CN113967226A

CN113967226A - Medicine for inhibiting bacterial quorum sensing system

Info

Publication number: CN113967226A
Application number: CN202111345426.7A
Authority: CN
Inventors: 郑俊霞; 夏志云; 陈婷婷; 陈浩铭; 顾子敏; 方静妍; 黄蕊; 梁佩云; 黄华容
Original assignee: Guangdong University of Technology
Current assignee: Guangdong University of Technology
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-01-25

Abstract

The invention discloses a medicament for inhibiting a bacterial quorum sensing system, which mainly comprises a mustard seed extract; aims to provide a medicine for inhibiting a bacterial quorum sensing system, which can effectively reduce the formation and the pathogenicity of a bacterial biofilm and has the effect of difficult generation of bacterial drug resistance; belongs to the field of medical biotechnology.

Description

Medicine for inhibiting bacterial quorum sensing system

Technical Field

The invention relates to the technical field of medicines, in particular to a medicine for inhibiting a bacterial quorum sensing system.

Background

Bacterial resistance has been a great threat and is currently growing as a global threat. Most powerful antibiotics are losing efficacy at an uncertain rate due to the rapid development of bacterial defenses, gene exchange and their multidrug resistance. Treatment of pathogenic microbial infections is commonly treated with drugs, such as antibiotics, but abuse of antibiotics can cause the appearance of superbacteria, making it difficult to kill the bacteria. The bacterial drug resistance has been a very serious problem in our human beings, and the increasing drug resistance causes the drug to be unable to be effectively treated. Therefore, the research on the traditional Chinese medicine active substances is to solve the fundamental problem of drug resistance of bacteria.

Pseudomonas Aeruginosa (PA), also known as Pseudomonas aeruginosa, produces a green color to the wound during infection. The pseudomonas aeruginosa is gram-negative aerobic bacillus which belongs to pseudomonas and is a gamma subclass of bacteria. The pseudomonas aeruginosa is not only widely distributed in various water, air, soil and other natural world, but also exists in the skin, respiratory tract and intestinal tract of normal people, has low requirement on nutrition, and is good at utilizing various carbon sources and ammoniated compounds as nitrogen sources. Pseudomonas aeruginosa can infect the blood, respiratory and urinary tracts.

Quorum Sensing (QS) is an information exchange mechanism used by microorganisms to control and regulate the behavior of populations, during which extracellular chemicals are produced and released as signals to seeds. QS is a bacterial cell density-dependent mechanism involving the production and release of several different chemical classes of small diffusible signal molecules, called Autoinducers (AIs), that mediate cell-cell communication. As the number of bacterial cells increases, the concentration of these auto-inducer (AI) in the surrounding medium also increases. Once the concentration of AI reaches a certain threshold, the bacteria initiate collective behavior.

The biofilm and virulence factors produced by pseudomonas aeruginosa are largely co-regulated by three systems of the QS system (las, rhl and pqs). Quorum sensing systems regulate different microbial behaviors such as biofilm and virulence factor seed under environmental stimuli, for example: elastase, pyocyanin, rhamnolipids and siderophores and their ability to form biofilms on biological (non-biological) surfaces. These features in P.aeruginosa are largely controlled by the QS system. Quorum sensing relies on a network of autoinducer synthase, autoinducer, associated autoinducer receptors, and downstream signal transduction elements, mediated by synthesis, release, accumulation, and a full complement of autoinducers. The las and rhl system primarily regulates elastase, pyocin, and rhamnolipids. The las and pqs system is closely related to the motility and pyocyanin production of P.aeruginosa. QS is involved in controlling the production of several virulence factors and therefore, modulation of the QS system would be an attractive target for resistance to antibiotics.

Mustard seeds (Brassicajuncea seeds) are seeds of Brassica plants of the Brassicaceae family (Cruciferae). The traditional medicine considers that: mustard seed, semen Brassicae Junceae, pungent in flavor and warm in nature, enters lung and stomach meridians. Excels at benefiting qi and eliminating phlegm, warming middle energizer and dispelling cold, unblocking collaterals and relieving pain, and is commonly used for treating phlegm-fluid retention cough and asthma, fullness and pain in chest and hypochondrium, numbness of limbs, arthralgia, deep-rooted carbuncle of yin type, pyogenic infections, etc. And the products of mustard seed processing are very popular condiments. Therefore, mustard seeds are receiving more and more attention and attention in terms of medicinal value. It can be used for controlling food-borne pathogens and improving food safety, and can be used for developing new drugs.

