Polyketone compound and application thereof

Abstract

The invention discloses a polyketone compound and application thereof, the compound is orange oily, can be easily dissolved in chloroform, methanol and DMSO, has a molecular formula of C21H32O3, and is obtained by fermentation, separation and extraction of Trichoderma citrinoviride, the obtained compound has an inhibition effect on methicillin-resistant staphylococcus aureus, and valuable compound resources are provided for development of new anti-MRSA drugs.

Description

Technical Field

[0001] The invention relates to the field of chemistry, in particular to polyketide compounds, and also to application of polyketide compounds in preparing drugs for resisting methicillin-resistant Staphylococcus aureus. Background Art

[0002] The global situation of antimicrobial resistance is severe, and the mortality rate of infectious diseases caused by multidrug-resistant bacteria is increasing year by year. In 2022, The Lancet published a "Study on Antibiotic Resistance", which showed that more than 1.2 million people died from antibiotic-resistant infections in 2019, which was higher than the deaths caused by HIV or malaria. By 2050, the annual death toll due to antibiotic resistance is expected to be 10 million. Methicillin-resistant Staphylococcus aureus (MRSA), known as a "superbug", is resistant to multiple types of antibiotics such as methicillin, quinolones, and vancomycin. Today, the number of MRSA infections has exceeded that of AIDS and hepatitis B, ranking first among the three most difficult infectious diseases in the world.

[0003] Mild MRSA infection can cause skin infection and soft tissue infection, including furuncle, abscess, pustule and cellulitis. Severe MRSA infection can cause sepsis, bacteremia and necrotizing pneumonia. Clinically, glycopeptides, oxazolidinones, cyclolipopeptides, glycine and fusidic acid antibiotics are mainly used to treat MRSA infection. Current problems: (i) Lack of new drugs. (ii) Large toxic and side effects. For example, glycopeptide drugs have the inevitable disadvantages of being difficult to absorb orally, requiring intravenous injection and having certain toxic and side effects; the oxazolidinone drug linezolid has the toxic and side effects mainly manifested in neurotoxicity and is expensive; the cyclolipopeptide drug daptomycin has the disadvantages of strong toxic and side effects and can only be administered by intravenous injection; monotherapy with glycine tigecycline will increase the mortality rate of patients; the fusidic acid drug fusidic acid has a low resistance barrier, and patients are prone to develop resistance to fusidic acid monotherapy for a long time, and it has also been shown in in vitro experiments that it is easy to develop resistance to Staphylococcus aureus. (III) Due to the high prevalence of MRSA and the widespread use of vancomycin, vancomycin-intermediate Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. Therefore, it is urgent to develop new anti-MRSA drugs with low toxicity and novel structure.

[0004] Natural products from microorganisms have good biological activity against MRSA. Vancomycin isolated from the secondary metabolites of Amycoticum orientalis, daptomycin isolated from the secondary metabolites of Streptomyces roseosporus, and fusidic acid isolated from the metabolites of Lipocystis have been developed as first-line drugs for the clinical treatment of MRSA infections. The pharmacological effects of Coptis chinensis are diverse, mainly broad-spectrum antibacterial, anti-inflammatory, antioxidant, antiviral and hypoglycemic effects. Coptis chinensis is generally distributed in high mountainous areas at 1200-1800. Its special growth environment makes its endophytic fungi potentially unique. It is expected that lead compounds with significant anti-MRSA activity will be discovered from the secondary metabolites of its endophytic fungi, providing valuable strains and compound resources for the development of new anti-MRSA drugs. In addition, Coptis chinensis has problems such as a long planting cycle and a low content of medicinal ingredients. The endophytic fungi of Coptis chinensis have the advantages of a short cultivation cycle, a high content of medicinal ingredients, easy processing, and low cost. It is of great significance to use plant endophytic fungi resources to make up for medicinal plant resources. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a polyketide compound; a second object of the present invention is to provide use of the polyketide compound in the preparation of a drug against methicillin-resistant Staphylococcus aureus.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. A polyketone compound, the compound having the structural formula as follows:

[0008]

[0009] 2. Use of the polyketide compound in the preparation of a drug for resisting methicillin-resistant Staphylococcus aureus.

[0010] Preferably, the methicillin-resistant Staphylococcus aureus is XN108.

[0011] The beneficial effects of the present invention are as follows: the polyketone compound provided by the present invention is an orange oily substance, which is easily soluble in chloroform, methanol and DMSO, and has a molecular formula of C 21 H 32 O3 is obtained by fermentation, separation and extraction of Trichoderma citrinoviride. The obtained compound has an inhibitory effect on methicillin-resistant Staphylococcus aureus, providing a valuable compound resource for the development of new anti-MRSA drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0013] Figure 1 It is the endophytic fungus HL-2 of Coptis chinensis;

[0014] Figure 2 For compound [M+H] - Mass spectra;

[0015] Figure 3 Fluorescence microscopy images (ac are MRSA treated with DMSO, df are MRSA treated with daptomycin, and gi are MRSA treated with compounds). DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0017] Example 1. Strain Isolation and Identification

[0018] Take the five-year-old Coptis root that has been cleaned with purified water and cut into small sections. Rinse with 75% ethanol for 20 seconds, rinse with sterile water 4 times, disinfect with 5% sodium hypochlorite solution for 3 minutes, and rinse with sterile water again 5 times. Drain the water, cut into small pieces, grind in a mortar, spread the homogenate on the PDA medium containing double antibodies, and culture in a 28°C constant temperature incubator. After the fungus grows, pick the hyphae and purify and culture to obtain a single strain, named HL-2. The culture results are as follows: Figure 1 For short-term storage, store at 4°C on PDA slants or at -80°C in 20% glycerol for long-term storage.

