CN114920717A - Pimarane diterpenoid compound and preparation method and application thereof - Google Patents
Pimarane diterpenoid compound and preparation method and application thereof Download PDFInfo
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- CN114920717A CN114920717A CN202210787670.7A CN202210787670A CN114920717A CN 114920717 A CN114920717 A CN 114920717A CN 202210787670 A CN202210787670 A CN 202210787670A CN 114920717 A CN114920717 A CN 114920717A
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- pimarane
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- -1 Pimarane diterpenoid compound Chemical class 0.000 title claims abstract description 23
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 38
- 241000894006 Bacteria Species 0.000 claims abstract description 22
- 230000018612 quorum sensing Effects 0.000 claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 17
- 241000223600 Alternaria Species 0.000 claims abstract description 13
- 229930004069 diterpene Natural products 0.000 claims abstract description 13
- 229930000776 pimarane Natural products 0.000 claims abstract description 13
- FCBUKWWQSZQDDI-UHFFFAOYSA-N rhamnolipid Chemical compound CCCCCCCC(CC(O)=O)OC(=O)CC(CCCCCCC)OC1OC(C)C(O)C(O)C1OC1C(O)C(O)C(O)C(C)O1 FCBUKWWQSZQDDI-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000028327 secretion Effects 0.000 claims abstract description 12
- 230000007923 virulence factor Effects 0.000 claims abstract description 11
- 239000000304 virulence factor Substances 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 8
- YNCMLFHHXWETLD-UHFFFAOYSA-N pyocyanin Chemical compound CN1C2=CC=CC=C2N=C2C1=CC=CC2=O YNCMLFHHXWETLD-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229940124350 antibacterial drug Drugs 0.000 claims abstract description 7
- 102000016387 Pancreatic elastase Human genes 0.000 claims abstract description 5
- 108010067372 Pancreatic elastase Proteins 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 39
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
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- GZHFBZCDMVGRTI-DIJYYDPMSA-N pimarane Chemical compound CC1(C)CCC[C@]2(C)[C@H]3CC[C@](CC)(C)C[C@@H]3CCC21 GZHFBZCDMVGRTI-DIJYYDPMSA-N 0.000 claims description 8
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a pimarane diterpenoid compound, a preparation method and application thereof; the pimarane diterpene compound is extracted from marine Alternaria fungusAlternaria porri 25# GDMCC NO: 62148 from the fermentation culture; the pimarane diterpenoid can inhibit the formation of a biological membrane of pseudomonas aeruginosa under the condition of not inhibiting the growth of the pseudomonas aeruginosa, can obviously inhibit the secretion of virulence factors of pyocyanin, rhamnolipid and elastase in a quorum sensing system, can slow down the generation of multiple drug resistance of bacteria, and provides a new quorum sensing inhibitor and an antibacterial drug for research and developmentA lead compound has potential application as an antibacterial drug.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a pimarane diterpenoid compound, a preparation method thereof and application thereof in preparation of antibacterial drugs.
Background
Research finds that bacteria can block the action of drugs through formed biological membranes to generate drug resistance, so that the traditional antibiotics lose curative effect and bring great difficulty to clinical treatment, and therefore, the research and development of novel drug-resistant bacteria drugs are urgent.
Quorum Sensing (QS) is a bacterial density-dependent intercellular signaling system involving the production and release of several small diffusible signal molecules of different chemical classes, intercellular communication, and precise regulation of bacterial population behavior in chemical communication, including immune escape of bacterial biofilm formation, virulence factor gene expression, drug resistance production, bacterial motility and secondary metabolism regulation. By blocking or interfering the quorum sensing phenomenon of bacteria, the expression of pathogenic factors of the bacteria is specifically inhibited under the condition of not influencing the growth of the bacteria, the formation of bacterial biofilms, the generation of virulence factors or the expression of pathogenic genes are interfered, and the occurrence of drug-resistant mutants is reduced to the maximum extent. Therefore, quorum sensing system inhibitors become hot spots for drug research and development in the anti-infection field at present, and become potential ways for treating drug-resistant bacterial infection.