Disclosure of Invention

In view of the above, the present invention provides a drug for inhibiting bacterial quorum sensing, which is capable of effectively reducing the formation and pathogenicity of bacterial biofilms and is not easy to generate bacterial drug resistance.

The technical scheme is as follows:

a medicine for inhibiting bacterial quorum sensing system mainly contains mustard seed extract.

Further, in the drug for inhibiting the bacterial quorum sensing system, the effective dose of the mustard seed extract is 10-80 wt%.

Furthermore, the medicine for inhibiting the bacterial quorum sensing system also comprises pharmaceutically acceptable auxiliary materials.

Furthermore, the dosage form of the medicament for inhibiting the bacterial quorum sensing system is tablets, capsules, granules or dropping pills.

Further, a medicament as described above for inhibiting a bacterial quorum sensing system, said bacterial quorum sensing system comprising las and pqs signaling pathways.

Further, the above drug for inhibiting a bacterial quorum sensing system, wherein the bacterial quorum sensing system is formed by a bacterial biofilm.

Further, the above drug for inhibiting bacterial quorum sensing system is produced by secretion of extracellular polysaccharide drug.

Furthermore, the drug for inhibiting the bacterial quorum sensing system is produced by inhibiting secretion of a bacterial pyocin drug.

Furthermore, in the above drug for inhibiting a bacterial quorum sensing system, the mustard seed extract is a mustard seed ethanol extract, a mustard seed chloroform extract, a mustard seed petroleum ether extract, a mustard seed ethyl acetate extract, a mustard seed n-butanol extract, or a mustard seed extract

Water extract, steam distilled extract of mustard seed, and CO of mustard seed₂At least one of the supercritical extracts.

The mustard seed extract is at least one of a mustard seed ethanol extract, a mustard seed chloroform extract, a mustard seed petroleum ether extract, a mustard seed ethyl acetate extract, a mustard seed n-butanol extract, a mustard seed water extract and a mustard seed volatile oil.

Preferably, the ethanol extract of mustard seeds is a 45% ethanol extract and/or a 75% ethanol extract of mustard seeds and/or a 95% ethanol extract of mustard seeds.

Preferably, the preparation method of the mustard seed extract is one of the following methods:

(A) taking dried mustard seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking for 0-12 hours, performing ultrasonic-assisted extraction, removing insoluble substances, and drying to obtain a mustard seed extract;

(B) taking dried mustard seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking for 0-12 hours, performing reflux extraction, removing insoluble substances, and drying to obtain a mustard seed extract;

(C) taking dried mustard seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking at 4-25 ℃ for 0-72 hours, removing insoluble substances, and drying to obtain a mustard seed extract;

(D) taking dried mustard seeds, crushing, sieving with a 40-100-mesh sieve, adding an extraction solvent, soaking for 0-12 hours, performing microwave-assisted extraction, removing insoluble substances, and drying to obtain a mustard seed extract;

(E) taking dried mustard seeds, crushing, sieving with a 40-100-mesh sieve, adding deionized water, soaking for 0-12 h, extracting for 15-60min at the temperature of 40-60 ℃ under the condition of 700W, collecting an extracting solution, and removing water by using anhydrous sodium sulfate to obtain the mustard seed volatile oil.

(F) Crushing dried mustard seeds, sieving with a 40-100 mesh sieve, performing supercritical carbon dioxide extraction by using absolute ethyl alcohol as an entrainer, collecting the extract, and freeze-drying to obtain the mustard seed CO₂Beyond the limit of the extract.

Preferably, the extraction solvent in the method is 30-100% ethanol solution, water, chloroform, petroleum ether, ethyl acetate, n-butanol, and CO₂At least one of (1).

Preferably, the insoluble matter removal is implemented by filtering and collecting filtrate or centrifuging and collecting supernatant.

Preferably, the drying is reduced pressure drying, vacuum drying or freeze drying.

Preferably, the adding amount of the extraction solvent is 10-30 times of the weight of the mustard seeds, the extraction temperature is 10-150 ℃, the extraction times are 2-6, and the extraction time is 1-72 hours each time.

Preferably, the bacteria comprise pseudomonas aeruginosa, more preferably pseudomonas aeruginosa PAO 1.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the dosage form of the medicament is an oral preparation, preferably granules.

Preferably, the effective dose of the mustard seeds in the medicine is 20-80 wt%, more preferably 40-70 wt%, and the auxiliary material is preferably 20-80 wt%, more preferably 35-65 wt%.

