CN104784160A - Bacterial quorum sensing inhibitor and antibacterial application thereof - Google Patents
Bacterial quorum sensing inhibitor and antibacterial application thereof Download PDFInfo
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Abstract
The invention relates to antibacterial application of small molecular bacterial quorum sensing inhibitor Falcarindiol in the condition of no inhibition on bacteria growth concentration (subinhibitory concentration). The active compound has a broad-spectrum bacterial quorum sensing resistant activity, can inhibit bacterial virulence in subinhibitory concentration, reduces the harm of bacteria to the host, and can prevent and treat quorum sensing bacterial infections. The compound can play the role of inhibition of bacterial virulence in subinhibitory concentration, so that pressure for survival of pathogenic bacteria is not brought, drug resistance is not easy to produce, the antibacterial application of the small molecular bacterial quorum sensing inhibitor Falcarindiol in the condition of no inhibition on bacteria growth concentration (subinhibitory concentration) is different from application of killing bacteria and bacteria growth inhibition of other antibacterial agent, is also is different from the antibacterial application of bacteria growth inhibition of Falcarindiol with the bacteria concentration (the use final concentration is greater than the minimum bacteriostasis concentration), and is a new antibacterial application. The antibacterial application of the small molecular bacterial quorum sensing inhibitor Falcarindiol in the condition of no inhibition on bacteria growth concentration (subinhibitory concentration) has the advantages that: raw materials are easy to get, compound separation and purification are simple and easy to control, security is high, use concentration is low, and drug resistance is easy to produce, and the like, so that the Falcarindiol can be used in preparation of a new antibacterial agent.
Description
Technical field
The invention belongs to microorganism field, relate to a kind of antibacterial applications of compound falcarindiol as quorum-quenching agent of wide material sources.
Background technology
The drug resistance of antibacterial is a global difficult problem.The object of current antibacterial kills antibacterial or bacteria growing inhibiting, under this huge selection pressure, creates the amount reproduction of drug-resistant bacteria.Therefore, traditional antibiotic can not solve drug resistance problems.
Another kind strategy antibacterial is at present antitoxin power (anti-virulence) strategy, namely not with kill or anti-bacteria be grown to object and target spot, when not bacteria growing inhibiting, the expression of direct anti-bacteria virulence factor, antibacterial is locked in avirulence or low virulence state, loses or reduce the injury ability for host.On the other hand, due to the growth of not anti-bacteria, antibacterial is not easily made to produce drug resistance.The virulence of antibacterial is largely by the signaling system of antibacterial---and bacterial community induction regulates and controls, and anti-bacteria quorum sensing, can reduce the virulence of antibacterial, reduces the injury of antibacterial for host cell, and not easily produces drug resistance.This quorum sensing inhibitor can be used alone, and also can reach the object of control bacteriological infection as antibiotic auxiliary therapeutical agent.
The antibacterial with quorum sensing (Quorum Sensing, QS) regulating system can be synthesized and be discharged the signaling molecule that one is called as Autoinducer (Auto Inducer, AI).Along with the increase of bacterial density and the concentration buildup of signaling molecule arrive certain threshold value, signaling molecule and cytoplasmic receptor protein binding form activity regulation complex, the expression of regulation and control pre biooxidation correlation gene, thus make antibacterial form a kind of group behavior.In analysis of clinic pathogenic microorganism, bacterial community induction modulin homologue and the discovery of Autoinducer make bacterial community induction receive to pay attention to widely.
Bacterial signal molecules is mainly divided into four classes: the first kind is the QS system mediated by AHL of gram negative bacteria, and its signaling molecule is acylated homoserine lactone (N-aeylhomoserine lactones, AHLs); Equations of The Second Kind is the QS system mediated by self-induction peptide (Autoinducing Peptide, AIP) of gram positive bacteria, and its signaling molecule is aminoacid or small peptide; 3rd class is the class signaling system all existed in gram negative bacteria and positive bacteria, and its signaling molecule is furan boronic acid diester (AI-2); 4th class is the signaling molecule transboundary (as norepinephrine and epinephrine) that antibacterial can utilize host.