[0019] PDA medium: 6.0g potato flour, 20.0g glucose, 20.0g agar, add 1000mL distilled water, pH value is 5.8±0.2, stir and heat to boil until completely dissolved. Autoclave at 121℃ for 20min, cool to 50℃, pour into sterilized plates or sterilized test tubes. PDA plate containing double antibodies is PDA heated and dissolved and cooled to 50℃, 150mg penicillin and 150mg streptomycin are added to 1000mL medium, and the plate is poured immediately after sufficient mixing.

[0020] Example 2, strain identification

[0021] The genome was extracted and PCR amplified using universal primers ITS4: 5'-TCCTCCGCTTATTGATATGC-3' and ITS5: 5'-GGAAGTAA AAGTCGTA ACAAGG-3'. The PCR product was detected by 1% agarose gel, and the target bands appeared at 500bp and 750bp. The PCR amplification was successful and sent for sequencing. The obtained sequence was compared in NCBI's BLAST. The strain had a similarity of 100.0% with the fungus Trichoderma citrinoviride (sequence number KY750459.1) in the GenBank gene library and was identified as Trichoderma citrinoviride.

[0022] Example 3, fermentation and extraction

[0023] Trichoderma citrinoviride was inoculated on a PDA plate and cultured in a 28°C incubator for 5 days to prepare a spore suspension. Each bottle of 200 mL PDB medium was inoculated with 1 mL of spore suspension and cultured in a shaker at 28°C and 220 rpm for 3 days to prepare a seed solution. Each bottle of rice solid medium was inoculated with about 20 mL of seed solution and cultured at 28°C for 40 days. The fermentation product was extracted with ethyl acetate, ultrasonically extracted 4 times, filtered, the extracts were combined, and concentrated under reduced pressure at 55°C to an extract.

[0024] PDB culture medium: 6.0 g potato flour, 20.0 g glucose, add 1000 mL distilled water, stir and heat to dissolve, sterilize at 121°C for 20 min, and set aside.

[0025] Rice solid culture medium: 200 g rice, 200 mL pure water, 0.2 g anhydrous glucose, 0.1 g copper sulfate, autoclave at 121°C for 20 min, set aside.

[0026] Example 4: Compound separation

[0027] Silica gel column chromatography: dissolve the extract with ethyl acetate and methanol, mix the sample with 200-300 mesh silica gel in an amount of 0.5-0.8 times the weight of the extract, evaporate the solvent and apply it to a silica gel column, and gradient elute with petroleum ether-ethyl acetate solution, ethyl acetate, and methanol in a volume ratio of 15:1, 10:1, 7:1, 3:1, 1:1, 1:3, and 1:5. Each gradient elution is 5 L. 1 L of the eluate is collected and concentrated each time, and 10 components (HLA, HLB, HLC, HLD, HLE, HLF, HLG, HLH, HLI, and HLJ) are obtained by thin layer spot plate merging.

[0028] MCI column chromatography: Dissolve the HLD component in ethyl acetate and methanol, mix the sample with 0.8-1 times the weight of the HLD component macroporous resin, evaporate the solvent and load it on the MCI column, elute with 30%-100% methanol-water and acetone to obtain 10 components from HLD-1 to HLD-10.

[0029] ODS column chromatography: HLD-6 component was dissolved in methanol, wet loaded, and eluted with 30%-100% methanol-water to obtain 36 components from HLD-6-1 to HLD-6-36.

[0030] Semi-preparative liquid chromatography separation: detection wavelengths are 200nm and 232nm, and the chromatographic column is COSMOSIL 5C 18 -MS-IIPacked Column 10IDx250mm, mobile phase is 78% methanol-water solution, flow rate is 3mL / min, prepare and separate HLD-6-32 components, and collect compounds with elution time of 55min.

[0031] Example 5. Identification of compound structures

[0032] The compound is an orange oil, which is easily soluble in chloroform, methanol and DMSO. HRESIMS is m / z 333.2415 [M+H] + (Calculated value C 21 H 33 O3,333.2430)( Figure 2 ), determine the molecular formula of the compound to be C 21 H 32 O3. Compounds 1 H NMR and 13 The CNMR data are shown in Table 1.

[0033] Table 1. Compounds 1 H NMR and 13 C NMR data (solvent: CDCl3)

[0034]

[0035] The identified compound structure is as follows:

[0036]

[0037] Example 6, MIC value determination

[0038] The MIC values ​​of the compounds against MRSA were determined using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI).