Pseudomonas aeruginosa, one of the leading pathogens of hospital infections todayPseudomonasaeruginosaPA), which has strong pathogenic ability and complicated and variable drug resistance mechanism, faces a great challenge in clinical treatment. The research finds that the verdigris sheetThere are 3 quorum-sensing systems for Cytospora, including the LasI/LasR signal system, the RhlI/RhlR signal system, and the Pqs quinolone signal system. The expression of various pathogenic virulence factors of pseudomonas aeruginosa (such as elastase, pyocin, rhamnolipid and the like) is regulated and controlled by a quorum sensing system. In addition, by inhibiting the quorum sensing of bacteria, the formation of a biofilm which causes one of the drug resistance reasons of pathogenic bacteria can be regulated and controlled, the movement behavior capability of the pathogenic bacteria can be regulated and controlled, the harm of the bacteria to a host is reduced, and the aim of preventing or assisting antibiotics in treating bacterial infection is fulfilled. Thus, it has been found that low toxicity, high activity inhibitors of bacterial quorum sensing provide more effective antibacterial agents in the control of Pseudomonas aeruginosa bacterial infections.
The marine fungi have unique metabolic pathways and genetic backgrounds due to the special growth environment of the marine fungi, metabolize and accumulate active secondary metabolites with unique structures, and occupy an important position in the research of natural products. With the progress of research, researchers discover more and more marine natural products with biological activity, lay a good foundation for the research and development of new marine drugs, and are a great important source for discovering new anti-drug-resistance antibacterial drugs.
Disclosure of Invention
It is a first object of the present invention to provide a pimarane diterpenoid compound having antibacterial activity.
The structure is shown as formula (І) (II):
the second object of the present invention is to provide the preparation method of the said pimarane diterpenoid compounds, which is characterized in that the said compounds of formula (І) and formula (II) are obtained from marine Alternaria fungusAlternaria porri25# fermentation culture.
The marine Alternaria fungi of the present inventionAlternaria porri25# was deposited at 31.12.2021 in the culture collection of microorganisms of Guangdong province (GDMCC), with accession numbers: GDMCC number 62148. Address: guangzhou, Guangdong provinceThe institute of microbiology, national institute of sciences, Guangdong province, No. 59, of Jud, Fall, Middleya, Mr. 100, in Xianrei.
Preferably, the specific steps are as follows:
A. preparation of marine fungiAlternaria porri25# solid fermentation culture.
B. Extracting the fermented product with chloroform and methanol (volume ratio of 1: 1) and concentrating to obtain chloroform-methanol extract, extracting the extract with water and ethyl acetate (volume ratio of 1: 1), and concentrating ethyl acetate layer to obtain ethyl acetate crude extract.
And carrying out silica gel column chromatography on the obtained crude extract, and using petroleum ether-ethyl acetate as an eluent in the volume ratio of 9:1, 4:1, 7:3, 3:2, 1:1, 3:7 and 0:1 in sequence to carry out gradient elution to obtain 7 components Fr.1-Fr.7.
Collecting an eluted component Fr.2 of petroleum ether and ethyl acetate in a volume ratio of 4:1, continuously adopting silica gel column chromatography, and performing gradient elution with a petroleum ether-ethyl acetate system in a volume ratio of 9:1, 9:2, 9:3, 4:1 and 1:1 to obtain 5 components Fr.2.1-Fr.2.5.
And continuously carrying out Sephadex LH-20 chromatographic separation on the component Fr.2.2, eluting by using dichloromethane to methanol (volume ratio is 1: 1) as an eluent, and separating and purifying to obtain the compound shown in the formula (II). The component Fr.2.3 is recrystallized and purified to obtain the compound of the formula (І).
The marine fungus is preparedAlternaria porri25# the fermentation product is marine funguslternaria porri Culturing 25# as fermentation strain in amplified fermentation culture medium to obtain marine fungusAlternaria porri25# fermentation product; the amplification culture medium is a rice solid culture medium: comprises rice 100g, self-made seawater 100mL (sea salt 3 g, water 100 mL), and pH 7.2-7.4.
Through the evaluation of the antibacterial activity of the compound of the formula (І) and the compound of the formula (II), the compound of the formula (І) and the compound of the formula (II) are found to have obvious inhibition effects on the expression of virulence factors pyocyanin, rhamnolipid and elastase in a pseudomonas aeruginosa quorum sensing system, and have obvious inhibition effects on the formation of a pseudomonas aeruginosa biofilm.
