Gamma-lactam alkaloid compound with activity of resisting methicillin-resistant staphylococcus aureus as well as preparation method and application of gamma-lactam alkaloid compound

Abstract

The invention discloses a gamma-lactam alkaloid compound with anti-methicillin-resistant staphylococcus aureus activity and a preparation method and application thereof, and is characterized in that the structural formula of the compound is shown as I, a preparation method of the gamma-lactam alkaloid comprises the following steps: performing fermentation culture on aspergillus ustus with the preservation number of CCTCC NO: M2014086 to obtain a gamma-lactam alkaloid fermentation product, soaking the fermentation product in ethyl acetate, extracting to obtain a crude extract, removing oil from the crude extract through normal hexane, removing water from the crude extract through dichloromethane to obtain an extract, and performing normal-phase silica gel column chromatography on the extract on the basis to obtain the gamma-lactam alkaloid. The gamma-lactam alkaloid compound has the advantages that the gamma-lactam alkaloid compound has anti-methoxyl staphylococcus aureus activity, the antibacterial mechanism of the gamma-lactam alkaloid compound is described, and the gamma-lactam alkaloid compound can be used as a potential drug for resisting food-borne pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to a gamma-lactam alkaloid compound, in particular to a gamma-lactam alkaloid compound with anti-methicillin-resistant Staphylococcus aureus activity extracted from marine fungus fermentation products, and a preparation method and application thereof. Background Art

[0002] Staphylococcus aureus is a common foodborne pathogen that is widely present in the natural environment. Under appropriate conditions, Staphylococcus aureus can produce enterotoxins and cause food poisoning. With the emergence of drug-resistant Staphylococcus aureus (MASA), infection with Staphylococcus aureus has become more serious. Methicillin-resistant Staphylococcus aureus is a common and highly toxic bacterium in clinical practice that can cause severe gastroenteritis, nausea, vomiting, diarrhea and abdominal pain within 1 to 6 hours after eating contaminated food. Methicillin-resistant Staphylococcus aureus (MRSA) refers to Staphylococcus aureus that carries the mecA gene and is resistant to all penicillins, cephalosporins and carbapenems.

[0003] γ-lactam alkaloids are an important class of secondary metabolites in natural products, with a unique 10-phenyl-substituted perhydroisoindole-1-one skeleton as the main structural skeleton. Most of the existing marketed antibacterial drugs are β-lactam antibiotics. The invention of this compound provides a new natural product for the development of drugs against foodborne pathogens. The inventors discovered a new natural product of γ-lactam alkaloids in a chemical investigation of ethyl acetate extracts fermented in the culture medium of marine fungus Aspergillus sp. NBU4698. At present, there is no report on the chemical structure and anti-MRSA mechanism of this compound, so there is no related drug on the market. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity and a preparation method and use thereof.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] 1. A γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity, the structural formula of the compound is shown in (I):

[0007]

[0008] 2. The method for preparing the above-mentioned γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity comprises the following steps:

[0009] (1) Fermentation production

[0010] Aspergillus ustus with a preservation number of CCTCC NO: M2014086 is streaked on a plate of PDA solid culture medium, inverted and cultured in an incubator at 28° C. for 3 days, a single colony is picked and inoculated into PDB liquid culture medium, and cultured on a shaker at a temperature of 28° C. and a speed of 150 rpm / min for 3 days to obtain a seed solution, a conical flask is filled with rice culture medium, and then sterilized at 121° C. for 15 minutes, and each bottle of rice culture medium is inoculated with 15 mL of the seed solution, and cultured at 28° C. for 30 days to obtain a fermentation product;

[0011] (2) Extraction of crude extract

[0012] Adding ethyl acetate in an equal volume to the fermented product obtained in step (1), extracting repeatedly until the extract is colorless, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract;