The invention utilizes the mustard seed extract to inhibit a biological membrane and a virulence factor generated by pseudomonas aeruginosa. First, the inhibitory effect of mustard seed extract on pseudomonas aeruginosa PAO1 biofilm was studied and analyzed using confocal laser microscopy. Secondly, the inhibition effect of the mustard seed extract on pseudomonas aeruginosa PAO1 secreted pyocin and exopolysaccharide was studied. Finally, the effect of mustard seed extract on the fluorescent reporter strains PAO1-lasB-gfp and PAO1-pqsA-gfp was investigated.

The invention has the following advantages:

the invention takes the pseudomonas aeruginosa as a research object to carry out the inhibition experiment of the biomembrane and the virulence factor, and the result shows that the mustard seed ethanol extract, the mustard seed chloroform extract, the mustard seed petroleum ether extract, the mustard seed ethyl acetate extract, the mustard seed n-butanol extract and the mustard seed volatile oil can inhibit the formation of the pseudomonas aeruginosa PAO1 biomembrane and the release of the virulence factor, the research on the inhibitory activity of a bacterial quorum sensing system is carried out by using a PAO1-lasB-gfp and a PAO1-pqsA-gfp green fluorescent protein reporter strain, and the result shows that the mustard seed extract can inhibit a las and pqs quorum sensing system of pseudomonas aeruginosa, which indicates that the mustard seed extract can further reduce the formation and pathogenicity of a bacterial biofilm by inhibiting the bacterial quorum sensing system, and is not easy to generate the effect of bacterial drug resistance.

The invention provides a mustard seed extract used as a medicament for inhibiting a bacterial quorum sensing system, which has inhibition effects on biomembranes, pyocyanin, exopolysaccharides, lasB, pqsA signal pathways and the like and application thereof. The drug containing the mustard seed extract can prevent the formation of early pseudomonas aeruginosa biomembranes, and the thickness and biomass of the biomembranes can be obviously reduced through a light-gathering confocal microscope. The extract also exhibits dose-concentration dependent inhibition of QS-regulated pyocins and exopolysaccharides. In the analysis of the lasB-gfp and pqsA-gfp reporter genes, the drug containing the mustard seed extract has better inhibition effect, which indicates that the extract can effectively regulate the las and pqs systems. In addition, treatment with mustard seed extract is a potential approach to reduce the pathogenesis of pseudomonas aeruginosa infection and inhibit bacterial growth at lower concentrations. The mustard seed extract is used as a potential anti-biofilm and quorum sensing inhibitor, and can reduce the drug resistance of bacteria so as to achieve the purpose of treating bacterial infection.

Drawings

FIG. 1 is a graph of the results of various extracts of mustard seeds on inhibition of PAO1 biofilm;

FIG. 2 is a graph of the inhibition of PAO1 biofilm by mustard seed extract observed by confocal laser microscopy;

FIG. 3 is a graph showing the results of inhibition of pyocin by mustard seed extract;

FIG. 4 is a graph showing the results of inhibition of exopolysaccharides by mustard seed extract;

FIG. 5 is a graph showing the results of the effect of mustard seed extract on the strains PAO1-lasB-gfp and PAO 1-pqsA-gfp.

FIG. 6 shows the protective effect of different dosage forms of mustard seed extract drugs on mice infected with Pseudomonas aeruginosa;

FIG. 7 is a graph of LC-MS total TIC in negative ion mode for mustard seed extract of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, technical solutions in embodiments of the present invention will be clearly and completely described below.

The raw materials used in the examples of the invention are all commercially or self-made, wherein the PAO1-pqsA-gfp, the PAO1-lasB-gfp green fluorescent protein reporter strain and the Pseudomonas aeruginosa PAO1 are all obtained from the university of Physician south America.

Example 1: preparation of 95% ethanol extract of mustard seed

(1) Weighing 100g of mustard seeds, fully crushing by using a crusher, sieving by a 60-mesh sieve,

(2) according to the material-liquid ratio of 1: 20, adding 2L of 95% (v/v) ethanol solution, performing ultrasonic-assisted extraction with ultrasonic power of 350W, extracting at 50 deg.C for 30min, repeating the extraction for 3 times,

(3) filtering to remove insoluble substances, mixing the three extractive solutions, and concentrating under reduced pressure to about 10mL

(4) Placing the concentrated solution in a refrigerator at-80 deg.C for pre-freezing, and freeze-drying to obtain powder, i.e. 1.95g of 95% ethanol extract of mustard seed.

Example 2: preparation of 75% ethanol extract of mustard seed

(1) Accurately weighing 100g of mustard seeds, crushing by a crusher, and sieving by a 60-mesh sieve

(2) According to the material-liquid ratio of 1: 20, adding 2L of 75% (v/v) ethanol solution, soaking for 8h, heating and refluxing for extraction for 3 times, wherein the extraction temperature is 80 ℃, and the extraction time is 2 h.