Therefore, the third object of the present invention is to provide the use of the compound of formula (І) and the compound of formula (II) for preparing drugs against drug-resistant bacteria as inhibitors of bacterial quorum sensing, and to provide the minimum effective concentration for the use thereof with practical value.
Preferably, the antibacterial drug is a drug for resisting pseudomonas aeruginosa.
The fourth object of the present invention is to provide an antibacterial agent comprising an effective amount of the compound of formula (І) and/or the compound of formula (II) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.
Preferably, the antibacterial drug is a drug for resisting pseudomonas aeruginosa.
Compared with the prior art, the technical scheme provided by the invention has the following technical advantages:
the pimarane diterpenoid compound provided by the invention has a remarkable inhibiting effect on a bacterial quorum sensing system at a concentration far less than the growth of pathogenic bacteria, can remarkably reduce the secretion and expression of virulence factors such as pyocyanin, rhamnolipid, elastase and the like mediated by the bacterial quorum sensing system, can remarkably reduce the formation of a bacterial biofilm, and can reduce the virulence of bacteria. The pimarane diterpenoid compound is derived from marine fungi, has the advantages of simple microbial fermentation, simple and easily-controlled compound separation, purification and preparation, high safety, low use concentration, difficulty in generating drug resistance and the like, can be used for preparing drug-resistant bacteria resistant drugs and is used for preventing and treating infection caused by drug-resistant bacteria. Therefore, the invention provides a candidate compound for developing a new drug-resistant bacterium drug and has important significance for developing Chinese marine drug resources.
Drawings
FIG. 1 is a diagram of Compound 1 1 H NMR spectrum.
FIG. 2 is a drawing of Compound 1 13 C NMR spectrum.
FIG. 3 is an ESI-MS spectrum of Compound 1.
FIG. 4 shows Compound 2 1 H NMR spectrum.
FIG. 5 is a schematic representation of Compound 2 13 C NMR spectrum.
FIG. 6 is an ESI-MS spectrum of Compound 2.
FIG. 7a is the effect of Compound 1 on the growth curve of Pseudomonas aeruginosa.
FIG. 7b is the effect of Compound 2 on the growth curve of P.aeruginosa.
FIG. 8a is the effect of Compound 1 and Compound 2 on the biofilm formation of PAO1 (Crystal Violet staining).
FIG. 8b is a photomicrograph analysis of the effect of Compound 1 and Compound 2 on Pseudomonas aeruginosa biofilm formation.
FIG. 9 is a graph of the inhibitory effect of compound 1 and compound 2 on Pseudomonas aeruginosa pyocin secretion.
FIG. 10 shows the inhibitory effect of compound 1 and compound 2 on the secretion of rhamnolipid from Pseudomonas aeruginosa.
FIG. 11 is a graph of the inhibition of Pseudomonas aeruginosa elastase activity by Compound 1 and Compound 2.
FIG. 12 is a graph of the effect of Compound 1 and Compound 2 on genes associated with the quorum sensing system of Pseudomonas aeruginosa.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The marine Alternaria fungus of the present inventionAlternaria porri25# was deposited at 31.12.2021 in the culture collection of microorganisms of Guangdong province (GDMCC), with accession numbers: GDMCC number 62148. Address: the institute of microbiology, national institute of sciences of Guangdong province, No. 59, Zhou, Mieli, Guangzhou, Guangdong province.
Example 1 preparation of the pimarane diterpenoid Compounds Diaportein A and Diaportein B
1. Marine fungiAlternaria porriSolid fermentation of GDMCC number 62148 # 25
(1) Seed culture:
extracting marine Alternaria fungusAlternaria porri25#, GDMCC number 62148, was inoculated into Potato Dextrose Agar (PDA) plate, and the activated strain was cultured at 28 deg.C for 6 days, followed by taking about 1.5X 1.5 cm 2 Inoculating agar block with mycelium to Potato Dextrose Broth (PDB)Shaking-culturing at 28 deg.C and 160 rpm for 4 days to obtain seed culture solution.
