Marine fungus Trichoderma, quorum sensing inhibitor alpha, beta-unsaturated cyclopentanone compound as well as preparation method and application of quorum sensing inhibitor alpha, beta-unsaturated cyclopentanone compound

Abstract

The invention discloses a marine fungus Trichoderma, a quorum sensing inhibitor alpha, beta-unsaturated cyclopentanone compound as well as a preparation method and application of the quorum sensing inhibitor alpha, beta-unsaturated cyclopentanone compound. The alpha, beta-unsaturated cyclopentanone compound is separated from a fermentation culture of a marine fungus Trichoderma sp.6791, and has bacterial biofilm formation inhibition and quorum sensing system related virulence factors, and tests prove that the alpha, beta-unsaturated cyclopentanone Hypocreone C can inhibit the formation of a bacterial biofilm and quorum sensing system related virulence factors under the condition of not inhibiting the growth of pseudomonas aeruginosa, and can inhibit the formation of the quorum sensing system related virulence factors under the condition of not inhibiting the growth of pseudomonas aeruginosa. The pseudomonas aeruginosa can effectively inhibit the formation of a pseudomonas aeruginosa biofilm and obviously inhibit the secretion of pyocyanine and rhamnolipid virulence factors in a quorum sensing system, so that the pathogenicity and virulence of bacteria are reduced, and the generation of multiple drug resistance of the bacteria is slowed down. When the compound Hypocrenone C provided by the invention is combined with antibiotic drugs such as amikacin, the inhibition effect of amikacin on pseudomonas aeruginosa can be remarkably enhanced; the cyclopentanone compound provided by the invention provides a lead compound for developing novel quorum sensing inhibitors and antibacterial drugs, and has potential application as antibacterial drugs.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical biotechnology, and specifically relates to an α,β-unsaturated cyclopentanone compound and a preparation method thereof and application thereof in the preparation of antibacterial drugs. Background Art

[0002] Pseudomonas aeruginosa (PA) is a common conditional pathogen in clinical practice. Due to the various infections caused by the virulence factors of its biofilm and quorum sensing system, the clinical treatment of hospital infections faces huge challenges. At present, many Pseudomonas aeruginosa are resistant to traditional antibiotics, and the difficulty of prevention and treatment is gradually increasing. Therefore, the development of new anti-resistant bacteria drugs is the focus of antimicrobial drug research.

[0003] Quorum sensing (QS) is a communication system between bacterial cells. When the density of bacterial population reaches a certain threshold, it can release specific signal molecules into the environment, showing a series of physiological functions and behaviors that a single cell cannot complete, such as biofilm formation and toxin secretion. Studies have found that the expression of multiple virulence factors of Pseudomonas aeruginosa (such as elastase, pyocyanin, rhamnolipid, etc.) are regulated by the quorum sensing system. Inhibiting the QS system of PA can downregulate the expression of its virulence genes and the secretion of virulence factors, thereby achieving the purpose of PA prevention and treatment. In addition, quorum sensing inhibitors can regulate the formation of biofilms without inhibiting or slightly inhibiting bacterial growth, effectively alleviating the development of drug resistance. Therefore, the discovery of low-toxic and highly active bacterial quorum sensing inhibitors can provide more effective antibacterial agents for the prevention and treatment of Pseudomonas aeruginosa bacterial infections.

[0004] Special growth environments such as high salt and high pressure give marine fungi unique metabolic pathways and genetic backgrounds, which enable them to metabolize and accumulate active secondary metabolites with unique structures. They occupy an important position in natural product research and are one of the important sources for discovering new anti-resistant antibacterial drugs.

[0005] Hypocrenone C is an α,β-unsaturated cyclopentanone compound isolated from the marine fungus Trichoderma sp.6791. Experiments have found that at concentrations that do not inhibit bacterial growth, Hypocrenone C has the effects of significantly inhibiting the activity of the QS system of Pseudomonas aeruginosa and inhibiting the formation of biofilms of Pseudomonas aeruginosa. When used in combination with amikacin, it can significantly enhance the antibacterial activity of amikacin, becoming a new type of quorum sensing inhibitor of Pseudomonas aeruginosa. It is expected to be developed into a new anti-resistant antibacterial drug to prevent or treat bacterial infections. Summary of the invention

[0006] In view of the above-mentioned shortcomings, the first object of the present invention is to provide a marine fungus Trichoderma sp.6791, which is a Trichoderma fungus derived from marine sediments. It has been deposited in the Guangdong Provincial Microbiological Culture Collection Management Center with a deposit number of GDMCC NO: 62264 and a deposit date of March 2, 2022.