(3) Filtering to remove insoluble substances, mixing the extractive solutions for 3 times, testing, and concentrating to about 10 mL;

(4) pre-freezing the concentrated solution at-80 deg.C, and freeze drying to obtain powder, to obtain mustard seed 75% ethanol extract 1.87 g.

Example 3: preparation of 45% ethanol extract of mustard seed

(1) Accurately weighing 100g of mustard seeds, crushing by using a crusher, and sieving by using a 60-mesh sieve;

(2) according to the material-liquid ratio of 1: 30, adding 3L of 45% (v/v) ethanol solution, leaching at 4-25 ℃ for 72h, filtering to remove insoluble substances, and concentrating under reduced pressure to about 10mL

(3) Pre-freezing the above concentrated solution at-80 deg.C, and freeze drying to obtain powder, to obtain mustard seed 45% ethanol extract 2.19 g.

Example 4: preparation of chloroform, petroleum ether, ethyl acetate and n-butanol extract of mustard seed

(1) Weighing 500g of mustard seeds, fully crushing by using a crusher, sieving by a 60-mesh sieve,

(2) taking 4 clean conical flasks, adding 100g of mustard seed powder into each conical flask,

(3) each conical flask is filled with a liquid material according to the ratio of 1: 10, respectively adding 1L of chloroform, petroleum ether, ethyl acetate and n-butanol, sealing, soaking for 8h, performing ultrasonic-assisted extraction with ultrasonic power of 350W, extracting at 50 deg.C for 30min, repeatedly extracting for 3 times,

(4) filtering to remove insoluble substances, mixing the three extractive solutions, and concentrating under reduced pressure to about 10mL

(5) Placing the concentrated solution in a refrigerator at-80 deg.C for pre-freezing, and freeze drying to obtain powder, to obtain mustard seed chloroform extract 1.76g, petroleum ether extract 1.65g, ethyl acetate extract 2.03g, and n-butanol extract 1.78 g.

Example 5: preparation of mustard seed volatile oil

(1) Accurately weighing mustard seed 1k g, pulverizing with a pulverizer, and sieving with a 60-mesh sieve;

(2) taking mustard seed powder in a round-bottom flask, and mixing the mustard seed powder and the round-bottom flask according to a material-liquid ratio of 1: 10 adding 10L of deionized water and adding zeolite;

(3) the round flask is connected with a volatile oil extractor and a condensing device, and is heated by a constant temperature electric heating jacket at 100 deg.C for 5 hr, and oil layer in the volatile oil extractive solution is collected

(4) Removing excessive water from the above volatile oil extractive solution with sodium sulfate to obtain mustard seed volatile oil 16.21g

Example 6: supercritical CO₂Mustard seed extract prepared by extraction method

(1) 100g of mustard seeds are crushed and sieved by a 60-mesh sieve.

(2) Adding mustard seed powder into an extraction kettle, setting the extraction pressure at 36MPa and the temperature at 45 ℃, and adding CO₂The flow rate is 2L/min, and supercritical carbon dioxide extraction is carried out for 60 min.

(3) Separating the extract, extracting for 60min to obtain the second extract, and mixing the two extracts to obtain 1.28g of semen Brassicae Junceae extract.

Example 7: microwave-assisted extraction method for preparing mustard seed extract

(1) 100g of mustard seeds are crushed, sieved by a 60-mesh sieve and distilled water.

(2) Placing the mustard seed powder in a round-bottom flask, adding distilled water with the weight 8-20 times that of the mustard seeds, oscillating and uniformly mixing, placing in a microwave digestion instrument, extracting at the extraction power of 300-700W and the temperature of 40-60 ℃, extracting for 15-60min respectively according to the set parameters, and performing reflux extraction for 2 h.

(3) After the refluxed extract was cooled, the extract was dried over anhydrous sodium sulfate overnight, and insoluble matter was removed by filtration.

(4) Concentrating the filtrate at 45 deg.C under reduced pressure, performing parallel experiments for three times, and mixing the three extractive solutions. To obtain 1.59g of mustard seed extract.

Example 8: simultaneous distillation and extraction method for preparing mustard seed extract

(1) 100g of mustard seeds are crushed and sieved by a 60-mesh sieve.