Pseudomonas aeruginosa (
pseudomonas aeruginosa) be a kind of common opportunist, its intervention school-based comprises based on AHL signaling molecule
lassystem and
rhloutside system, the false unit cell quinolone signal system (Nature, 2000,406:959-964) that also to comprise with 2-heptyl-3-hydroxyl-4-quinolinones (PQS) be signaling molecule.The multiple pathogenic virulence of Pseudomonas aeruginosa is all subject to quorum sensing regulation and control.Chromabacterium biolaceum (
chromobacterium violaceum) colony induction signaling molecule belongs to homoserine lactone class, it can regulate and control the synthesis of the virulence factors such as secondary metabolite mycetin, therefore, has important regulating and controlling effect to the infection ability of antibacterial.Other animals and plants cause of diseases such as vibrio, Agrobacterium all have intervention school-based.
The expression of the multiple virulence factor such as extracellular protease (as elastoser), toxin (as pyo), rhamnolipid of current discovery bacterial community induction regulation and control pathogen, and regulation and control cause the biofilm of bacterial resistance (biofilm) to be formed relevant, and the motor behavior ability of pathogen can be regulated and controled.By the quorum sensing of anti-bacteria, also suppress the virulence that these bacterial community induction systems regulate and control, reduce antibacterial for the harm of host, reach and control in advance or the object of auxiliary antibiotic therapy bacteriological infection.Therefore quorum-quenching agent has broad application prospects in control bacteriological infection.
Falcarindiol (falcarindiol) is a kind of carbene compound extensively existed in Umbelliferae and Araliaceae, has been separated in Rhizoma Et Radix Notopterygii, Radix Angelicae Sinensis, the Radix Angelicae Dahuricae, Crithmum maritimum and Radix Dauci Sativae etc. at present and has obtained, and can synthetic, commercialization.The safety of falcarindiol is higher, has anticancer, antiinflammatory, analgesia, the growth of check melanin tumor, antifungal, antibacterial effect, but does not relate to zoopery for the detection of these activity.
Falcarindiol has bacteriostasis for gram positive bacteria staphylococcus aureus, falcarindiol has faint bacteriostasis for gram negative bacteria mycobacterium, Salmonella, Pseudomonas aeruginosa etc., but because the minimal inhibitory concentration of falcarindiol is too low relative to antibiotic traditional at present, so there is no the research as antibacterial.
Our Late Cambrian is under the concentration of not bacteria growing inhibiting, falcarindiol can anti-bacteria quorum sensing, also being different from the antibacterial applications of the bacteria growing inhibiting of the falcarindiol of Mlc (using final concentration to be greater than minimal inhibitory concentration MIC), is a kind of new antibacterial applications.
Summary of the invention
The new active and novelty teabag of one of micromolecular compound falcarindiol provided by the present invention (Falcarindiol, formula I).Under the concentration of not bacteria growing inhibiting (subinhibitory concentration); to having N-acyl homoserine lactones class signaling molecule; or quinolones signaling molecule; or the bacterial community induction system of Furanosyl borate diester class signaling molecule has remarkable suppression; and can the expression of virulence factor of anti-bacteria quorum sensing regulation and control, reduce the virulence of antibacterial, for preventing and treating the bacteriological infection with intervention school-based.
(I), wherein 1-17 is carbon atoms numbered.
Falcarindiol in the present invention is natural extract, also can the compound of chemosynthesis.
In the present invention, falcarindiol is prepared into separately antibiotic preparation or becomes the antibiotic preparation of compound with existing anti-infective drugs.
In order to prevent and treat pathogen or harmful bacteria generation drug resistance in the present invention, the falcarindiol concentration of employing does not suppress the concentration of this bacterial growth, adopts the effective dose of falcarindiol in antibiotic preparation.
In the present invention, the antibacterial applications scope of quorum-quenching agent falcarindiol is the control of the infection of the inside and outside microorganism of human or animal, or the process of breeding water body, or for the control of phytopathogen.