[0007] The second object of the present invention is to provide a quorum sensing inhibitor α, β-unsaturated cyclopentanone compound prepared and separated from the secondary metabolites of the marine fungus Trichoderma sp. 6791.

[0008] The third object of the present invention is a method for preparing the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound.

[0009] The fourth object of the present invention is the use of α,β-unsaturated cyclopentanone compounds as quorum sensing inhibitors.

[0010] To this end, the first technical solution provided by the present invention is as follows:

[0011] The marine fungus Trichoderma sp.6791 in the present invention is a strain of Trichoderma fungus derived from marine sediment, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Management Center with the deposit number GDMCC NO: 62264 and the deposit date of March 2, 2022.

[0012] The second technical solution provided by the present invention is as follows:

[0013] A quorum sensing inhibitor α, β-unsaturated cyclopentanone compound, wherein the α, β-unsaturated cyclopentanone compound is prepared and separated from the secondary metabolites of the marine fungus Trichoderma sp. 6791, and its structural formula is shown in formula (I):

[0014] The α,β-unsaturated cyclopentanone compound with antibacterial activity has a structure as shown in formula (I):

[0015]

[0016] The third technical solution provided by the present invention is a method for preparing the above-mentioned quorum sensing inhibitor α, β-unsaturated cyclopentanone compound, and the specific steps are as follows:

[0017] A. Preparation of liquid fermentation broth extract of marine fungus Trichoderma sp.6791.

[0018] B. The fermentation broth extract was separated by silica gel column chromatography, using petroleum ether-ethyl acetate-methanol as the mobile phase system, with a volume ratio of 4:1:0, 1:1:0, 1:4:0, 0:1:0, 0:4:1 as the eluent, and gradient elution was performed in sequence to obtain 5 components Fr.1-Fr.5. The component Fr.2 was eluted with a volume ratio of 1:1:0 of ​​petroleum ether: ethyl acetate: methanol, and silica gel column chromatography was continued, using a petroleum ether-ethyl acetate system, with a volume ratio of 4:1, 7:3, 2:3, 1:1, 1:4, 0:1 gradient elution sequence to obtain 6 components Fr.2.1-Fr.2.6. The component Fr.2.3 was further separated by Sephadex LH-20 chromatography, using water: methanol (volume ratio 1:1) as the eluent, and the compound α, β-unsaturated cyclopentanone Hypocrenone C was separated and purified.

[0019] The method for preparing the fermentation broth extract of marine fungus Trichoderma sp.6791 is to use fungus Trichoderma sp.6791 as a fermentation strain, perform a large-scale fermentation culture (50 L), concentrate the fermentation broth to about 5 L, and then extract and concentrate it with ethyl acetate (volume ratio, water: ethyl acetate 1:1) to obtain the fermentation broth extract.

[0020] Through the evaluation of the antibacterial activity of the compound α,β-unsaturated cyclopentanone Hypocrenone C, it was found that the compound α,β-unsaturated cyclopentanone Hypocrenone C can significantly inhibit the secretion of virulence factors pyocyanin and rhamnolipid by downregulating the expression of LasB, RhlR, and PqsR genes in the quorum sensing system of Pseudomonas aeruginosa without affecting the growth of Pseudomonas aeruginosa, and has a significant effect on its biofilm formation. Toxicity experimental evaluation shows that Hypocrenone C is not toxic to rat cardiomyocytes.

[0021] Therefore, the third technical solution of the present invention is to provide the use of the compound α, β-unsaturated cyclopentanone Hypocrenone C in the preparation of bacterial quorum sensing inhibitors against drug-resistant bacteria, and to provide a minimum effective concentration with application value for the application.

[0022] Preferably, the antibacterial drug is an anti-Pseudomonas aeruginosa drug.

[0023] The fourth technical solution of the present invention is to provide an antibacterial drug, characterized in that it includes an effective amount of the compound α, β-unsaturated cyclopentanone Hypocrenone C or its pharmaceutically acceptable salt as an active ingredient, and a pharmaceutically acceptable carrier.

[0024] The mass ratio of Hypocrenone C to the pharmaceutically acceptable excipient is (1-9): (1-9).

[0025] Preferably, the drug is an oral preparation, a capsule, a tablet or a granule.

[0026] Preferably, the antibacterial drug is an anti-Pseudomonas aeruginosa drug.