[0039] The tested strain was the MRSA standard strain: ATCC43300.

[0040] MRSA-resistant strain: XN108 (Diagnostic Microbiology and Infectious Disease 77 (2013) 64–68), the first ST239 vancomycin-intermediate Staphylococcus aureus (VISA) reported in China, was isolated from a wound secretion sample of a steam burn patient in Chongqing, China.

[0041] Preparation of compound stock solution: 1-2 mg of compound was dissolved in DMSO to prepare a 25.6 mg / mL stock solution, which was stored at -20°C.

[0042] Preparation of bacterial solution:

[0043] (1) Take a small amount of the strain frozen at -80℃, streak it on an MHA plate, and culture it at 37℃ for 16-18h.

[0044] (2) Pick a single colony and inoculate it into 3 mL of CAMHB medium. Cultivate at 37°C, 220 rpm for 5 h until the logarithmic growth phase.

[0045] (3) The bacterial suspension was first diluted to 0.5 McFarland turbidity, and then diluted 100 times with CAMHB medium. The bacterial suspension concentration was about 10 6 CFU / mL.

[0046] Drug dilution: dilute the compound to 256μg / mL with CAMHB medium, add 200μL of drug solution to the first row of wells in a 96-well plate, add 100μL of CAMHB medium to the second to eighth rows, use an eight-channel pipette to draw 100μL of drug solution from the first row to the second row, mix, then draw 100μL from the second row to the third row, mix. Perform a two-fold gradient dilution to the eighth row, draw out 100μL after mixing and discard, the drug concentrations are 256, 128, 64, 32, 16, 8, 4, 2μg / mL.

[0047] Mixing of drug solution and bacterial solution: Add 100 μL of bacterial suspension to each 100 μL drug solution well. The final concentrations of the compound are 128, 64, 32, 16, 8, 4, 2, and 1 μg / mL, and the final concentration of the bacterial solution is 5x10 5 CFU / mL. The 96-well plate was placed in a 37°C incubator for 16-18 hours. The lowest concentration at which no bacteria grew was observed by naked eye as the MIC value of the compound.

[0048] Results: The blank control (CAMHB) wells were clear, and the negative control (bacterial solution) wells were turbid. The MICs of the compounds and vancomycin against MRSA are shown in Table 2.

[0049] Table 2. MICs of compounds and vancomycin against MRSA strains (μg / mL)

[0050]

[0051] CAMHB medium: 3.0 g beef extract powder, 17.5 g acid hydrolyzed casein, 1.5 g soluble starch, 0.05 g calcium chloride, add 1000 mL distilled water, pH value is 7.3 ± 0.1, heat to dissolve. Autoclave at 121℃ for 20 min and set aside.

[0052] MHA medium: 2.0g beef powder, 17.5g acid hydrolyzed casein, 1.5g soluble starch, 17.0g agar, add 1000mL distilled water, heat to dissolve. Autoclave at 121℃ for 20min, cool to 55℃ and pour into a plate for later use.

[0053] Example 7: Fluorescence microscopy

[0054] The bacterial suspension of MRSA resistant strain (XN108) was diluted to 0.5 McFarland turbidity, and then diluted 10 times with CAMHB medium. The bacterial suspension concentration was about 10 7 CFU / mL. Set up a drug-treated group (compound concentration of 32μg / mL), a positive control group (daptomycin concentration of 32μg / mL) and a negative control group (DMSO), and the total sample volume is 1mL. Cultivate at 37℃, 220rpm for 6h, centrifuge at 4000rpm, 4℃ for 3min, remove the supernatant, wash twice with PBS, resuspend in 160μL PBS, add 20μL DAPI (4',6-diamidino-2-phenylindole, 10μg / mL) and 20μL PI (propidium iodide, 20μg / mL), respectively, and incubate in the dark for 30min. Centrifuge at 4000rpm, 4℃ for 3min, remove the supernatant, wash three times with PBS, and resuspend in an appropriate amount of PBS. Pipette 3μL of bacterial solution to prepare slides, image under a 100× objective lens of a fluorescence microscope, and take pictures. The results are as follows. Figure 3 shown.

[0055] DAPI can pass through intact or damaged cell membranes, bind to double-stranded DNA in the cell nucleus, and emit blue fluorescence. PI can only pass through damaged cell membranes and bind to DNA, showing red fluorescence. The DMSO negative control group only showed blue fluorescence, indicating that the cell membrane of MRSA was intact. In contrast, at a concentration of 32μg / mL, blue and red fluorescence were observed after the compound and daptomycin were treated with MRSA, respectively, indicating that the compound can destroy the cell membrane and has the potential to be studied and developed as an anti-MRSA drug.

[0056] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A polyketone compound, characterized in that: The compound structural formula is as follows:

2. Use of the polyketide compound according to claim 1 in the preparation of a drug for resisting methicillin-resistant Staphylococcus aureus.

3. The use according to claim 2, characterized in that: The methicillin-resistant Staphylococcus aureus is XN108.

Images