(2) Amplification culture:
the amplification culture medium is a rice solid culture medium: according to the proportion of a single culture medium, a 1000 mL triangular flask comprises 100g of rice, 100mL of self-made seawater (3 g of sea salt and 100mL of water), and the pH value is 7.2-7.4.
Under aseptic operation, the cultured seed culture solution is respectively inoculated into the solid culture medium for amplification fermentation, 5mL of the seed culture solution is added into each 1000 mL conical flask (containing 100g of rice and 100mL of self-made seawater), and 70 bottles are counted. Standing and culturing at 25 deg.C for 28 days to obtain marine Alternaria fungusAlternaria porri25# fermentation product.
3) Preparation of the Compounds
Extracting the fermented product with chloroform and methanol (volume ratio of 1: 1) for 3 times, concentrating to obtain chloroform methanol extract, extracting the extract with water and ethyl acetate (volume ratio of 1: 1) for 3 times, mixing organic phases, and concentrating ethyl acetate layer to obtain ethyl acetate crude extract 50.1 g.
Subjecting the ethyl acetate crude extract to silica gel column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate as eluent in a volume ratio of 9:1, 4:1, 7:3, 3:2, 1:1, 3:7 and 0:1 in sequence to obtain 7 components Fr.1-Fr.7.
Collecting an eluted component Fr.2 (10.8 g) of petroleum ether and ethyl acetate in a volume ratio of 4:1, continuously performing silica gel column chromatography, and performing gradient elution with a petroleum ether-ethyl acetate system in a volume ratio of 9:1, 9:2, 9:3, 4:1 and 1:1 to obtain 5 components Fr.2.1-Fr.2.5.
The fraction Fr.2.2 (500 mg) was further chromatographed on Sephadex LH-20, eluting with dichloromethane: methanol (volume ratio 1: 1) as eluent, and isolated and purified to give compound 2 (50.1 mg).
Component Fr.2.3 (1.50 g) was purified by recrystallization to give compound 1 (1.06 g).
4) Structural characterization of Compound 1 and Compound 2
1 H-NMR、 13 C-NMR spectra were determined on a Bruker-Advance 400M NMR spectrometerTetramethylsilane (TMS) is used as an internal standard; ESI-MS data were measured on a ThermoFisher LTQ Qrbitrap mass spectrometer.
As shown in FIGS. 1-6, FIG. 1 is a schematic representation of Compound 1 1 H NMR Spectrum (DMSO-d) 6 ) (ii) a FIG. 2 is a diagram of Compound 1 13 C NMR Spectroscopy (DMSO-d) 6 ) (ii) a FIG. 3 is an ESI-MS spectrum of Compound 1; FIG. 4 is a drawing of Compound 2 1 H NMR Spectrum (DMSO-d) 6 ) (ii) a FIG. 5 is a schematic representation of Compound 2 13 C NMR Spectrum (DMSO-d) 6 ) (ii) a FIG. 6 is an ESI-MS spectrum of Compound 2.
The compound 1 was subjected to structural analysis and testing to obtain the following physicochemical property data, the nuclear magnetic data of which are shown in table 1:
compound 1 colorless crystal, ESI-MSm/z 365.3 [M-H] + . The search literature compares that the nuclear magnetic data of the compound 1 and the compound Diaporthein A are basically consistent, and the compound 1 is identified as the pimarane diterpene Diaporthein A.
The compound 2 was subjected to structural analysis and testing to obtain the following physicochemical property data, the nuclear magnetic data of which are shown in table 1:
Of the compound 1 H NMR and 13 the C NMR data was very similar to compound 1. The searched literature compares that the nuclear magnetic data of the compound 2 is basically consistent with that of Diporthein B, and the compound 2 is determined to be a pimarane diterpene Diaporthein B.
Table 1: of compounds 1 and 2 1 H (400 MHz) and 13 c NMR (100 HMz) data
Instruments and reagents used for compound activity testing:
ThermoFisher VARIOSKANLUX microplate reader (Sammer fly, USA);
roche LightCycler96 real-time fluorescent quantitative PCR instrument (Roche Switzerland);
the Kit EnzChekfi Protease Assay Kit E6638 (Sammer fly, USA);
kit LIVE/DEAD back background Viability Kit (U.S. seemer fly);
kit HiScript II One Step SYBR Green Kit (Nanjing Novozam).