[0027] Through the evaluation of the antibacterial activity of the compound α, β-unsaturated cyclopentanone Hypocrenone C and the antibiotic amikacin, it was found that the compound α, β-unsaturated cyclopentanone Hypocrenone C can significantly improve the antibacterial ability of amikacin.

[0028] The fifth technical solution of the present invention is to provide a combination of antibacterial drugs. When the compound α,β-unsaturated cyclopentanone Hypocrenone C and the antibiotic amikacin are used in combination, the molar ratio of the two is (40-160 μM): (10-20 μM), both of which show good bactericidal effects.

[0029] Preferably, the antibacterial drug is an anti-Pseudomonas aeruginosa drug.

[0030] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0031] 1. The α,β-unsaturated cyclopentanone compound α,β-unsaturated cyclopentanone Hypocrenone C provided by the present invention has a significant inhibitory effect on the bacterial quorum sensing system at a concentration far lower than the growth of pathogenic bacteria, and can significantly reduce the secretion and expression of virulence factors such as pyocyanin and rhamnolipid mediated by the bacterial quorum sensing system, as well as the formation of bacterial biofilms, thereby reducing the virulence of bacteria.

[0032] 2. The α,β-unsaturated cyclopentanone compounds provided by the present invention are derived from marine fungi, and have the advantages of simple microbial fermentation, simple and easy control of compound separation and purification, high safety, low concentration, and low resistance, etc., and can be used to prepare drugs against drug-resistant bacteria, and to prevent and treat infections caused by drug-resistant bacteria. Therefore, the present invention provides candidate compounds for the development of new drugs against drug-resistant bacteria, which is of great significance to the development of China's marine drug resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the compound α,β-unsaturated cyclopentanone Hypocrenone C 1 HNMR spectrum;

[0034] Figure 2 It is the compound α,β-unsaturated cyclopentanone Hypocrenone C 13 C NMR spectrum;

[0035] Figure 3This is the ESI-MS spectrum of the compound α,β-unsaturated cyclopentanone Hypocrenone C;

[0036] Figure 4 The effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on the growth curve of Pseudomonas aeruginosa;

[0037] Figure 5 The effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on the biofilm formation of PAO1 (crystal violet staining method);

[0038] Figure 6 Laser confocal microscopy was used to analyze the effect of α,β-unsaturated cyclopentanone Hypocrenone C on the biofilm formation of Pseudomonas aeruginosa.

[0039] Figure 7 The inhibitory effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on the secretion of pyocyanin by Pseudomonas aeruginosa;

[0040] Figure 8 The inhibitory effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on rhamnolipid secretion by Pseudomonas aeruginosa;

[0041] Fig. 9 The effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on the genes related to the quorum sensing system of Pseudomonas aeruginosa;

[0042] Fig.10 The effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on rat cardiomyocytes H9C2;

[0043] Fig.11 The effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C combined with amikacin on Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0044] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0045] The marine fungus Trichoderma sp.6791 provided by the present invention is a Trichoderma fungus derived from marine sediment, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Management Center with a deposit number of GDMCC NO: 62264 and a deposit date of March 2, 2022.

[0046] Example 1 Preparation method of α,β-unsaturated cyclopentanone compound Hypocrenone C

[0047] 1. Liquid fermentation of marine fungus Trichoderma sp.6791

[0048] (1) Seed culture:

[0049] The mycelium of marine fungus Trichoderma sp.6791 was inoculated into potato dextrose agar (PDA) plates and incubated at 28°C for 6 days to activate the strain. 2 The agar block with mycelium was inoculated into potato dextrose broth (PDB) medium and cultured in a shaking incubator at 28°C and 160 rpm for 4 days to obtain seed culture solution.

[0050] (2) Scale-up culture:

[0051] The fermentation was scaled up to a liquid culture medium: 200 g glucose, 100 g peptone, 100 g yeast extract, 1000 mL homemade seawater (3 g sea salt, 1000 mL water), pH 7.2-7.4.

[0052] Under aseptic operation, the cultured seed culture solution was inoculated into the enlarged fermentation medium (each 1000mL conical flask contained 500mL of liquid culture medium), a total of 100 bottles. The culture was statically cultured at 25°C for 28 days to obtain the fermentation solution of marine fungus Trichoderma sp.6791.

[0053] (3) Preparation of compounds

[0054] The fermented product was extracted three times with ethyl acetate: water (volume ratio 1:1), and the ethyl acetate phase was concentrated to obtain 20.3 g of ethyl acetate crude extract.