[0013] (3) Removal of oil and water from crude extract

[0014] The crude extract obtained in step (2) is first dissolved in 95% methanol, and then the same volume of n-hexane as the methanol is added to extract and remove oil. After repeating the operation three times, the methanol solution is vacuum evaporated to dryness; then it is dissolved in dichloromethane, and the same volume of water as the dichloromethane is added to extract and remove water. The dichloromethane solution is vacuum evaporated to dryness to obtain an extract;

[0015] (4) Isolation and preparation of compounds

[0016] The extract obtained in step (3) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 100:1 as an eluent, and the eluate is collected; the collected eluate is subjected to reverse phase medium pressure column chromatography, and linear gradient elution is performed using methanol-water with a methanol volume percentage of 45-100% as an eluent, and the eluted fractions are collected, and the fractions are arranged in descending order according to polarity, and 6 components are obtained by merging; the fourth component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution composed of acetonitrile-water in a volume ratio of 53:47 as a mobile phase to obtain a monomer compound, whose structure is shown in (I):

[0017]

[0018] Furthermore, the PDA solid culture medium described in step (1) is prepared as follows: 6 g of potato powder, 20 g of glucose and 20 g of agar are added to 1000 mL of distilled water.

[0019] Furthermore, the preparation method of the PDB liquid culture medium described in step (1) is as follows: 6 g of potato extract powder and 20 g of glucose are dissolved in 1000 mL of water.

[0020] Furthermore, the rice culture medium described in step (1) is prepared as follows: 90 g of rice and 3 g of sea salt are dissolved in 110 mL of water.

[0021] Furthermore, in the linear gradient elution of the reversed-phase medium-pressure column chromatography in step (4), the volume percentage of methanol ranges from 45 to 100%, and the elution time is 120 min.

[0022] Furthermore, the flow rate of the compound separation preparation by semi-preparative reverse-phase HPLC described in step (4) is 2.0 mL / min.

[0023] 3. Use of the above-mentioned γ-lactam alkaloid compounds in the preparation of anti-methoxy-resistant Staphylococcus aureus inhibitors.

[0024] Compared with the prior art, the advantages of the present invention are: a γ-lactam alkaloid compound and its preparation method and use are obtained by separating and culturing the obtained microorganisms, and then the fermented product is extracted by soaking the fermented product with ethyl acetate to obtain a crude extract, and then the crude extract is deoiled by n-hexane and dehydrated by dichloromethane to obtain an extract, and then the extract is separated and purified by medium-pressure normal phase column chromatography, medium-pressure reversed phase column chromatography, and semi-preparative high-performance liquid chromatography to obtain a monomer compound, which has anti-methicillin-resistant Staphylococcus aureus activity and low toxicity. The antibacterial mechanism study found that the compound can cause the bacterial surface to be irregular and wrinkled, thereby destroying the structure of the bacterial cell wall and cell membrane, and can be used for the development of drugs to inhibit related diseases caused by methicillin-resistant Staphylococcus aureus. .

[0025] The Aspergillus ustus is a strain DJ003, with a deposit number of CCTCC NO: M2014086, and was deposited in China Center for Type Culture Collection on March 14, 2014, at Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the HR-ESI-MS spectrum of the compound of the present invention;

[0027] Figure 2 is the UV spectrum of the compound of the present invention;

[0028] Figure 3 is the nuclear magnetic resonance IR spectrum of the compound of the present invention;

[0029] Figure 4 is the hydrogen nuclear magnetic resonance spectrum of the compound of the present invention;

[0030] Figure 5 is the carbon nuclear magnetic resonance spectrum of the compound of the present invention;

[0031] Figure 6 is the nuclear magnetic resonance DEPT-135 spectrum of the compound of the present invention;

[0032] Figure 7 is the COSY nuclear magnetic resonance spectrum of the compound of the present invention;

[0033] Figure 8 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;

[0034] Fig. 9 is the nuclear magnetic resonance HMBC spectrum of the compound of the present invention;

[0035] Fig.10 is the nuclear magnetic resonance NOESY spectrum of the compound of the present invention;

[0036] Fig.11 is a single crystal diffraction pattern of the compound of the present invention;