The object of the invention is to overcome the shortcoming existed in prior art, the antibacterial new opplication mode of quorum-quenching agent falcarindiol is provided, falcarindiol extensively exists in Umbelliferae and Araliaceae, and content is higher, also can synthetic or directly buy.Therefore the present invention has that raw materials easily obtains, compound separation purification is simple and easy to control, safety is high, working concentration is low and not easily produces the advantages such as drug resistance.
detailed description of the invention:
Embodiment 1: falcarindiol Isolation and characterization in Chinese Medicine " qianghuo "
Methanol crude extract preparation method: take 10 g powder after being pulverized by Rhizoma Et Radix Notopterygii rhizome, every 2.5 g powder, 200 mL methanol extractions, soak 24 h after supersound process 1 h; Filter paper filtering obtains leachate, and Rotary Evaporators evaporate to dryness filter liquor is also weighed; Again rear utilization self-control Cotton Gossypii frit removing insoluble matter is dissolved with 30 mL methanol; Evaporate to dryness filter liquor weighing again.
The preparation method of compound: after above-mentioned extractum proper silica gel H stirs, be separated with vacuum liquid phase column chromatography, with the eluent petroleum ether of different ratio, dichloromethane, dichloromethane: methanol (100:1), dichloromethane: methanol (50:1), dichloromethane: methanol (20:1), methanol rushes post respectively, and each elution fraction carries out eluting with 3 column volumes.Collect dichloromethane: the component of methanol (50:1) eluting, be separated with Sephadex LH-20 gel filtration chromatography after concentrated, 100% methanol is that eluent is separated, silica gel plate thin layer chromatography TLC combine with technique vanillin and universal visualization agent is utilized to dye, analyze each outflow component, merge active stream part, utilize preparative high performance liquid chromatography instrument with methanol: water is that 70% mobile phase carries out purification, obtains monomer falcarindiol.TLC general stain colour developing (commercial falcarindiol in contrast) of falcarindiol and Rhizoma Seu Radix Notopterygii extract determines the peak collected.The purity of the falcarindiol obtained by said method analyzes (retention time of commercial falcarindiol in contrast) through HPLC.
Experimental result: with commercial falcarindiol for contrast, the component through separation and purification shows single stain at TLC, and position is consistent with contrast, and HPLC analyzes simple spike, identical with contrast retention time, purity reaches more than 98%.
Compound structure identify: utilize mass spectrum (MS), nuclear-magnetism (
1hNMR,
13c NMR, DEPT) Structural Identification is carried out to compound, in conjunction with this compound
1h spectrum and
13c analysis of spectrum.
Experimental result: molecular formula is C
17h
24o
2;
1h NMR and
13c NMR(DEPT) nuclear-magnetism composes the data obtained and document (Archives of Biochemistry and Biophysics, 2009,488:34 – 41) compares, and determines that it is compound: falcarindiol.
Compound spectral data:
1H NMR (CDCl
3, 600 MHz) δ: 0.85 (3H, t-like, J = 6.8 Hz, 17-H), 1.24 (2H, m, 13, 14, 15, 16-H), 1.30 (2H, m, 12-H), 2.04 (2H, m,11-H), 5.08 (1H, dd, J = 7.7,5.4 Hz, 8-H), 4.83 (1H, dd, J =5.4, 5.4 Hz,3-H), 5.12 (1H, d, J = 10.3,1.7 Hz, 1-Htrans),5.30 (1H, d, J = 17.2,1.6 Hz, 1--Htrans), 5.40 (1H, m, 9-H),5.45 (1H, m, 10-H), 5.85 (1H, ddd, J =17.2, 10.3, 5.1 Hz, 2-H).
13C NMR (CDCl
3, 150 MHz) δ：138.0(C-2), 132.3(C-10),130.0(C-9),116.0(C-1), ,82.0(C-4), 80.7(C-7), 69.0(C-5), 67.6(C-6), 62.2(C-3) ,57.5(C-8), 31.9(C-11),29.3(C-12),29.1(C-13), 29.1(C-14),27.5(C-15),22.7(C-16),14.5(C-17).