[0055] The extract of the fermentation broth was separated by silica gel column chromatography, using petroleum ether-ethyl acetate-methanol as the mobile phase system, with a volume ratio of 4:1:0, 1:1:0, 1:4:0, 0:1:0, 0:4:1 as the eluent, and gradient elution was performed in sequence to obtain 5 components Fr.1-Fr.5. The component Fr.2 was eluted with a volume ratio of 1:1:0 of ​​petroleum ether: ethyl acetate: methanol, and silica gel column chromatography was continued, using a petroleum ether-ethyl acetate system, with a volume ratio of 4:1, 7:3, 2:3, 1:1, 1:4, 0:1 gradient elution sequence to obtain 6 components Fr.2.1-Fr.2.6. The component Fr.2.3 was further separated by Sephadex LH-20 chromatography, and eluted with water: methanol (volume ratio 1:1) as the eluent, and the compound α, β-unsaturated cyclopentanone Hypocrenone C was separated and purified.

[0056] (4) Structural identification of the compound α,β-unsaturated cyclopentanone Hypocrenone C

[0057] 1. Instruments and reagents used for compound activity testing:

[0058] ThermoFisher VARIOSKANLUX microplate reader (Thermo Fisher Scientific, USA);

[0059] Zeiss LSM 800WithAirscan high-resolution laser confocal microscope (Carl Zeiss, Germany);

[0060] Roche LightCycler 96 real-time fluorescence quantitative PCR instrument (Roche, Switzerland);

[0061] Kit: LIVE / DEAD BacLight Bacterial Viability Kit (Thermo Fisher Scientific, USA);

[0062] Kit: HiScriptⅡOne Step SYBR Green Kit (Nanjing Novozymes Co., Ltd.).

[0063] 2. Test result analysis

[0064] 1 H-NMR, 13 C-NMR spectra were measured using a Bruker-Advance 400 MHz NMR spectrometer with tetramethylsilane (TMS) as the internal standard; ESI-MS data were measured using a ThermoFisher LTQ Qrbitrap mass spectrometer. Figure 1-3 As shown, Figure 1 It is the compound α,β-unsaturated cyclopentanone Hypocrenone C 1 HNMR spectrum (DMSO-d6); Figure 2 It is the compound α,β-unsaturated cyclopentanone Hypocrenone C 13 C NMR spectrum (DMSO-d6); Figure 3 This is the ESI-MS spectrum of the compound α,β-unsaturated cyclopentanone Hypocrenone C.

[0065] The compound α,β-unsaturated cyclopentanone Hypocrenone C was subjected to structural analysis to obtain the following physical and chemical data. Its NMR data are shown in Table 1.

[0066] Compound α,β-unsaturated cyclopentanone Hypocrenone C: pale yellow solid, ESI-MS m / z 151[MH] + . Search the literature, compound α, β-unsaturated cyclopentanone Hypocrenone C1 H (400MHz, DMSO-d6) and 13 C NMR (100HMz, DMSO-d6) was basically consistent with the NMR data of compound Hypocrenone C, confirming that the compound α,β-unsaturated cyclopentanone Hypocrenone C was α,β-unsaturated cyclopentanone Hypocrenone C.

[0067] Table 1: 1D NMR data of compound α,β-unsaturated cyclopentanone Hypocrenone C

[0068]

[0069] Example 2 Determination of the Minimum Inhibitory Concentration (MIC) of Compound α,β-Unsaturated Cyclopentanone Hypocrenone C against Pseudomonas aeruginosa

[0070] Samples to be tested: Compound Hypocrenone C of the present invention is prepared into 200 mM with DMSO and azithromycin (AQ, 200 mM) for use;

[0071] The MIC value of the sample against Pseudomonas aeruginosa PAO1 (ATCC15692) was determined by the two-fold dilution method. Bacterial LB culture medium (peptone 10.0 g, sodium chloride 5.0 g, glucose 1.0 g, yeast extract powder 5.0 g, water 1000 mL, pH 7.0). In a 96-well plate, add OD 600 100 μL of PAO1 bacterial solution with a concentration of 0.01 was added, and then 100 μL of the sample compound α, β-unsaturated cyclopentanone Hypocrenone C was added to make the final concentrations 640 μM, 320 μM, 160 μM, 80 μM, 40 μM, and 20 μM; no drug was added to the positive control group, and no bacteria was added to the negative control group. Each concentration was paralleled to 6 replicate wells. The cells were cultured at 37°C for 24 hours, and then 20 μL of 0.2% red tetrazolium solution was added to each well. After continuing to culture at 37°C for 4 hours, the bacterial growth was observed. The lowest sample concentration without red production was the minimum inhibitory concentration MIC.