(2) One end of the distillation extraction device is connected with a round-bottom flask containing 100g of mustard seed samples and 800ml of distilled water, and a constant-temperature electric heating jacket is used for heating; the other end of the device is connected with a round-bottom flask containing 80ml of dichloromethane, and the round-bottom flask is placed in a constant-temperature water bath kettle to be heated, the temperature of the water bath is 60 ℃, and meanwhile, the round-bottom flask is distilled and extracted for 2 hours.

(3) After extraction was complete, filtration was carried out and the filtrate was concentrated under reduced pressure at 45 ℃. The experiments are carried out in parallel for three times, and the extracting solutions obtained by three times are combined. To obtain 1.49g of mustard seed extract.

Example 9: inhibition effect of mustard seed extract extracted by different solvents on pseudomonas aeruginosa biomembrane

Test samples: mustard seed extracts extracted with different solvents were weighed out and prepared into a 32mg/mL stock with 100% DMSO, Azithromycin (AZM) as a positive control and 0.2% DMSO as a solvent control (DMSO + Blank).

The experimental method comprises the following steps:

(1) selecting bacteria, performing monoclonal culture in LB broth culture medium at 37 deg.C overnight for about 12 h;

(2) measuring OD with 200 μ L of cultured bacterial liquid for 12h₆₀₀Diluting the bacterial liquid to OD with ABTGC culture medium₆₀₀＝0.02；

(3) The samples (extracts and antibiotics) were dissolved with molecular biological DMSO reagents and diluted to the measured concentration with ABTGC medium. The DMSO group, Blank group, and drug group were set up, wherein 100. mu.L of drug and 100. mu.L of diluted bacterial suspension were added to the corresponding wells. The wells were plated with 6-10 replicates of 200. mu.L of ABTGC medium to prevent edge effects due to evaporation. Culturing overnight in an incubator at 37 ℃ for 24 h;

(4) carefully sucking out the bacterial liquid, washing each hole with 200 μ L PBS for 2 times, and drying; adding 200 μ L of methanol solution into each well, and fixing at room temperature for 20 min;

(5) sucking methanol away, drying, adding 200 μ L of filtered 0.1% crystal violet, and dyeing for 15 min;

(6) the dye was aspirated off, washed three times with PBS until no purple color was present;

(7) after drying, 200. mu.L of 33% glacial acetic acid is added to dissolve the crystal violet dye, and OD is measured after shaking up₅₇₀。

(8) Calculating the formula:

biofilm inhibition rate [ (DMSO)_{OD value}-Blank_{OD value}) - (drug group)_{OD value}-Blank_{OD value})]/(DMSO_{OD value}-Blank_{OD value})×100％

The experimental results are as follows: as can be seen from the experimental results of FIG. 1, the mustard seed extracts extracted with different solvents inhibited the formation of Biofilm (Biofilm). Wherein the extract of mustard seed with 95% ethanol, the extract of mustard seed with petroleum ether, the extract of mustard seed with ethyl acetate, and the CO of mustard seed₂The inhibition effect of the supercritical extract is most remarkable.

Example 10: laser confocal microscope observation of inhibition effect of mustard seed extract on biological membrane

Test samples: mustard seed extract was weighed out and made up into 32mg/mL stock with 100% DMSO, with 0.2% DMSO as a solvent control (DMSO + Blank).

The experimental method comprises the following steps:

(2) measuring OD with 200 μ L of liquid-cultured bacterial liquid₆₀₀The bacterial liquid was diluted to OD by adding LB broth medium₆₀₀Is 0.06;

(3) the samples (extracts and antibiotics) were dissolved with molecular biological DMSO reagents and diluted to the determined concentration with LB medium. And setting a DMSO group and a drug group, wherein 1000 mu L of drugs and 1000 mu L of bacteria liquid are respectively added into corresponding glass bottom culture dishes. Culturing overnight in an incubator at 37 ℃ for 24 h;

(4) after culturing for 24h, using a pipette gun to absorb the bacterial liquid in the pore plate, taking 2mL of PBS buffer solution to wash off the redundant bacterial liquid, and repeating for 3 times;

(5) mu.L of SYTO9/PI dye was stained in the dark for 30min, washed off with 2000. mu.L of PBS buffer and repeated 3 times. Biofilm formation was observed by CLSM.

The experimental results are as follows: the effect of mustard seed extract on pseudomonas aeruginosa biofilm formation was observed by laser scanning confocal microscopy (CLSM), staining with SYTO9/PI dye, SYTO9 being a green fluorescent nucleic acid stain and PI being an propidium iodide red fluorescent nucleic acid stain.