Embodiment 2: the anti-chromabacterium biolaceum quorum sensing inhibit activities test of falcarindiol
Chromabacterium biolaceum screening model CV026 composition and method: the solid LB culture medium that 19 mL melt is cooled to 40 DEG C, add the chromabacterium biolaceum CV026 bacterium liquid of 600 μ L incubated overnight, kanamycin that signaling molecule hexanoyl homoserine lactone (C6-HSL) that 15 μ L concentration are 500 μm of ol/L and 15 μ L concentration are 30 mg/mL, mix in culture dish.After flat board solidifies, punch with the card punch of sterilizing, in each hole, add 5 μ L compounds, cultivate 12h in 30 DEG C of incubators, observe well surrounding bacterial growing state.If this compound is quorum sensing inhibitive factor, then can suppress the generation of mycetin, show muddiness on a lbmc agar plate but opaque circle; The larger proof compound activity of muddy circle is higher.
Experimental result: with positive control furanone C
30(6.5 μ g) compares, and 6.5 μ g, 13 μ g, 26 μ g and 65 μ g falcarindiol all can suppress the output of chromabacterium biolaceum mycetin, and increases with concentration and increase, and occurs without transparent inhibition zone.
Embodiment 3: the quorum sensing inhibit activities test of the resisting pseudomonas aeruginosa of falcarindiol
Suppress Pseudomonas aeruginosa AHL intervention school-based:
Pseudomonas aeruginosa detection model QSIS2 composition and method: the solid LB culture medium that 17.5 mL melt is cooled to 40 DEG C, adding 2 mL concentration is the sucrose of 60%, the Pseudomonas aeruginosa QSIS2 bacterium liquid of 250 μ L incubated overnight, 8 μ L concentration are signaling molecule 3-oxygen dodecanoyl homoserine lactone (3-oxo-C12-HSL) of 100 μm of ol/L, 200 μ L concentration are 5%(w/v) viable bacteria stain red tetrazolium (TTC) and 32 μ L concentration be the gentamycin of 50 mg/mL, pour in culture dish after mixing.After flat board solidifies, punch with the card punch of sterilizing, in each hole, add the compound of 5 μ L variable concentrations respectively, be placed in 37 DEG C of incubators and cultivate, observe thalli growth situation around well.If compound has quorum sensing inhibit activities, then occur red bacterial growth circle around well, bacterium loop diameter larger expression compound suppresses quorum sensing activity stronger,
Experimental result: with positive control C
30(6.5 μ g) compares, and 6.5 μ g, 13 μ g, 26 μ g and 65 μ g falcarindiol present positive findings, and becomes concentration dependent, and occurs without transparent inhibition zone.
Pseudomonas quinolone signal system (PQS) intervention school-based inhibit activities is suppressed to detect:
The detection composition of PQS intervention school-based signaling molecule PQS and method: Pseudomonas aeruginosa PAO1 monoclonal is in fresh 5ml LB culture fluid, shaking table is cultured to exponential phase, diluted by bacterium liquid 1:100 with fresh LB culture fluid, subpackage 4 groups, often group 3 is parallel; Often organize and add the falcarindiol that final concentration is 0,12.5 μm of ol/L, 25 μm of ol/L and 50 μm ol/L respectively, the ethyl acetate of 100 μ L acid is added in the culture fluid supernatant of 300 μ L, abundant mixing, draws organic facies in 1.5ml centrifuge tube, as 37 DEG C of natural dryings; Prepare lamellae: get lamellae, with the KH of 10%
2the activation of PO4 solution is placed in baking oven dries 1h, after carrying out preview experiment with developing solvent, lamellae is continued to dry, after point sample dissolve with methanol, get 5 μ L point samples in ready lamellae, thoroughly dry up, carry out in expansion system observing under thin layer chromatography experiment is placed on UV.
Experimental result: Pseudomonas aeruginosa PQS signaling molecule not only has the effect of regulation and control downstream virulence gene expression, and directly can produce toxic action to human body cell.TLC method is utilized to carry out qualitative detection to the output of signaling molecule under falcarindiol inhibitory action, add the generation that 12.5 μm of ol/L can suppress signaling molecule PQS, under 50 μm of ol/L concentration, PQS output is suppressed and plays a role clearly, but does not cause antibacterial growth phenomenon.