[0072] The experimental results showed that the minimum inhibitory concentration (MIC) of Hypocrenone C against Pseudomonas aeruginosa was greater than 640 μM.

[0073] Example 3 Effect of α,β-unsaturated cyclopentanone Hypocrenone C on the growth of Pseudomonas aeruginosa 50 mL of Pseudomonas aeruginosa (final bacterial concentration: OD 600=0.01) were added to conical flasks, respectively, to make the final concentration of α, β-unsaturated cyclopentanone Hypocrenone C 160 μM (<1 / 4MIC), and the control group was not added with drugs; the cells were placed at 37°C and cultured on a shaking platform for 24 h; samples were taken every 2 h to measure OD 600 Values ​​are plotted as curves.

[0074] The test results show that ( Figure 4 ): Hypocrenone C had no significant effect on the growth curve of Pseudomonas aeruginosa at a concentration of 160 μM, indicating that Hypocrenone C had no bactericidal effect on Pseudomonas aeruginosa at a concentration of 160 μM and did not affect the growth of Pseudomonas aeruginosa.

[0075] Example 4 Effect of Compound α,β-Unsaturated Cyclopentanone Hypocrenone C on Biofilm Formation of Pseudomonas aeruginosa Crystal violet staining method was used to determine the inhibitory effect of the compound on biofilm

[0076] Methods: 500 μL of Pseudomonas aeruginosa PAO1 bacterial solution was inoculated in a 24-well plate, and the compound α,β-unsaturated cyclopentanone Hypocrenone C was added at a final concentration of 40 μM, 80 μM, and 160 μM, and incubated at 37°C for 24 hours. After removing the surface bacterial solution in the well, the 24-well plate was carefully washed with distilled water, and the floating bacteria were washed away and dried. Then 1 mL of 1% crystal violet solution was added to the 24-well plate, and the staining was allowed to stand at room temperature for 15 minutes. The excess crystal violet solution was aspirated, and the biofilm was carefully washed 3 times with distilled water, and then allowed to stand and dry. Then 33% glacial acetic acid was added to dissolve, oscillated evenly, and the OD value was measured at a wavelength of 570 nm on an ELISA reader.

[0077] The test results show that ( Figure 5 ): The compound α,β-unsaturated cyclopentanone Hypocrenone C has a certain inhibitory effect on the formation of Pseudomonas aeruginosa biofilm. The inhibition rate of biofilm production is 25.2%-57.3% at a concentration of 40μM-160μM.

[0078]

[0079] Microscopic imaging analysis to detect the effect of α,β-unsaturated cyclopentanone Hypocrenone C on biofilm formation

[0080] Inoculate 1mL of Pseudomonas aeruginosa PAO1 bacterial solution in a 35mm wave bottom culture dish, add 1mL of compound α,β-unsaturated cyclopentanone Hypocrenone C to a final concentration of 160μM, 80μM, and 40μM, incubate at 37℃ for 24h, aspirate the surface bacterial solution in the well, wash the culture dish 3 times with distilled water to wash away the floating bacteria. Then add the dye of bacterial viability kit to stain, and observe the biofilm under laser confocal microscope, green fluorescence Syt9: excitation wavelength 483nm, emission wavelength 500nm; red fluorescence PI: excitation wavelength 305nm, emission wavelength 617nm.

[0081] The experimental results were analyzed using ImageJ software ( Figure 6 ): When the concentrations were 160 μM, 80 μM, and 40 μM, the inhibition rates of the compound α, β-unsaturated cyclopentanone Hypocrenone C on the biofilm production of Pseudomonas aeruginosa were 59.5%, 49.0%, and 27.9%, respectively.

[0082] The experimental results of crystal violet staining and microscopic imaging analysis showed that the compound α,β-unsaturated cyclopentanone Hypocrenone C had a significant inhibitory effect on the formation of Pseudomonas aeruginosa biofilm.