The growth of the biofilm was further verified under CLSM. The bacteria were incubated with mustard seed extract for 24 hours and stained with SYTO9 and PI. As shown in FIG. 2, it was observed that Pseudomonas aeruginosa can form thick biofilm after 24 hours of culture in the control group (DMSO group), green fluorescence is high, and total amount of organisms is high. However, the addition of the mustard seed extract significantly reduced biofilm formation and produced significantly thinner, flaky biofilms, and the mustard seed extract was very effective at 32 μ g/mL treatment, with significantly less biofilm mass, dispersed bottom biofilm, reduced green fluorescence, and more red fluorescence observed from the 3D plot. Laser confocal experiments show that the mustard seed extract can effectively inhibit the generation of a biological film.

Example 11: inhibition of pyocyanin by mustard seed extract

Test samples: mustard seed extract was weighed out and made into a 32mg/mL stock with 100% DMSO, Azithromycin (AZM) as a positive control and 0.2% DMSO as a solvent control (DMSO + Blank).

The experimental method comprises the following steps:

(2) measuring OD with 200 μ L of liquid-cultured bacterial liquid₆₀₀The bacterial liquid was diluted to OD by adding LB broth medium₆₀₀Is 0.02;

(3) diluting the compound to working concentration with prepared bacterial liquid, adding 50mL into sterilized conical flask, respectively adding 50 μ L of medicinal solution, and covering with air-permeable plug. Shaking and culturing in a shaker at 37 deg.C and 200rpm/min for 24 hr;

(4) after 24h of culture, the bacterial liquid is measured for 3 times by an ultraviolet spectrophotometer, wherein the wavelength is 600 nm;

(5) respectively putting 8.5mL of the bacterial liquid into 10mL centrifuge tubes (4 tubes are taken for each sample), and centrifuging for 10min (10000rpm/min) at 4 ℃;

(6) the supernatant of each sample was collected in 8mL and placed in a 50mL centrifuge tube, and 8mL chloroform was added as required (chloroform: supernatant 1: 4), shaken well, vortexed for 5min, and centrifuged at 2000r/min for 10 min. Then, 2mL of the chloroform layer (lower layer) was removed by using a pipette, and 0.2M HCl in an amount of 0.3 time the volume of chloroform (0.6mL) was added to a 10mL centrifuge tube, and 0.6mL of the upper layer was removed, and OD was measured using an ultraviolet spectrophotometer₅₂₀。

The experimental results are as follows:

as can be seen from fig. 3, the mustard seed extract inhibited the secretion of pyocin (Pyocyanin) by pseudomonas aeruginosa. A reduction in the production of pyocins was observed and was dose-dependent when different concentrations of mustard seed extract were added to the pseudomonas aeruginosa culture compared to the drug-free treatment group. The effect is best when the concentration is 32 mug/mL, thereby proving that the mustard seed extract has the effect of resisting pseudomonas aeruginosa and can inhibit the secretion of pseudomonas aeruginosa pyocin.

Example 12: inhibition of exopolysaccharides by mustard seed extracts

The experimental method comprises the following steps:

(2) measuring OD with 200 μ L of liquid-cultured bacterial liquid₆₀₀The bacterial liquid was diluted to OD by adding LB broth medium₆₀₀Is 0.01;

(4) after the bacteria are cultured for 24 hours, centrifuging 6mL of bacterial liquid in a high-speed centrifuge for 10min, controlling the temperature at 4 ℃ and the rotating speed at 10000 rpm/min;

(5) sucking 5mL of supernatant with a pipette gun, placing in a 50mL centrifuge tube, adding 15mL of anhydrous ethanol, placing in a 4 ℃ refrigerator, standing for 12h, centrifuging in a high-speed centrifuge for 15min at 4 ℃ and 10000rpm/min, and separating polysaccharide. Removing the supernatant, gently washing the residual supernatant with anhydrous ethanol (taking care not to wash out the precipitate), collecting polysaccharide precipitate and drying for later use;

(6) measuring the content of polysaccharide generated by pseudomonas aeruginosa by a phenol-sulfuric acid method, accurately weighing 5g of phenol powder, adding 100mL of deionized water, and preparing for later use;

(7) adding dried polysaccharide into 5mL deionized water for redissolving, adding 0.5mL phenol, 1mL redissolved solution, adding 0.5mL phenol solution (polysaccharide: phenol: 2:1), adding 2.5mL concentrated sulfuric acid, shaking, placing in 90 deg.C water bath, adding for 30min, cooling, and determining OD₄₉₀。

The experimental results are as follows:

as can be seen from fig. 4. In comparison to the inhibition by DMSO, an effective reduction in Exopolysaccharide (EPS) production was observed when treated with 4, 8, 16 and 32. mu.g/mL mustard seeds. The mustard seed extract is proved to have the effect of resisting pseudomonas aeruginosa and inhibiting the secretion of extracellular polysaccharide of bacteria, thereby reducing the pathogenicity of the bacteria and being capable of being used as a novel quorum sensing inhibitor.

