of the drug were set up in 96-well plates (FHTP group: 200–1.5625 μg / mL; vancomycin group: 100–0.78125 μg / mL), with three replicates for each concentration. A blank control (containing bacterial suspension and culture medium) and a solvent control (containing an equal volume of DMSO and bacterial suspension) were also included. Each well was inoculated with an equal volume of bacterial suspension to bring the final volume to 200 μL. After incubation at 37°C for 20 h, the minimum inhibitory concentration (MIC) was determined by visual inspection of the wells for clarity and no turbidity (inhibition rate >80%). The minimum bactericidal concentration was determined using agar titration. 10 μL of the drug-containing bacterial suspension from each of the MIC, 2×MIC, 4×MIC, and 8×MIC wells was dropped onto MH agar plates and incubated at 37°C for 24 h. Bacterial growth was then observed. The minimum drug concentration at which no colony growth was observed was defined as the MBC value of FHTP and vancomycin against Staphylococcus aureus ATCC 43300.

[0056] The experimental results for MIC and MBC are shown in Tables 1 and 2 below.

[0057] Table 1. Antibacterial results of different concentrations of antibacterial compounds and vancomycin

[0058]

[0059] Table 2. Bactericidal results of different concentrations of compounds and vancomycin

[0060]

[0061] According to Tables 1 and 2, the MICs of the antibacterial compound FHTP and the positive control vancomycin are 25 μg / mL and 3.125 μg / mL, respectively, and the MBCs are 50 μg / mL and 6.25 μg / mL, respectively.

[0062] Experimental Example 2: Determination of the Time-Bactericidal Curve of Antimicrobial Compounds Against MRSA

[0063] method:

[0064] The antibacterial efficacy of antimicrobial compounds against MRSAATCC 43300 strain was evaluated using bactericidal kinetics. Based on the MICs obtained in previous experiments, final concentration gradients of the antimicrobial compounds FHTP and vancomycin were set at 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC. A solvent control (containing the same volume of DMSO as the highest drug concentration) and a blank control (containing bacterial suspension and MH medium, but without the drug) were also included. Sterile 96-well plates were used for the experiments, with three independent replicates per group. The plates were incubated at 37℃ and 200 rpm in a constant temperature shaking incubator. The OD600 values ​​of each well were measured using a microplate reader at 15 time points: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 24 hours (the plates were shaken for 10 seconds before detection to ensure uniform bacterial suspension). Using detection time as the x-axis and OD600 measurement values ​​(mean ± standard deviation of three replicate wells) as the y-axis, the time-bacterial curve of bacteria under drug action was plotted using GraphPad Prism 10.0 software.

[0065] The results are shown in Table 3.

[0066] Table 3. Kinetics of the antibacterial compounds FHTP or vancomycin

[0067]

[0068]

[0069] Table 3 shows that both the blank control and solvent control groups rapidly increased in the logarithmic phase after 2 hours, and then slowly entered the plateau phase after 8 hours. After treatment with the antibacterial compounds FHTP or vancomycin, at concentrations of 0.5×MIC and 1×MIC, the drugs could inhibit bacterial growth for a certain period; however, at concentrations of 2×MIC and 4×MIC, bacterial growth was inhibited throughout the entire growth process, indicating that drug concentrations above 2×MIC can have a bactericidal effect.

[0070] Experiment Example 3: Antimicrobial resistance assessment of the antimicrobial compound FHTP

[0071] method:

[0072] The resistance of the antimicrobial compound FHTP to MRSA ATCC 43300 was evaluated. Vancomycin, linezolid, and teicoplanin were selected as positive control drugs. First, the minimum inhibitory concentrations (MICs) of FHTP, vancomycin, linezolid, and teicoplanin against MRSA were determined to be 25, 3.125, 3.125, and 1.5625 μg / mL, respectively. 5 mL of MRSA was incubated at sub-MIC concentrations for 21 h, centrifuged, washed three times with PBS buffer, and resuspended in PBS buffer. 50 μL of the resuspended cells were transferred to 5 mL of blank MH broth and activated for 3 h to reach the logarithmic growth phase. Subsequently, the logarithmic growth phase bacteria were used for the next subculture and MIC determination was performed. A total of 14 subcultures were performed.

[0073] The results are shown in Table 4.

[0074] Table 4. Results of resistance to the antimicrobial compound FHTP and other positive control agents.

[0075]

[0076]

[0077] As shown in Table 4, during the 14-day passage process, teicoplanin showed resistance earliest (second generation), and the resistance increased continuously, with the MIC value increasing by 128 times in the eighth generation; linezolid showed resistance in the fourth generation, and the resistance increased continuously until the MIC value increased by 64 times in the sixth generation; vancomycin showed resistance in the sixth generation, and the resistance increased in the eighth generation, with the MIC value increasing by 16 times; while FHTP began to show resistance in the seventh generation, and the resistance increased slowly until the MIC value increased by only 8 times in the eleventh generation.