[0083] Example 5 Effect on the expression of virulence factors regulated by the quorum sensing system

[0084] Pseudomonas aeruginosa was cultured overnight in LB medium. 50 mL of activated Pseudomonas aeruginosa was inoculated into a conical flask (final bacterial concentration: OD 600 =0.01), and at the same time, the compound α,β-unsaturated cyclopentanone Hypocrenone C was added to the final concentrations of 64μM, 32μM, and 16μM, respectively, and erythromycin was added to make the final concentration 10μM; after culturing on a shaking table at 37°C, 200r / min for 24 hours, 200uL of the bacterial solution was taken to a 96-well plate, and the OD was measured at a wavelength of 600nm. 600 value.

[0085] 1) Inhibitory effect of the compound α,β-unsaturated cyclopentanone Hypocrenone C on the secretion of the virulence factor pyocyanin

[0086] Test method: Transfer the above cultured bacterial solution to a 50mL centrifuge tube and centrifuge at 10000rpm / min and 4℃ for 10min. After centrifugation, take 40mL of the supernatant and add 32mL of chloroform to fully shake for extraction. After standing, take 1mL of the chloroform layer and transfer it to a new tube. Add 0.6mL of 0.2mol / L hydrochloric acid for extraction. After centrifugation and stratification, take 200uL of the hydrochloric acid layer and place it in a 96-well plate. Measure its OD value at a wavelength of 520nm on an ELISA reader.

[0087]

[0088] Note: A1 is the pyocyanin content in the drug group (OD 520 / OD 600 ); A0 is the blank group pyocyanin content (OD 520 / OD 600 )

[0089] Depend on Figure 7 As shown in the results of the experiment, it can be seen that the inhibition rate of the compound α, β-unsaturated cyclopentanone Hypocrenone C on the secretion of the virulence factor pyocyanin is 41.9%-61.8% at 16μM-64μM, and the inhibition rate increases with increasing concentration, indicating that the compound α, β-unsaturated cyclopentanone Hypocrenone C has a significant inhibitory effect on the secretion of the virulence factor pyocyanin in the quorum sensing system, and the positive control is 10μM erythromycin (Ery).

[0090] 2) Inhibitory effect of α,β-unsaturated cyclopentanone Hypocrenone C on rhamnolipid secretion by Pseudomonas aeruginosa

[0091] Test method: Take 5mL of cultured bacterial liquid, centrifuge at 10000rpm / min, 4℃ for 10min. Take 2mL of supernatant in a 10mL EP tube, add 4mL of ether and shake to mix and extract. Take 1mL of ether layer in a 1.5mL EP tube, put it in a fume hood, and remove the ether. Add 40μL of ultrapure water to dissolve the extracted rhamnolipid, add 360μL of orcinol solution (0.19g orcinol powder is added to 50% concentrated sulfuric acid, and the volume is adjusted to 100ml), mix well and boil in a water bath at 80℃ for 30min. After cooling, take 200μL and place it in a 96-well plate, and measure the OD value at a wavelength of 421nm on the microplate reader.

[0092]

[0093] Note: B1 is the rhamnolipid content of the drug group (OD 421 / OD 600 ); B0 is the rhamnolipid content OD of the control group 421 / OD 600

[0094] The test results found that ( Figure 8 ): The inhibition rate of the compound α, β-unsaturated cyclopentanone Hypocrenone C on rhamnolipid secretion was 10.2%-21.5% at a concentration of 16μM-64μM; the compound α, β-unsaturated cyclopentanone Hypocrenone C has the effect of inhibiting the secretion of virulence factor rhamnolipid in the quorum sensing system of Pseudomonas aeruginosa, and the positive control is 10μM erythromycin (Ery).

[0095] Example 6 Effect of the Compound α,β-Unsaturated Cyclopentanone Hypocrenone C on the Expression of Genes Related to the Quorum Sensing System of Pseudomonas aeruginosa

[0096] Pseudomonas aeruginosa was cultured overnight in LB medium. 50 mL of activated Pseudomonas aeruginosa was inoculated into a conical flask (final bacterial concentration: OD 600 =0.01), and then added the compound α,β-unsaturated cyclopentanone Hypocrenone C with a final concentration of 64 μM and 32 μM, respectively, and cultured on a shaking platform for 24 hours.

[0097] Take 1mL of bacterial solution, centrifuge at 8000rpm for 3min, remove the supernatant, add 500μL PBS solution, shake gently to wash, centrifuge, and discard the supernatant. Then add 200μL lysozyme, shake and mix, add 800μL RNA extraction solution, blow gently with a pipette to break the cells, then add 250μl chloroform to mix thoroughly, let stand for 3min, and centrifuge for 10min.