Embodiment 4: the quorum sensing inhibit activities test of anti-Vibro harveyi (Vibirio harveyi) AI-2 of falcarindiol
Vibrio harveyi AI-2 quorum sensing screening model Vibirio harveyi BB170 composition and method: the Vibirio harveyi BB170 bacterium liquid diluting incubated overnight with AB culture fluid 1:100, adds the kanamycin that final concentration is 100 μ g/mL; Be sub-packed in culture test tube by the bacterium liquid of dilution, often pipe 5mL, add the falcarindiol that final concentration is 0 μ g/mL, 26 μ g/mL, 65 μ g/mL and 130 μ g/mL, 30 DEG C, 140rpm cultivates 18h simultaneously; The luminous situation of thalline in each culture fluid is observed in dark surrounds.This compound effectively can reduce the luminescent behavior of quorum sensing regulation and control under 26 μ g/mL concentration; Under 65 μ g/mL and 130 μ g/mL concentration, vibrio luminescent behavior is effectively suppressed, but this concentration is far below minimal inhibitory concentration.
Embodiment 5: the falcarindiol of subinhibitory concentration is tested pathogen virulence factor elastase activity
The impact that falcarindiol grows wild type Pseudomonas aeruginosa PAOI:
Picking wild type Pseudomonas aeruginosa PAO1 in fresh LB culture fluid, 37 DEG C, 140rpm is cultured to logarithmic (log) phase; With fresh LB, the bacterium liquid being cultured to logarithmic (log) phase is diluted to OD
600≈ 0.01, subpackage 5 groups, adds final concentration final compound concentration 0 μ g/mL, 65 μ g/mL, 130 μ g/mL, 260 μ g/mL, 520 μ g/mL respectively, and often group 3 is parallel; 37 DEG C, the cultivation of 140rpm shaking table; Each pipe OD is measured every 2 hours
600light absorption value, measures 24h altogether; With incubation time time(h) be abscissa, with the optical absorbance at 600nm place for vertical coordinate, draw the growth curve of wild type Pseudomonas aeruginosa PAO1 under falcarindiol effect.
Experimental result: this compound does not suppress the growth of wild type Pseudomonas aeruginosa PAO1 in 0-520 μ g/mL concentration range, OD
600do not decline.
The falcarindiol of subinhibitory concentration is tested pathogen virulence factor elastase activity:
Picking wild type Pseudomonas aeruginosa PAO1 monoclonal is inoculated in fresh LB, 37 DEG C, 140 rpm incubated overnight; With fresh LB culture fluid, bacterium liquid 1:100 is diluted, be sub-packed in 50 mL point end culture tubes, often pipe 5 mL; Often organize simultaneously add final concentration respectively and be 0 μ g/mL, 6.5 μ g/mL, 13 μ g/mL, 26 μ g/mL and 52 μ g/mL falcarindiol, often organize 3 parallel; 37 DEG C, 140 rpm cultivate 12 h.
Elastase activity assay method: draw 1 mL culture fluid and manage in 1.5 mL Eppendorf, 4 DEG C, 12000 rpm are after centrifugal 15 minutes, Aspirate supernatant, with disposable filter (0.22 μm) filtration sterilization; 5 mg elastase substrate are added in 2 mL shaken cultivation pipes---Congo red (EcR) and l mL reaction buffer (0.1 M Tris-HCl; 1mM CaC1
2, pH 7.2), add 0.2 mL bacterial supernatant, 37 DEG C, 140 rpm oscillating reactions 8 hours; Add the EDTA cessation reaction of 0.12 mol/L of 0.l mL, reactant is placed on ice.4 DEG C, centrifugal 10 min of 12000 rpm, remove insoluble ECR; Draw 200 μ L supernatant and read absorbance at OD495 nm place.
Experimental result: 6.5 μ g/mL and 26 μ g/mL falcarindiol reduce by 40.5% and 70.2% to Pseudomonas aeruginosa PAO1 elastin laminin production of enzyme respectively.