Example 2: determination of Minimal Inhibitory Concentration (MIC) of Pseudomonas aeruginosa by Compounds 1 and 2
A sample to be tested: the compounds Diaportein A and Diaportein B of the invention are prepared into 200 mM by DMSO and azithromycin (AQ, 200 mM) for standby;
the MIC values for the samples to inhibit Pseudomonas aeruginosa PAO1 (ATCC 15692) were determined by the double dilution method. Bacterial LB broth (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 96-well plates, OD was added 600 100 mu L of 0.01 PAO1 bacterial liquid, then respectively adding 100 mu L of tested sample compound 1, compound 2 and azithromycin to make their final concentration be 320 mu M, 160 mu M, 80 mu M, 40 mu M, 20 mu M and 10 mu M; the positive control group was free of drug and the negative control group was free of bacteria, each concentration being in duplicate of 6 wells. Culturing at 37 deg.C for 24 hr, adding 20 μ L of 0.2% red tetrazolium solution into each well, and culturing at 37 deg.C for 4 hr; and observing the growth condition of bacteria, wherein the concentration of the lowest sample to be detected without red generation is the Minimum Inhibitory Concentration (MIC).
The test result shows that: the minimum inhibitory concentrations MIC of Diaportein A and Diaportein B to pseudomonas aeruginosa are both greater than 320 mu M; the minimum inhibitory concentration MIC value of the azithromycin to the pseudomonas aeruginosa is 80 mu M.
Example 3: effect of Compound 1 and Compound 2 on the growth of Pseudomonas aeruginosa
Containing 50 mL of Pseudomonas aeruginosa (final bacterial concentration: OD) 600 = 0.01) into each of the Erlenmeyer flasks, compound 1 and compound 2 were added to give a final concentration of 40. mu.M: (<1/8 MIC), control group did not add drug; culturing at 37 deg.C for 24 h; sampling every 2h respectively to measure OD 600 Values, plotted as a graph.
The test results show (figures 7a and 7B) that Diaportein A and Diaportein B have no obvious influence on the growth curve of the pseudomonas aeruginosa at the concentration of 40 mu M, and that Diaportein A and Diaportein B have no bactericidal effect on the pseudomonas aeruginosa at the concentration of 40 mu M and do not influence the growth of the pseudomonas aeruginosa.
Example 4: effect of Compounds 1 and 2 on quorum-sensing regulated biofilms
Method for determining inhibition effect of compound on biological membrane by crystal violet staining method
The method comprises the following steps: the 24-well plate is inoculated with 500 mu L of pseudomonas aeruginosa PAO1 bacterial liquid, and simultaneously, the compound 1 and the compound 2 with the final concentration of 40 mu M are respectively added, and the incubation is carried out for 24 h at 37 ℃. After removing the surface bacteria liquid in the pores, carefully washing the 24-pore plate with distilled water, washing off floating bacteria, and drying. Then 1mL of 1% crystal violet solution was added to a 24-well plate, the plate was left to stand and stain at room temperature for 15min, the excess crystal violet solution was aspirated, the biofilm was carefully washed 3 times with distilled water, and then left to stand and dry. Then adding 33% glacial acetic acid for dissolving, oscillating uniformly, and measuring the OD value at the wavelength of 570 nm of an enzyme labeling instrument.
The test result shows that: the compound 1 and the compound 2 have certain inhibiting effect on the formation of the pseudomonas aeruginosa biomembrane, and the inhibiting rate on the biomembrane production is 33 percent and 32 percent respectively.
Microscopic imaging analysis to examine the effects of Compound 1 and Compound 2 on biofilm formation
(1) Common optical microscopy imaging analysis: inoculating 500 mu L of pseudomonas aeruginosa PAO1 bacterial liquid into a 24-pore plate, simultaneously respectively adding a compound 1 and a compound 2 with final concentration of 40 mu M, incubating for 24 h at 37 ℃, sucking surface bacterial liquid in the pore, washing the 24-pore plate with distilled water, washing floating bacteria, washing for three times, and drying; then 1mL of 1% crystal violet solution was added to a 24-well plate, and the plate was left to stand and stain at room temperature for 15min, and the excess crystal violet solution was aspirated, washed carefully with distilled water for 3 times, dried, and placed in a normal optical microscope to observe the film.