[0098] Transfer 400 μL of supernatant to a new centrifuge tube, add 0.8 times the volume of isopropanol, mix well, place at -20°C for 15 minutes, centrifuge for 10 minutes, remove the supernatant, add 1.5 ml of 75% ethanol to wash the precipitate, centrifuge, and the precipitate is RNA. Add 15 μL of sterile enzyme-free water to the centrifuge tube to dissolve the RNA precipitate, and use an ultra-micro nucleic acid protein quantifier (Nanodrop 2000) to detect its concentration and purity. Finally, use sterile enzyme-free water to prepare an RNA solution with a final concentration of 200 ng / μL.

[0099] According to the instructions of the SYBR Green qPCR kit, the specific primers of the gene to be tested, the RNA solution and the dye reagent were mixed, the reaction conditions were set, and the real-time quantitative PCR detection was performed in the LightCycler96 fluorescent quantitative PCR instrument. The 16S ribosomal RNA gene was used as the internal reference to normalize the PCR data. -ΔΔCT The gene expression level was calculated.

[0100] △CT=CT target gene-Ct internal reference gene

[0101] △△CT=△CT gene in drug treatment group-△CT gene in control group

[0102] Use 2 -(△△Ct) Relative quantification was performed to calculate the fold change ( Fig. 9), the compound α,β-unsaturated cyclopentanone Hypocrenone C at concentrations of 64μM and 32μM had a significant down-regulatory effect on the expression of LasA, LasB, LasI, LasR, RhlI, RhlR, PqsA, and PqsR genes.

[0103] Example 7 Experiment on the toxicity of Hypocrenone C on rat myocardial cells H9C2

[0104] Cell pretreatment: Rat cardiomyocytes H9C2 cells were revived in a -80°C refrigerator and cultured in a cell culture incubator until the cells grew well.

[0105] The compound Hypocrenone C of the present invention was prepared into a 200 mM stock solution with 100% DMSO, and 0.2% DMSO was used as a solvent control.

[0106] Experimental method: cells with good growth status were cultured in 96-well plates, 50,000 cells / well, 100 μL, and after 24 hours of culture, 100 μL of Hypocrenone C with different concentrations (final concentrations were 320 μM, 160 μM, and 80 μM) was added, and control wells were set up, with 3 parallels for each concentration, and the drug was allowed to act for 72 hours. Then the culture medium was removed, and 200 μL of serum-free MTT solution (0.5 mg / ml) was added to each well. After 3 hours of action, the solution was removed, and 150 μL of DMSO was added to each well, and the cells were incubated in the incubator for 10 minutes, and then the OD value was measured at 570 nm on a microplate reader.

[0107] Calculation formula: Cell survival rate (%) = 100-[(OD Control -OD Blank )-(OD 药物组 -OD Blank )] / (OD Control -OD Blank )×100%

[0108] Experimental data showed that Hypocrenone C had no significant toxicity to mouse cardiomyocytes ( Fig.10 ), indicating that Hypocrenone C has no obvious toxicity to cells at a certain dose, but has a certain effect on the quorum sensing of Pseudomonas aeruginosa. Therefore, Hypocrenone C can be used as a candidate drug for inhibiting quorum sensing.

[0109] Example 8 Anti-Pseudomonas aeruginosa Quorum Sensing Granules

[0110]

[0111] The preparation steps of the anti-Pseudomonas aeruginosa quorum sensing granules are as follows:

[0112] (1) Hypocrenone C is first mixed with lactose, mannitol and CMS-Na;

[0113] (2) then adding the prepared 10% starch slurry to the above mixture and passing it through a 16-mesh sieve to prepare a soft material;

[0114] (3) drying in a vacuum drying oven at 60° C. and granulating to obtain anti-Pseudomonas aeruginosa quorum sensing granules.

[0115] Example 9 Anti-Pseudomonas aeruginosa Quorum Sensing Capsules

[0116]

[0117] The preparation steps of anti-Pseudomonas aeruginosa quorum sensing capsules are as follows:

[0118] (1) Mix Hypocrenone C and lactose for 10-15 minutes;

[0119] (2) adding microcrystalline cellulose and mixing for 10-15 minutes;

[0120] (3) Add talcum powder and mix for 3-5 minutes;

[0121] (4) The mixture is filled into a gelatin capsule shell to obtain the anti-Pseudomonas aeruginosa quorum sensing capsule of the present invention.