[0037] Fig.12 It is a COSY and HMBC intuitive schematic diagram of the compound of the present invention;

[0038] Fig.13 This is a schematic diagram of the bactericidal curve of the compound of the present invention;

[0039] Fig.14 The cytotoxicity analysis results of the compounds of the present invention;

[0040] Fig.15 are indicators of the antibacterial mechanism of the compounds of the present invention, wherein A is the scanning electron microscopy (SEM) analysis of MRSA ATCC43300 treated with compound I, B is the PI staining flow cytometric analysis of MRSA ATCC43300 treated with compound I for 30 or 120 minutes, red line: no treatment as a negative control, blue line: 30 minutes of treatment with 1×MIC compound, orange line: 30 minutes of treatment with 2×MIC compound, pink line: 120 minutes of treatment with 2×MIC compound, purple line: 120 minutes of treatment with 4×MIC compound; C is the interaction between compound I and MRSA ATCC43300 genomic DNA, M: DNA marker, CK: untreated DNA. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below with reference to the accompanying drawings.

[0042] Example 1: A γ-lactam alkaloid compound having anti-methoxy-resistant Staphylococcus aureus activity has a structural formula as shown in (I):

[0043]

[0044] Example 2: A method for preparing a γ-lactam alkaloid compound represented by structural formula (I) in Example 1, comprising the following steps:

[0045] (1) Fermentation production

[0046] The Aspergillus ustus with a preservation number of CCTCC NO: M2014086 was streaked on a plate of PDA solid medium (prepared as follows: 6 g of potato powder, 20 g of glucose and 20 g of agar were added to 1000 mL of distilled water), inverted and cultured in a 28° C. incubator for 3 days, and then a single colony was picked and inoculated in PDB liquid medium (prepared as follows: 6 g of potato powder and 20 g of glucose were dissolved in 1000 mL of water), and placed on a shaking table at a temperature of 28° C. and a speed of 150 rpm / min and cultured for 3 days to obtain seed liquid, and a rice medium (90 g of rice, 3 g of sea salt, and 110 mL of water) was loaded into a 1 L conical flask, and then autoclaved at 121° C. for 15 minutes; finally, each bottle of rice medium was inoculated with 15 mL of seed liquid, and cultured at 28° C. for 30 days to obtain a fermentation product;

[0047] (3) Extraction of crude extract

[0048] Adding ethyl acetate in an equal volume to the fermented product obtained in step (1), extracting repeatedly until the extract is colorless, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract;

[0049] (3) Removal of oil and water from crude extract

[0050] The crude extract obtained in step (2) is first dissolved in 95% methanol, and then the same volume of n-hexane as the methanol is added to extract and remove oil. After repeating the operation three times, the methanol solution is vacuum evaporated to dryness; then it is dissolved in dichloromethane, and the same volume of water as the dichloromethane is added to extract and remove water. The dichloromethane solution is vacuum evaporated to dryness to obtain an extract;

[0051] (4) Isolation and preparation of compounds

[0052] The extract obtained in step (3) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 100:1 as an eluent, and the eluent is collected; the collected eluent is subjected to reverse phase medium pressure column chromatography, and linear gradient elution is performed using methanol-water with a methanol volume percentage of 45-100% as an eluent, and the elution time is 120min, and the eluted fractions are collected, arranged in descending order according to the polarity of the fractions, and 6 components are obtained by merging; the fourth component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution composed of acetonitrile-water in a volume ratio of 53:47 as a mobile phase at a flow rate of 2.0mL / min to obtain a monomer compound, whose structure is shown in (I):

[0053]

[0054] Example 3: Structural identification of the γ-lactam alkaloid compounds prepared by the method of Example 2.