The results of the assay, as shown in FIG. 8a, were analyzed by ImageJ software and the inhibition of Pseudomonas aeruginosa biofilm formation by Compound 1 and Compound 2 was 39% and 42%, respectively, at a concentration of 40. mu.M.
(2) Laser confocal imaging analysis: inoculating 1mL of pseudomonas aeruginosa PAO1 bacterial liquid into a 35mm wave-bottom culture dish, respectively adding 1mL of compound 1 and compound 2 until the final concentration is 40 mu M, incubating at 37 ℃ for 24 h, sucking off bacteria on the surface layer in the hole, washing the culture dish for 3 times by distilled water, and washing off planktonic bacteria. Then adding a bacterial viability kit for dyeing, observing the biological membrane under a laser confocal microscope, wherein the green fluorescence Syt 9: excitation wavelength 483 nm and emission wavelength 500 nm; red fluorescent PI: excitation wavelength 305 nm, emission wavelength 617 nm.
The results of the test are analyzed by ImageJ software, as shown in FIG. 8b, the inhibition rate of compound 1 and compound 2 on the generation of Pseudomonas aeruginosa biofilm at a concentration of 40. mu.M is 33% and 44%, respectively.
The results of the above 2 microscopic imaging analyses showed that: the compound 1 and the compound 2 have obvious inhibiting effect on the formation of the pseudomonas aeruginosa biofilm.
Example 5: influence on expression level of virulence factor regulated by quorum sensing
And culturing the pseudomonas aeruginosa in the LB culture solution overnight. 50 mL of activated Pseudomonas aeruginosa was inoculated into Erlenmeyer flasks (final concentration of bacteria: OD) 600 = 0.01) while adding compound 1 and compound 2, respectively, at final concentrations of 10 μ M, 20 μ M, 40 μ M, respectively; culturing at 37 deg.C and 200 r/min for 24 hr, respectively placing 200 uL of bacterial liquid in 96-well plate, and measuring OD at 600 nm wavelength 600 The value is obtained.
1) Inhibition of virulence factor pyocyancin secretion by compounds 1 and 2
The test method comprises the following steps: transferring the cultured bacterial liquid into a 50 mL centrifuge tube, centrifuging at 10000 rpm/min and 4 ℃ for 10 min. Centrifuging, adding 32 mL chloroform into 40 mL of supernate after centrifugation, fully oscillating for extraction, standing, transferring 1mL chloroform layer to a new tube, adding 0.6 mL 0.2 mol/L hydrochloric acid for extraction, centrifuging and layering, placing 200 uL hydrochloric acid layer in a 96-well plate, and measuring the OD value at the wavelength of 520 nm of an enzyme-labeling instrument.
Note: a. the 1 The content (OD) of pyocin in the drug group 520 /OD 600 ）；A 0 Is a blank group of pyocin containing (OD) 520 /OD 600 ）
The experimental results shown in fig. 9 show that the inhibition rates of the compounds 1 and 2 on the secretion of virulence factor pyocyancin are 10% -30%, and the inhibition rates increase with the increase of concentration, which indicates that the compounds 1 and 2 play a role in inhibiting the secretion of pseudomonas aeruginosa pyocyancin.
2) Inhibition of P.aeruginosa rhamnolipid secretion by compounds 1 and 2
The test method comprises the following steps: taking 5mL of the cultured bacterial liquid, centrifuging at 10000 rpm/min and 4 ℃ for 10 min. 2 mL of the supernatant was placed in a 10 mL EP tube, and 4 mL of diethyl ether was added thereto and shaken to mix well for extraction. The ether layer (1 mL) was placed in a 1.5mL EP tube in a fume hood and the ether was removed. Adding 40 μ L of ultrapure water to dissolve the extracted rhamnolipid, adding 360 μ L of orcinol solution (0.19 g of orcinol powder is added into 50% concentrated sulfuric acid, and the volume is determined to 100 ml), mixing, and decocting in 80 deg.C water bath for 30 min. After cooling, 200. mu.L of the suspension was placed in a 96-well plate, and OD was measured at a wavelength of 421nm using a microplate reader.