[0122] Example 10 Anti-Pseudomonas aeruginosa Quorum Sensing Tablet

[0123]

[0124] The preparation steps of the anti-Pseudomonas aeruginosa quorum sensing tablet are as follows:

[0125] (1) Mix Hypocrenone C and lactose for 10-15 minutes;

[0126] (2) adding 10% starch slurry to make a soft material, passing through a 14-mesh sieve, granulating, drying, and passing through a 12-mesh sieve to size the particles;

[0127] (3) then adding crospovidone and magnesium stearate and mixing for 3-5 minutes;

[0128] (4) After mixing evenly, the mixture is pressed into tablets to obtain the anti-Pseudomonas aeruginosa quorum sensing tablets of the present invention.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A marine fungus, Trichoderma, It is characterized in that It is a strain of Trichoderma fungus derived from marine mud. It was deposited in the Guangdong Provincial Microbiological Culture Collection Management Center on March 2, 2022, with the deposit number GDMCC NO: 62264.

2. A quorum sensing inhibitor α,β-unsaturated cyclopentanone compound, It is characterized in that The α,β-unsaturated cyclopentanone compound is prepared and separated from the secondary metabolites of the marine fungus Trichoderma sp.6791, and its structural formula is as described in formula (I):

3. The method for preparing the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound according to claim 2, It is characterized in that The steps are as follows: (1) preparing a liquid fermentation culture of marine fungus Trichoderma sp. 6791, concentrating the fermentation broth to about 5 L, and then extracting and concentrating with ethyl acetate to obtain an ethyl acetate extract; (2) The obtained extract was subjected to silica gel column chromatography, using petroleum ether-ethyl acetate-methanol as the mobile phase system, with a volume ratio of 4:1:0, 1:1:0, 1:4:0, 0:1:0, 0:4:1 as the eluent, and gradient elution was performed in sequence to obtain 5 components Fr.1-Fr.

5. Component Fr.2 was collected with a volume ratio of 1:1:0 of ​​petroleum ether: ethyl acetate: methanol, and silica gel column chromatography was continued, using a petroleum ether-ethyl acetate system, with a volume ratio of 4:1, 7:3, 2:3, 1:1, 1:4, 0:1 gradient elution sequence to obtain 6 components Fr.2.1-Fr.2.

6. Component Fr.2.3 was further separated by Sephadex LH-20 chromatography, using water: methanol (volume ratio 1:1) as the eluent, and the quorum sensing inhibitor α, β-unsaturated cyclopentanone compound Hypocrenone C was separated and purified.

4. Use of the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound according to claim 2 as a preparation of antibacterial drugs.

5. Use of the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound according to claim 4 as a preparation of antibacterial drugs, It is characterized in that The quorum sensing inhibitor α, β-unsaturated cyclopentanone compound is used as an antibacterial drug for inhibiting bacterial quorum sensing system; or, the quorum sensing inhibitor α, β-unsaturated cyclopentanone compound is used as an antibacterial drug for inhibiting bacterial biofilm formation.

6. Use of the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound according to claim 5 as a preparation of antibacterial drugs, It is characterized in that The bacteria is Pseudomonas aeruginosa.

7. Use of the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound according to claim 6 as a preparation of antibacterial drugs, It is characterized in that The use of quorum sensing inhibitor α, β-unsaturated cyclopentanone compounds in the preparation of antibacterial drugs that inhibit the virulence factors pyocyanin and rhamnolipid in the quorum sensing system of Pseudomonas aeruginosa; or the use of quorum sensing inhibitor α, β-unsaturated cyclopentanone compounds in the preparation of antibacterial drugs that downregulate the expression of LasB, RhlR, and PqsR genes in the quorum sensing system of Pseudomonas aeruginosa. 8.An antibacterial drug, It is characterized in that It comprises the quorum sensing inhibitor α,β-unsaturated cyclopentanone compound or a pharmaceutically acceptable salt thereof as claimed in claim 2, and pharmaceutically acceptable excipients; The mass ratio of Hypocrenone C to the pharmaceutically acceptable excipient is (1-9): (1-9).

9. An antibacterial drug according to claim 1, It is characterized in that The medicine is an oral preparation, a capsule, a tablet or a granule.

10. An antibacterial drug according to claim 1, It is characterized in that The invention also comprises an antibiotic amikacin; a quorum sensing inhibitor α,β-unsaturated cyclopentanone compound and the amikacin, and the molar ratio thereof is (40-160 μM): (10-20 μM).

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