Embodiment 6: the falcarindiol of subinhibitory concentration is tested pathogen virulence factor pyo output
Picking Pseudomonas aeruginosa PAO1 monoclonal in the LB culture fluid of fresh 5 mL, 37 DEG C, 140 rpm incubated overnight; With fresh LB culture fluid, bacterium liquid 1:100 is diluted, be sub-packed in 50 mL point end culture tubes, often pipe 5mL; Often organize simultaneously add final concentration respectively and be 0 μ g/mL, 6.5 μ g/mL, 13 μ g/mL, 26 μ g/mL and 52 μ g/mL falcarindiol, often organize 3 parallel; 37 DEG C, 140rpm cultivates 12h.
Pyo quantitative approach: draw 1 mL culture fluid and manage in 1.5 mL Eppendorf, with 4 DEG C, 12000 rpm centrifugation medium 15 min, gentle aspiration culture fluid supernatant; With chloroform with pyo in the ratio of 5:3 extraction culture fluid supernatant, turbine mixer is utilized to make extraction process more thorough; To extract mixed liquor with centrifugal 2 min of 12000 rpm, organic facies is thoroughly separated with aqueous phase; Draw organic facies and add hydrochloric acid (0.2 N) with the ratio of 3:1 wherein, 37 DEG C of shaking tables hatch to make it fully react; With 12000 rpm centrifugal 2 min, organic facies is thoroughly separated with aqueous phase mixed liquor; Draw 200 μ L aqueous phases and measure light absorption value in 520 nm places.
Experimental result: the output of falcarindiol to pyo has obvious inhibitory action, when 6.5 μ g/mL, inhibitory action reaches 50%, and suppression degree is 70% under the concentration of 26 μ g/mL.
Embodiment 7: the falcarindiol of subinhibitory concentration is for the suppression of bacterial biof iotalm
Picking Pseudomonas aeruginosa PAO1 monoclonal in the LB culture fluid of fresh 5 mL, 37 DEG C, 140 rpm incubated overnight; With fresh LB culture fluid, bacterium liquid 1:100 is diluted, be divided into 4 groups often organize 6 parallel, add different final concentration falcarindiol respectively; Be sub-packed in after 2 h cultivated by 37 DEG C of shaking tables in 96 orifice plates, 200ul/ hole, be placed in 37 DEG C of constant incubator quiescent culture 12 h, 24 h and 36h;
With after pipettor slow sucking-off bacterium liquid with 250 μ L PBS(pH7.2,0.01mM) wash thoroughly to remove thalline and leave standstill for 2-3 time and dry; Add 250 μ L crystal violets (0.01%) to dye 15 min, again wash gently with the PBS that 250 μ L are aseptic and go out removal floating color 2-3 time, leave standstill and dry; Add 200 μ L glacial acetic acids (33%) and dissolve biomembrane, measure absorbance value at 595nm.
Experimental result: falcarindiol is formed with obvious suppression for biofilm under above-mentioned concentration, and wherein suppression degree is about 60% under 20uM concentration.
Embodiment 8: the falcarindiol of subinhibitory concentration is applied for the infection of transplant infection animal
Picking Pseudomonas aeruginosa PAOI, cultivates 18-20h; The centrifugal 10min of 3000rpm, collects thalline, and the NaCl with 0.9% is by bacterium liquid dilution OD
600=0.1; 5mm × 5mm the silica gel sheared is placed in 0.5%NaClO solution soaked overnight, and after sterilized water rinse, the NaCl with aseptic 0.9% soaks; Every 10 silica gel pieces put into the bacterium liquid of 20ml; 37 DEG C, 110rpm cultivates 20h, makes bacterial adhesion on silica gel piece; Take out silica gel piece, at 2ml sterilized water mesoscale eddies vibration 1min, get liquid diluting and be coated in blue agar culture medium, select 10
-3under concentration, nearly 50-300 monoclonal silica gel piece modeling.The otch of about 1cm is cut at female KM mice groin place, after little silica gel piece being moved into intraperitoneal transplanting, with the cotton thread of sterilizing, by wound suture with scalpel.Animal is grouped into: PAO1 wild mushroom infects matched group (ethanol-NaCl solution of equivalent), P furanone C30 positive compound treatment group (1.2 μ g/g), falcarindiol treatment group (1.2 μ g/g), falcarindiol (2.4 μ g/g), falcarindiol (6 μ g/g), individual 10 of falcarindiol (12 μ g/g), intraperitoneal injection: every 24h gives once.