Note: b is 1 Is the rhamnolipid content (OD) of the drug group 421 /OD 600 ）；B 0 The rhamnolipid content OD is a control group 421 /OD 600
The test result shows that (figure 10), the inhibition rate of the compound 1 on the secretion of the rhamnolipid of the pseudomonas aeruginosa is between 40% and 61%; the inhibition rate of the compound 2 on the secretion of the rhamnolipid of the pseudomonas aeruginosa is 19-42%, and the inhibition rate is obviously increased along with the increase of the concentration, which shows that the compound 1 and the compound 2 have the function of obviously inhibiting the secretion of the rhamnolipid of the virulence factor of the pseudomonas aeruginosa.
3) Inhibition of Pseudomonas aeruginosa elastase activity by Compounds 1 and 2
Preparing an elastin solution: elastin solution was prepared according to the Kit EnzChekfi Protease Assay Kit E6638 (care protected from light).
0.2 mL of PBS was added to a vial containing the lyophilized substrate for BODIPY FL elastin, and after sufficient solubilization, the concentration of BODIPY FL elastin was 1.0 mg/mL, followed by dilution of the elastin solution to 10 μ g/mL with pH 7.8 Tri-HCl buffer.
Centrifuging the cultured bacterial liquid for 10min at 10000 rpm/min in 5mL, filtering with a 0.22 μm filter membrane, adding 100 μ L filtrate into 100 μ L BODIPY FL elastin solution of 10 μ g/mL, mixing, reacting for 4 hr, measuring fluorescence intensity RFU (excitation wavelength 400 nm, emission wavelength 450 nm), and calculating the inhibition rate.
As shown in FIG. 11, the test results show that the inhibition rate of the compound 1 on the activity of the pseudomonas aeruginosa elastase is between 20% and 55%, and the inhibition rate of the compound 2 on the activity of the pseudomonas aeruginosa elastase is between 19% and 67%, and the inhibition rate is increased along with the increase of the concentration. The compound 1 and the compound 2 have obvious inhibition effect on the pseudomonas aeruginosa elastase.
Example 7: effect of Compounds 1 and 2 on expression of genes associated with the quorum sensing System of Pseudomonas aeruginosa
And culturing the pseudomonas aeruginosa in the LB culture solution overnight. 50 mL of activated Pseudomonas aeruginosa was inoculated in a conical flask (final bacterial concentration: OD) 600 = 0.01), and then compounds 1 and 2 were added to a final concentration of 40 μ M, respectively, and shake-cultured for 24 hours.
Taking 1mL of bacterial liquid, centrifuging at 8000 rpm for 3min, removing supernatant, adding 500 mu L of PBS liquid, washing by gentle shaking, centrifuging, and removing supernatant. Then adding 200 mu L of lysozyme, shaking and mixing uniformly, adding 800 mu L of RNA extracting solution, blowing and beating the broken cells by a gun head lightly, then adding 250 mu L of trichloromethane, mixing uniformly, standing for 3min, and centrifuging for 10 min.
Transferring 400 μ L of supernatant into a new centrifuge tube, adding 0.8 times volume of isopropanol, mixing, standing at-20 deg.C for 15min, centrifuging for 10min, removing supernatant, adding 1.5ml of 75% ethanol, washing precipitate, centrifuging, and collecting precipitate as RNA. Adding 15 mu L of sterile enzyme-free water into a centrifuge tube to dissolve the RNA precipitate, detecting the concentration and the purity of the RNA precipitate by using an ultramicro nucleic acid protein quantifier (Nanodrop 2000), and finally preparing the RNA solution with the final concentration of 200 ng/. mu.L by using the sterile enzyme-free water.
According to the specification of a SYBR Green qPCR kit, a specific primer of a gene to be detected, an RNA solution and a dye reagent are mixed, reaction conditions are set, and real-time quantitative PCR detection is carried out in a LightCycler96 fluorescent quantitative PCR instrument. The PCR data were normalized with the 16S ribosomal RNA gene as an internal reference. By 2 -ΔΔCT The expression level of the gene was calculated.