Example 13: effect of mustard seed extract on the strains PAO1-lasB-gfp and PAO1-pqsA-gfp

Test samples: mustard seed extract was weighed out and made up into 32mg/mL stock with 100% DMSO, with 0.2% DMSO as solvent control (DMSO).

The experimental method comprises the following steps:

(1) selecting single colonies of PAO1-lasB-gfp and PAO1-pqsA-gfp, putting the single colonies into LB broth added with gentamycin resistance, and incubating overnight at 37 ℃ for culturing for about 12 hours;

(2) measuring OD with 200 μ L of liquid-cultured bacterial liquid₆₀₀Adding culture medium to dilute the bacterial liquid to OD₆₀₀Is 0.05;

(3) preparing the compound to working concentration by using diluted bacterial liquid, adding the compound into a 96-well plate, simultaneously setting a Control group and a Blank group, placing the compound in an enzyme-linked immunosorbent assay (ELISA), incubating the compound for at least 16h at 37 ℃, and measuring OD once every 15min₆₀₀To determine whether the extract has an effect on fluorescence.

The experimental results are as follows:

as can be seen from FIG. 5, in the experiments of inhibiting the PAO1-lasB-gfp and PAO1-pqsA-gfp fluorescent reporter strains, the mustard seed extract has an inhibitory effect on the expression of the lasB-gfp and pqsA-gfp reporter strains. After 4h of culture, the expression levels of lasB-gfp and pqsA-gfp in each group began to increase; in the 5-16h of the logarithmic growth phase of the strain, the mustard seed extract has obvious inhibition effect on the fluorescence of the strains lasB-gfp and pqsA-gfp, which shows that the mustard seed extract can further inhibit the secretion of pyocyanin and reduce the pathogenicity of bacteria by inhibiting the bacterial quorum sensing las system and pqs system.

The experiment results of the invention prove that the mustard seed extract has better effect on inhibiting the growth of bacterial biofilms, and can be used as a potential anti-biofilm and QS inhibitor.

Example 14: anti-pseudomonas aeruginosa quorum sensing granules

The present example provides a pseudomonas aeruginosa resistant quorum sensing particle, which has the following formula:

the preparation steps of the pseudomonas aeruginosa resistant granules are as follows:

(1) firstly, uniformly mixing a mustard seed extract sample with lactose, mannitol and CMS-Na;

(2) then, adding the prepared 10% starch slurry into the mixture, and sieving the mixture by a 16-mesh sieve to prepare a soft material;

(3) drying at 60 ℃ in a vacuum drying oven, and finishing granules to obtain the anti-pseudomonas aeruginosa quorum sensing granules.

Example 15: anti-pseudomonas aeruginosa quorum sensing capsule

The embodiment of the invention provides a pseudomonas aeruginosa-resistant capsule, which comprises the following formula:

the preparation method of the pseudomonas aeruginosa resistant capsule comprises the following steps:

(1) firstly, mixing the mustard seed extract with lactose for 8-20 minutes;

(2) adding microcrystalline cellulose and mixing for 7-18 minutes;

(3) adding talcum powder and mixing for 4-8 min;

(4) and filling the mixture into a gelatin capsule shell, namely the pseudomonas aeruginosa resistant capsule.

Example 16: anti-pseudomonas aeruginosa quorum sensing dropping pill

The embodiment of the invention provides a pseudomonas aeruginosa quorum sensing resistant dropping pill, which comprises the following components:

30mg of mustard seed extract;

60mg of polyethylene glycol;

the preparation method of the anti-pseudomonas aeruginosa dropping pill comprises the following steps:

(1) respectively weighing the mustard seed extract and polyethylene glycol, heating the polyethylene glycol to be molten, adding the mustard seed extract under stirring, and uniformly mixing to obtain a mixed solution;

(2) dripping pills: adding the mixed solution into a dripping device of a dripping pill machine, dripping into condensate through a dripper, adjusting a temperature control system of the dripping pill machine, keeping the temperature of the dripper of the dripping pill machine at 75 ℃, the gradient temperature of the condensate at 45 ℃, and the inner diameter of a dripping pipe opening at 4 mm;

(3) wiping pills: taking out the pills, and removing surface condensate to obtain the anti-pseudomonas aeruginosa dropping pills.