[0055] The compound I of the present invention is a white solid. Figure 1 The positive ion HR-ESI-MS gave a quasi-molecular ion peak of m / z 464.2424 [M+H] + , its molecular formula is determined to be C 28 H 34 O5 (calculated molecular weight is 464.2437), indicating that its degree of unsaturation is 13. 1 H and 13 C NMR spectra, 2D data and single crystal data are available at Figure 4-12 and Table 1: Figure 6-10 is the main two-dimensional correlation of the compounds of the present invention; Figure 4 of 1 The HNMR spectrum showed four methyl signals (CH3-11; CH3-12; CH3-22; CH3-23), two methylene signals (CH2-10; CH2-15), a monosubstituted phenyl group (δ H 7 . 14, 7.19, 7.26), 2 pairs of olefin hydrogen signals (CH-13 / 14; CH-19 / 20), 13-ene group and 19-ene group have trans configuration, and their chemical shifts have large coupling constants (J H -13 / H-14 = 15.2 Hz and J H -19 / H-20=15.5Hz), 2 oxygen-containing sp 3 Hybridized carbon signal (δ C 88.7, C-9; δ C 80.4, C-18), two sp 2Quaternary carbon signal (δ C 140.5, C-6; δ C 137.5, C-1′), a conjugated carbonyl signal (δ C 213.1, C-17) and two ester or amide carbonyl signals (δ C 166.2, C-21; δ C 171.5, C-1); Figure 5 and Figure 6 It can be seen that the compound contains 28 carbon atoms, of which 13 C and DEPT 135 spectra show that there are 7 quaternary carbons (C-1, C-6, C-9, C-17, C-18, C-21, C-1′), 2 methylene groups (C-10, C-15), and 15 methine groups (C-3, C-4, C-5, C-7, C-8, C-13, C-14, C-16, C-19, C-20, C-2′ / 3′, C-5′ / 6′, C-4′) in the compound. Figure 4 of 1 The H NMR high-field region signal analysis revealed four methyl carbon signals (C-11, C-12, C-22, and C-23); Figure 7 The COSY spectrum shows that H-19 and H-20, H-2′ / H-3′ / H-4′ / H-5′ / H-6′, H-3 / H-4 / H-5 / H2-10 / H3-11, H-7 / H-8 / H-13 / H-14 / H2-15 / H-16 / H3-22 are COSY-correlated. Figure 8 The HSQC spectrum of Ⅰ shows the corresponding relationship between the chemical shifts of protons and atoms. These characteristics indicate that Ⅰ has a 10-phenyl-substituted perhydroisoindole-1-one skeleton. Fig. 9 As shown, HMBC correlations from H3-11 to C-4, C-6 and from H3-12 to C-5, C-7 indicate that C-5, C-6 in Ⅰ are connected to methyl groups (CH3-11, CH3-12). HMBC correlations of CH3-22 to C-15 and C-17 confirm that CH3-22 is connected to C-16, and HMBC correlations of CH3-23 to C-18 and C-19 confirm that CH3-23 is connected to C-18. In addition, the key COSY correlations of H-7 / H-8 / H-13 / H-14 / H2-15 / H-16 / H3-22 show that the olefin carbon (C-7) in Ⅰ is connected to C-8, and H-3 / H-4 / H-5 / H2-10 / H3-11 confirm that the methylene carbon (C-10) in Ⅰ is connected to C-3, thus confirming the planar structure of Ⅰ and finally determining that the compound is a new compound. Fig.10 The NOESY spectrum lacks key NOESY correlations. Fig.11The absolute configuration of Ⅰ was determined by the X-ray single crystal diffraction pattern.

[0056] Table 1. Compound I 1 H and 13 C NMR data (DMSO-d6)

[0057]

[0058]

[0059] Note 1: s-singlet, d-doublet, t-triplet, dd-quartet, m-multiplet.

[0060] Note 2: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.

[0061] Fig.11 It is the single crystal diffraction pattern of the compound of the present invention. Fig.12 COSY and HMBC are intuitive examples of the compounds of the present invention. Fig.12 All the information of the above NMR spectrum can be known.

[0062] Example 4: Determination of the activity of the γ-lactam alkaloid compounds of Example 1 against methicillin-resistant Staphylococcus aureus.