Experimental result: in initial infection, each group Mice Body weight average reduces 4%-8%, and after 3 days, except C30 positive compound treatment group, all the other Mouse Weights all start to rise.To infecting the later stage, before PAO1 wild mushroom infection matched group weight recovery has arrived experiment, C30 positive compound treatment group body weight is lower than before experiment, and all the other falcarindiol treatment group body weight all higher than before experiment, illustrate that furanone C30 has hypotoxicity in Mice Body.
Take out the silica gel piece moved in body, silica gel piece sterile scissors is cut into equal two parts (diagonally cutting), a part is for observing the biofilm situation on its silica gel piece, and another part is for adding up the clump count situation (clump count situation method is with the method in thalline preparation) on silica gel piece; And according to the situation of biofilm, calculate thickness and the Biomass of biofilm on silica gel piece.When infection 4 days, the clump count of positive compound C30 reduced 3 times than PAOI infected group, and falcarindiol is within the scope of 1.2 μ g/g-12 μ g/g, and the clump count on silica gel piece than the few 2-6 of PAOI infected group doubly.When infection 7 days, fewer than PAOI infected group 90 times of the clump count of positive compound C30, the best one group of clump count also reached on silica gel piece of falcarindiol reduced 50-100 doubly than PAOI infected group.Falcarindiol effectively can suppress Biomass 30-80% on silica gel piece within the scope of 1.2 μ g/g-12 μ g/g, higher than the suppression ratio of positive compound C30 by 20%.
Dissect mice, get its liver, falcarindiol group nodeless mesh occurs, falcarindiol can make the clump count in every gram of liver reduce 2-10 doubly.
Get mouse blood, the murine interleukin quantity compared with normal murine interleukin quantity that mice hemogram display PAOI wild mushroom infects matched group increases by 1.5 times, and falcarindiol treatment group, number of white blood cells is made to add 2-4 doubly than normal group at the treatment initial stage, showing falcarindiol is not reach therapeutic effect by anti-inflammatory activity, but by lowering pathogen virulence, enable host identify pathogen, induction of the removing of host for pathogen.
Embodiment 9: the falcarindiol of subinhibitory concentration is applied for the infection of scald infection animal
Picking Pseudomonas aeruginosa PAOI monoclonal, overnight incubation; In 1% ratio switching, by strain culturing to OD600=0.6, dilute 100 times, get 100 μ l and be coated with, now connecing bacterium amount is 1.24 × 10
4.Animal grouping is as embodiment 8.Scald process: injection 1% pentobarbital sodium (5 μ l/g) in every mouse peritoneal before depilation; With shaver by the depilation process of its back; Angle iron calcination under alcohol burner is scalded the mouse back lost hair or feathers immediately after 20 seconds and is caused scald in 5 seconds, causes III level to burn (all being destroyed to subcutaneous from epidermis).Often organize the scalded skin getting 2 mices at random simultaneously and carry out tissue slice qualification scald rank.Subcutaneous injection 0.3mL normal saline after modeling.Get 100 μ l bacterium liquid coatings, after bacterium liquid absorbs, be coated with 100 μ l testing compounds, matched group is coated with 0.9% normal saline; After compound doubly absorbs, bind up a wound with three layers of sterile gauze, burned mouse puts sub-cage rearing in the environment of room temperature 25 DEG C, ad lib water.Every 48h, change a medicine, again bandage wound;
Experimental result: the mortality rate measuring each group of mice from 48h; Falcarindiol treatment group can make the mortality rate of mice scald infection reduce 10-50%, makes the clump count of mice every gram liver reduce 10-50% according to infected group.Mouse thymus, the index and spleen index of infected group all reduce 50% according to normal group mice, and falcarindiol group can make thymus, index and spleen index all reduce 40% according to normal group mice.Falcarindiol treatment group, the expression that Mice Mice can be made to scald surface bacteria virulence gene reduces 30-70%.