Example 17: anti-pseudomonas aeruginosa group tablet of mustard seed extract

The invention provides a pseudomonas aeruginosa-resistant tablet, which comprises the following components:

the preparation steps of the pseudomonas aeruginosa resistant tablet are as follows:

(1) mixing the mustard seed extract with lactose for 5-15 min;

(2) adding 10% starch slurry to make soft material, sieving with 14 mesh sieve, granulating, drying, sieving with 12 mesh sieve, and grading;

(3) then adding the crospovidone and the magnesium stearate, and mixing for 3-6 minutes;

(4) and tabletting after uniformly mixing to obtain the pseudomonas aeruginosa resistant tablet.

Implementation 18: the 4 preparations have the protective effect on mice infected by pseudomonas aeruginosa by anti-pseudomonas aeruginosa medicines.

Test samples: the mustard seed extract anti-pseudomonas aeruginosa quorum sensing granules prepared in example 14, the mustard seed extract anti-pseudomonas aeruginosa quorum sensing capsules prepared in example 15, and the mustard seed extract anti-pseudomonas aeruginosa quorum sensing dropping pills prepared in example 16 were taken, the mustard seed extract anti-pseudomonas aeruginosa quorum sensing tablets prepared in example 17 were taken, and a DMSO solution was prepared into a 32mg/mL mother liquor for standby.

The experimental method comprises the following steps: the pseudomonas aeruginosa is inoculated to LB culture medium containing the drugs, after overnight culture, thalli are collected by centrifugation, PBS thalli are used for resuspending and diluting to 1 multiplied by 108CFU/mL, DMSO is used as a negative control group, 60 male mice with the age of 7 weeks are randomly divided into 6 groups, 10 mice in each group, 100 mu L of bacterial resuspension liquid after different drug forms of drug treatment is injected into the abdominal cavity of each group of mice, pure PBS solution is used as a blank control group for injection, and the survival rate of the mice is observed within 1 week.

The experimental results are as follows: as shown in FIG. 6, the survival rate of the mice in the blank control group is 100%, the survival rate of the mice in the negative control group is only 20%, and different mustard seed anti-pseudomonas aeruginosa drugs play a certain role in protecting infected mice, wherein the effect of the mustard seed extract anti-pseudomonas aeruginosa quorum sensing granules is the best, and the survival rate of the mice reaches 80%.

Example 19: LC-MS analysis of mustard seed extract

The experimental method comprises the following steps: qualitative analysis of the mustard seed extract of example 1 was performed by Q-active quadrupole-electrostatic orbitals high resolution mass spectrometer using a hyperspil GOLD C18 column, mobile phase a: 0.1% aqueous formic acid, mobile phase B: acetonitrile solution, chromatographic conditions: 0-5 min, 5% → 15% acetonitrile, 5-15 min, 15% → 30% acetonitrile, 15-25 min, 30% → 100% acetonitrile, 25-30 min, 100% acetonitrile, the mass spectrum scanning mode is a positive and negative ion scanning mode, the scanning range is as follows: 70-1000 m/z. The experimental results are as follows: as shown in fig. 7.

Claims

1. A medicament for inhibiting a bacterial quorum sensing system, wherein the medicament consists essentially of a mustard seed extract.

2. The drug for inhibiting a bacterial quorum sensing system according to claim 1, wherein the effective amount of the mustard seed extract is 10-80 wt%.

3. The medicament for inhibiting a bacterial quorum sensing system according to claim 1, further comprising a pharmaceutically acceptable excipient.

4. The medicament for inhibiting a bacterial quorum sensing system according to claim 1, wherein the dosage form is tablets, capsules, granules or dropping pills.

5. The medicament of claim 1, wherein the bacterial quorum sensing system comprises las and pqs signaling pathways.

6. The agent of claim 1, wherein the bacterial quorum sensing system is formed by a bacterial biofilm.

7. The agent of claim 1, wherein the bacterial quorum sensing system is produced by secretion of a bacterial exopolysaccharide drug.

8. The agent of claim 1, wherein the bacterial quorum sensing system is one that inhibits secretion of a bacterial pyocin drug.

9. The drug for inhibiting a bacterial quorum sensing system according to claim 1, wherein the mustard seed extract is a mustard seed ethanol extract, a mustard seed chloroform extract, a mustard seed petroleum ether extract, a mustard seed ethyl acetate extract, a mustard seed n-butanol extract, a mustard seed aqueous extract, a mustard seed water vapor distillation extract, a mustard seed CO extract₂At least one of the supercritical extracts.