[0078] Example 4: Evaluation of the antibacterial activity of the compound FHTP against other bacteria

[0079] method:

[0080] Following the performance standards for antimicrobial susceptibility testing established by CLSI, the antimicrobial efficacy of compound FHTP at a concentration of 100 μM against Staphylococcus aureus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa was evaluated using the microbroth dilution method.

[0081] The results are shown in Table 5.

[0082] Table 5. Antibacterial results of the antimicrobial compound FHTP

[0083]

[0084]

[0085] As shown in Table 5, the antibacterial compounds at 100 μM all exhibit antibacterial activity against the aforementioned bacteria.

[0086] Experimental Example 5: Evaluation of the antifungal activity of the antimicrobial compound FHTP

[0087] To evaluate the antimicrobial activity of the antimicrobial compound FHTP against plant pathogenic fungi, the following fungi were selected as experimental subjects: Alternaria alternata, Fusarium circinatum, Fusarium perseae, Fusarium solani, and Colletotrichum acutatum and Colletotrichum nymphaeae.

[0088] The antibacterial compound FHTP was dissolved in DMSO, diluted with PBS buffer, and uniformly mixed into PDA medium at 50°C in a molten state. The final concentration of the compound was set at 50 μg / mL, which served as the drug-containing medium. PDA medium containing only DMSO was used as a blank control. A 6 mm wide fungal disc was inoculated in the center of the medium and incubated at 25°C in the dark for 5 days. The growth of each colony was then observed. Three replicates were performed. The diameter of the colonies was measured using the cross-cross method, and the inhibition rate was calculated using the following formula:

[0089] Inhibition rate (%) = (AB) / (AC) × 100%. Where A represents the fungal growth area of ​​the blank control, B represents the fungal growth area of ​​the compound-treated fungus, and C represents the area of ​​a 6 mm wide mycelial cake.

[0090] The results are shown in Table 6.

[0091] Table 6. Antifungal results of the antimicrobial compound FHTP

[0092]

[0093] As shown in Table 6, the antibacterial compound FHTP exhibited an inhibition rate of over 50% against fungi at a concentration of 50 μg / mL.

[0094] In summary, the antibacterial compounds provided in the embodiments of the present invention have the following properties:

[0095] (1) It has anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, with a MIC of 25 μg / mL and an MBC of 50 μg / mL.

[0096] (2) It has broad-spectrum antibacterial activity, specifically, it can inhibit Staphylococcus aureus, Escherichia coli, Salmonella and Pseudomonas aeruginosa at a concentration of 100 μM.

[0097] (3) It has broad-spectrum antifungal activity. Specifically, at a concentration of 50 μg / mL, it can inhibit Alternaria, Fusarium and Anthrax fungi.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibacterial compound, characterized in that, It is selected from compounds with the following structural formulas:

2. A method for preparing the antibacterial compound according to claim 1, characterized in that, include: Fermentation of Podocarpus macrophyllus was carried out to form fermentation products; then the fermentation products were extracted and separated by column chromatography.

3. The preparation method according to claim 2, characterized in that, include: The fermentation products were extracted using ester solvents to form ester extracts; Then, an alcohol-water system is used for elution.

4. The preparation method according to claim 3, characterized in that, include: The fermentation product was extracted with ethyl acetate to form an ethyl acetate extract; The ethyl acetate extract was eluted isocratically with a first mixed solvent, and then eluted isocratically with a second mixed solvent. Wherein, the first mixed solvent is a mixed solvent formed by mixing methanol and water in a volume ratio of (60-78):(22-40); The second mixed solvent is a mixed solvent formed by mixing methanol and water in a volume ratio of (80-87):(13-20).

5. The use of the antibacterial compound of claim 1 in the preparation of a medicament against drug-resistant bacteria.

6. The application according to claim 5, characterized in that, The drug-resistant bacteria include multidrug-resistant bacteria; preferably methicillin-resistant Staphylococcus aureus.

7. The use of the antibacterial compound of claim 1 in the preparation of an antifungal agent.

8. The application according to claim 7, characterized in that, The fungi include Alternaria, Fusarium, and Anthrax fungi.

9. A pesticide formulation, characterized in that, It includes the antibacterial compound as described in claim 1.

10. The use of the antibacterial compound of claim 1 in the preparation of an antibacterial agent. Preferably, the bacteria include Staphylococcus aureus, Escherichia coli, Salmonella, and Pseudomonas aeruginosa.

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