[0063] 1. Determination of minimum inhibitory concentration (MIC)

[0064] Experimental materials: methicillin-resistant Staphylococcus aureus ATCC43300 (MRSAATCC43300), MH medium, vancomycin, sterile water, dimethyl sulfoxide (DMSO).

[0065] Experimental steps: transfer and activate methicillin-resistant Staphylococcus aureus ATCC43300 overnight, incubate the activated bacteria to the logarithmic phase and dilute to 5 log 10 CFU / ml, compound I was diluted with DMSO to a final concentration of 128, 64, 32, 16, 8 (μg / ml) and added to a 96-well plate. Vancomycin was diluted with sterile water to a final concentration of 2×MIC and added to a 96-well plate to form a positive control group. The diluted bacterial solution was added to the experimental wells, incubated in a 37°C incubator for 24 hours, and then observed for bacterial growth. The determination of the minimum inhibitory concentration showed that the MIC of compound I against MRSAATCC43300 was 64μg / mL.

[0066] 2. Sterilization curve

[0067] Experimental materials: MRSAATCC43300, MH medium, vancomycin, sterile water, dimethyl sulfoxide (DMSO), PBS phosphate buffer, agar powder.

[0068] Experimental steps: MRSAATCC43300 was transferred overnight for activation and then transferred to the logarithmic phase. MH solid medium was poured into the bacterial culture dish and cooled to solidify. The bacteria were diluted to a final concentration of 5 log 10 CFU / ml, add 5ml of MH medium to a conical flask, add bacterial solution, add 1×MIC, 2×MIC, 4×MIC compounds respectively according to calculation, add DMSO with the same volume as the highest drug concentration to the blank control group, and add 2×MIC vancomycin to the antibiotic control group according to calculation. After mixing, dilute ten times to the appropriate concentration, dilute to the appropriate multiple, apply the plate, and perform three parallel experiments. Repeat the operation at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h after treatment. Count and plot after 24h.

[0069] The results are as follows Fig.13 As shown by Fig.13 It can be seen that for the MRSAATCC43300 control group, the colony count (log 10 cfu / mL) increased rapidly within 2 to 4 hours and maintained growth within 12 hours, eventually reaching 7.0log 10 cfu / mL. Under 1×MIC concentration treatment, the number of bacteria increased linearly to 7.4log 10 cfu / mL. Within 2-12h, the number of bacteria continued to decrease under the 4×MIC concentration treatment, and the number of bacteria was less than 1.0log 10 cfu / mL, and slightly increased within 6-8h under 2×MIC concentration treatment, and the colony count was close to 2.9log 10 cfu / mL, and finally stabilized at 2.5log10 cfu / mL. Therefore, 2×MIC and 4×MIC have certain antibacterial effects.

[0070] 2. Cytotoxicity

[0071] Experimental materials: RAW264.7 macrophages, DMEM culture medium, PBS phosphate buffer, dimethyl sulfoxide (DMSO), CCK-8 solution.

[0072] Experimental steps: RAW264.7 macrophages were cultured in DMEM medium in an incubator at 37°C, 5% CO2 and saturated humidity. When the cells reached the logarithmic phase, they were resuspended in DMEM medium containing 10% fetal bovine serum and cultured at 2.5×10 5The cells / well density was inoculated in a 96-well plate, 100 μL per well, and placed in a CO2 constant temperature incubator for 24 hours. After the culture was completed, the DMEM medium was discarded, and then washed twice with PBS buffer. 100 μL of compounds with concentrations of 128, 64, 32, 16, 8, and 4 μg / mL were added to each well according to the concentration gradient. At the same time, only the culture medium was added as a blank control, and the cells were incubated for another 24 hours. After the incubation was completed, 10 μL / well of CCK-8 solution was added to the 96-well plate, incubated at 37°C for 4 hours, and the absorbance of each well was measured at 460nm by an enzyme reader. Each sample was repeated three times.

[0073] The results are as follows Fig.14 As shown by Fig.14 It can be seen that when the drug concentration is 128μg / mL, it has a certain toxicity, and the survival rate is about 65%. As the drug concentration decreases, the toxicity also decreases. Therefore, compound I is safe to use in food processing and storage environments, and has minimal impact on the host.