Delta CT = CT target gene-Ct reference gene
Delta CT =deltaCT drug treatment group gene-delta CT control group gene
Fold changes were calculated for relative quantitation using 2- (. DELTA.. DELTA.Ct) (FIG. 12), and compound 1 and compound 2 showed significant down-regulation of expression of the LasB, RhlR, PqsR genes at a concentration of 40. mu.M.
Claims (10)
1. A pimarane diterpene compound characterized by having a pimarane diterpene compound represented by the formula (І) and/or (II):
2. the pimarane diterpenoid compound according to claim 1, wherein the pimarane diterpenoid compound is derived from a marine alternaria fungusAlternaria porri Preparation and separation of 25# Secondary metaboliteAnd (4) obtaining the product.
3. The pimarane diterpenoid compound according to claim 2, wherein the marine alternaria fungusAlternaria porri 25# is a alternaria fungus from mangrove rhizosphere soil, which is preserved in Guangdong province microbial strain preservation management center with the preservation number of GDMCC NO: 62148, preservation date is 2021, 12 months and 31 days.
4. The process for producing a pimarane diterpene compound according to claim 1, which comprises the following steps in order:
(1) preparation of marine Alternaria fungiAlternaria porri 25# solid fermentation culture
The marine Alternaria fungusAlternaria porri Extracting and concentrating the 25# fermentation product by using chloroform and methanol in a volume ratio of 1:1 to obtain a chloroform-methanol extract, distributing and extracting the extract by using water and ethyl acetate in a volume ratio of 1:1, and concentrating an ethyl acetate layer to obtain an ethyl acetate crude extract;
(2) subjecting the obtained crude extract to silica gel column chromatography, and gradient eluting with petroleum ether-ethyl acetate at volume ratio of 9:1, 4:1, 7:3, 3:2, 1:1, 3:7, and 0:1 sequentially as eluent to obtain 7 components Fr.1-Fr.7;
(3) collecting an elution component Fr.2 of petroleum ether and ethyl acetate in a volume ratio of 4:1, continuously adopting silica gel column chromatography, and performing gradient elution with a petroleum ether-ethyl acetate system in a volume ratio of 9:1, 9:2, 9:3, 4:1 and 1:1 to obtain 5 components Fr.2.1-Fr.2.5;
(4) continuously carrying out Sephadex LH-20 chromatographic separation on the component Fr.2.2, eluting by using dichloromethane and methanol with the volume ratio of 1:1 as an eluent, and separating and purifying to obtain a compound shown in the formula (II); the component Fr.2.3 is recrystallized and purified to obtain the compound of the formula (І).
5. The method of claim 4, wherein the fungus belonging to the genus Alternaria is a marine alternariaAlternaria porri The preparation method of 25# fermentation product is from seaFungus of the genus AlternariaAlternaria porri Culturing 25# as fermentation strain in amplified fermentation culture medium to obtain marine Alternaria fungusAlternaria porri 25# fermentation product;
the amplification fermentation culture medium is a rice solid culture medium: comprises rice 100g, self-made seawater 100mL, and pH 7.2-7.4.
6. Use of the pimarane type diterpene compound according to claim 1 for the preparation of antibacterial medicaments.
7. The use of a pimarane type diterpene compound according to claim 6 for the preparation of an antibacterial medicament, wherein the pimarane type diterpene compound is used for the preparation of an antibacterial medicament for inhibiting a bacterial quorum sensing system and inhibiting the formation of a bacterial biofilm.
8. The use of a pimarane type diterpene compound according to claim 7 for the preparation of an antibacterial medicament, wherein the bacteria of the bacterial quorum sensing system is pseudomonas aeruginosa.
9. The use of a pimarane type diterpene compound according to claim 8 for the preparation of an antibacterial medicament, wherein the pimarane type diterpene compound is used for the preparation of an antibacterial medicament for inhibiting the secretion of virulence factors pyocyanin, rhamnolipid and elastase in the quorum sensing system of pseudomonas aeruginosa; or in the preparation of antibacterial drugs for down-regulating LasB, RhlR and PqsR gene expression in a pseudomonas aeruginosa quorum sensing system.
10. An antibacterial agent comprising an effective amount of the pimarane diterpene compound or its pharmaceutically acceptable salt according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.
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