Embodiment 10: bacteriological infection is treated in falcarindiol and the existing antibacterial coupling of subinhibitory concentration
Pseudomonas aeruginosa is adopted to carry out zoografting infection experiment step as embodiment 8, but grouping is different: PAOI wild mushroom infects matched group (ethanol-NaCl solution of equivalent), falcarindiol (2.4 μ g/g), gentamycin group (0.2 μ g/g), each 10 of falcarindiol+gentamycin group, administering mode is lumbar injection: every 24h gives once.Often organize execution 3 mices respectively the 4th, 7, get silica gel piece respectively and do bacteriology checking, biofilm detection, eyeball is got blood and is done analysis on hemogram.
Experimental result: before infection, mid-term gentamycin processed group about reduce by 10 times and 150 times of silica gel piece bacterium colonies, then substantially cannot observe colony growth after falcarindiol and gentamycin coupling.Mice hemogram index quantification measures: treating initial infection: make number of white blood cells add 3 times according to normal group during falcarindiol infects in the early stage, falcarindiol and gentamycin coupling group number of white blood cells also increase about 3 times, but alone gentamycin group number of white blood cells is normal range, the direct bacteria growing inhibiting of gentamycin is described, different from quorum-quenching agent falcarindiol, do not raise leukocytic effect.And the antibacterial applications mechanism of falcarindiol is different from gentamycin, it is a kind of novel antibacterial applications.
Claims (5)
1. a micromolecular compound falcarindiol (Falcarindiol) can as having N-acyl homoserine lactones class signaling molecule, or quinolones signaling molecule, or the inhibitor of the bacterial community induction system of Furanosyl borate diester class signaling molecule, preparation control has the application of the pathogen of respective fine bacteria quorum sensing system or the antibacterial of harmful bacteria; It is characterized in that molecular formula is C
17h
24o
2, structural formula is:
（I），
Wherein 1-17 is carbon atoms numbered.
2. according to the application described in claim 1, it is characterized in that: described falcarindiol can be the compound of natural extract or synthetic.
3. according to the application described in claim 1, it is characterized in that: described antibacterial is that falcarindiol is prepared into separately antibiotic preparation or becomes the antibiotic preparation of compound with existing anti-infective drugs.
4. according to the application described in claim 1, it is characterized in that: described antibacterial comprises the falcarindiol of effective dose, use final concentration not suppress corresponding pathogen or harmful bacteria growth.
5. according to the application described in claim 1, it is characterized in that: described antibacterial range of application is the control of the infection of the inside and outside microorganism of human or animal, or the process of breeding water body, or for the control of phytopathogen.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105287622A (en) * | 2015-10-23 | 2016-02-03 | 陕西省微生物研究所 | Method, target spot and application for reducing invasiveness of pseudomonas aeruginosa through NO accumulation |
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| CN113368137A (en) * | 2021-05-06 | 2021-09-10 | 华南农业大学 | Natural bacterial quorum sensing inhibitor and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105287622A (en) * | 2015-10-23 | 2016-02-03 | 陕西省微生物研究所 | Method, target spot and application for reducing invasiveness of pseudomonas aeruginosa through NO accumulation |
| CN105287622B (en) * | 2015-10-23 | 2019-05-17 | 陕西省微生物研究所 | NO accumulation reduces method, target spot and the application of pseudomonas aeruginosa invasiveness |
| CN105418428A (en) * | 2015-11-13 | 2016-03-23 | 四川医科大学 | Method for synthesizing chiral falcarindiol analogues |
| CN105769831A (en) * | 2016-04-26 | 2016-07-20 | 暨南大学 | Application of polyacetylene compounds to preparation of medicines for resisting drug-resistance bacteria |
| CN110590599A (en) * | 2019-09-30 | 2019-12-20 | 大连民族大学 | Quorum sensing signal molecular structure analogue and preparation method thereof |
| CN110590599B (en) * | 2019-09-30 | 2022-07-26 | 大连民族大学 | Quorum sensing signal molecular structure analogue and preparation method thereof |
| CN113368137A (en) * | 2021-05-06 | 2021-09-10 | 华南农业大学 | Natural bacterial quorum sensing inhibitor and application thereof |
| CN113368137B (en) * | 2021-05-06 | 2023-08-18 | 华南农业大学 | Natural bacterial quorum sensing inhibitor and application thereof |
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