[0074] 3. Scanning electron microscopy to observe the morphology of biofilm

[0075] Experimental materials: MRSAATCC43300, MH medium, dimethyl sulfoxide (DMSO), PBS buffer, anhydrous ethanol, and glutaraldehyde solution.

[0076] Experimental steps: Activate MRSAATCC43300 overnight, then transfer and culture to the mid-logarithmic phase, and dilute the bacteria with PBS to a concentration of 1×10 8 CFU / mL bacterial suspension and 1mL / tube were added to the centrifuge tube. Compound I was added to the bacterial solution to make the final concentration 4×MIC, and the untreated bacterial suspension was used as a negative control, and incubated at 37℃ for 2h. After the incubation, centrifuge at 5000rpm for 5min at room temperature, discard the supernatant and wash the bacteria twice with PBS buffer, add 1mL 2.5% glutaraldehyde solution to the bacterial sediment, gently blow to suspend the bacteria and fix the bacteria for 15min. After glutaraldehyde fixation, wash twice with PBS buffer again, and then use different concentrations of ethanol solution (20%-50%-70%-85%-95%×2-100%×2) for gradient dehydration, each time for 5min, then drop the sample on the silicon wafer and dry it at room temperature overnight. Finally, use an ion sputtering instrument to form a metal film on the surface of the sample, and then use S4800 scanning electron microscope to observe the morphology of the bacteria after gold palladium sputtering.

[0077] The results are as follows Fig.15As shown in Figure A, the results of scanning electron microscopy showed that the bacterial surface of the blank control group was smooth and intact. However, after treatment with compound I at a concentration of 4×MIC, obvious irregular wrinkles appeared on the bacterial surface, the cell membranes adhered to each other to form filaments, and formed multiple holes of varying sizes, leading to cytoplasm leakage and cell death, indicating that compound I may have the ability to destroy bacterial cell walls and cell membranes.

[0078] 4. Effect of Compound Ⅰ on bacterial cell membrane

[0079] Experimental materials: MRSAATCC43300, MH medium, dimethyl sulfoxide (DMSO), PBS phosphate buffer, propidium iodide (PI) solution.

[0080] Experimental steps: MRSAATCC43300 was cultured overnight and inoculated to the logarithmic phase. The bacteria were diluted to 1×10 8 CFU / mL, 1mL was added to a centrifuge tube, and compound I was added to make the final concentrations 1×, 2× and 4×MIC, respectively, and then incubated at 37°C for 30min and 120min, respectively. After the incubation, centrifuge at 5000rpm for 5min, and then the bacteria were washed twice with PBS buffer. Finally, PI solution was added to make the final concentration 50μg / mL and incubated at room temperature for 15min. The untreated group was used as a negative control. Finally, fluorescence was detected by flow cytometry.

[0081] The results are as follows Fig.15 As shown in B, Fig.15 From Figure B, it can be seen that the fluorescence intensity of MRSA ATCC43300 without compound I was 1.78%, indicating that the bacterial cell membrane was intact before compound I was used. As the drug concentration increased, the fluorescence increased from 13.8% (1×MIC) to 16.9% (2×MIC), while at a concentration of 4×MIC, the fluorescence decreased, which may be due to the difficulty in capturing bacterial fragments under high concentration drug treatment, resulting in low values. The fluorescence intensity decreased after 120 minutes of treatment with compound I (1×MIC: 2.09%, 2×MIC: 2.37%, 4×MIC: 5.48%). This may be due to the fact that long-term high-concentration drug use caused the bacteria to be completely broken and unable to maintain a certain morphology, so some PI-stained cells were not captured, resulting in a decrease in fluorescence intensity. Therefore, compound I has a strong effect of destroying cell walls and cell membranes.

[0082] 5. Effect of Compound Ⅰ on bacterial DNA

[0083] Experimental materials: MRSA ATCC43300, bacterial genome extraction kit, DMSO.

[0084] Experimental steps: MRSAATCC43300 was cultured to the logarithmic phase, and bacterial genomic DNA was collected and extracted using a bacterial genome extraction kit. After that, compound I diluted to different concentrations (8-128 μg / mL) was mixed with bacterial DNA in a volume ratio of 1:1, cultured at room temperature for 10 minutes, and then 1% agarose electrophoresis was used to analyze the migration of bacterial genomic DNA. DNA not treated with compound I was used as a negative control. After the electrophoresis, the electrophoresis results were observed using a gel imager.

[0085] The results are as follows Fig.15 As shown in C, Fig.15 From C, we can see that the blocking effect on the bacterial genome is very small, so compound I has little effect on changing the structure of the bacterial genome and is more difficult to bind to DNA.

[0086] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity, characterized in that The structural formula of the compound is shown in (I); 2. A method for preparing the γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 1, characterized in that The steps include: (1) Fermentation production Aspergillus ustus with a preservation number of CCTCC NO: M2014086 is streaked on a plate of PDA solid culture medium, inverted and cultured in an incubator at 28° C. for 3 days, a single colony is picked and inoculated into PDB liquid culture medium, and cultured on a shaker at a temperature of 28° C. and a speed of 150 rpm / min for 3 days to obtain a seed solution, a conical flask is filled with rice culture medium, and then sterilized at 121° C. for 15 minutes, and each bottle of rice culture medium is inoculated with 15 mL of the seed solution, and cultured at 28° C. for 30 days to obtain a fermentation product; (2) Extraction of crude extract Adding ethyl acetate in an equal volume to the fermented product obtained in step (1), extracting repeatedly until the extract is colorless, and then vacuum evaporating the ethyl acetate extract to obtain a crude extract; (3) Removal of oil and water from crude extract The crude extract obtained in step (2) is first dissolved in 95% methanol, and then the same volume of n-hexane as the methanol is added to extract and remove oil. After repeating the operation three times, the methanol solution is vacuum evaporated to dryness; then it is dissolved in dichloromethane, and the same volume of water as the dichloromethane is added to extract and remove water. The dichloromethane solution is vacuum evaporated to dryness to obtain an extract; (4) Isolation and preparation of compounds The extract obtained in step (3) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 100:1 as an eluent, and the eluate is collected; the collected eluate is subjected to reverse phase medium pressure column chromatography, and linear gradient elution is performed using methanol-water with a methanol volume percentage of 45-100% as an eluent, and the eluted fractions are collected, and the fractions are arranged in descending order according to polarity, and 6 components are obtained by merging; the fourth component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a mixed solution composed of acetonitrile-water in a volume ratio of 53:47 as a mobile phase to obtain a monomer compound, whose structure is shown in (I):

3. The method for preparing a γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 2, characterized in that The PDA solid culture medium described in step (1) is prepared as follows: 6 g of potato powder, 20 g of glucose and 20 g of agar are added to 1000 mL of distilled water.

4. The method for preparing a γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 2, characterized in that The preparation method of the PDB liquid culture medium described in step (1) is as follows: 6 g of potato extract powder and 20 g of glucose are dissolved in 1000 mL of water.

5. The method for preparing a γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 2, characterized in that The rice culture medium described in step (1) is prepared as follows: 90 g of rice, 3 g of sea salt, and 110 mL of water.

6. The method for preparing a γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 2, characterized in that: In the linear gradient elution of the reverse phase medium pressure column chromatography in step (4), the volume percentage of methanol ranges from 45 to 100%, and the elution time is 120 min.

7. The method for preparing a γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 2, characterized in that: The flow rate of the semi-preparative reversed-phase HPLC compound separation preparation described in step (4) is 2.0 mL / min.

8. Use of the γ-lactam alkaloid compound having anti-methicillin-resistant Staphylococcus aureus activity according to claim 1 in the preparation of an anti-methicillin-resistant Staphylococcus aureus inhibitor.

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