Application of alpha-mangostin or pharmaceutically acceptable salt thereof in preparation of pseudomonas aeruginosa quorum sensing system inhibitor

Abstract

The invention belongs to the technical field of biological medicine, and relates to application of alpha-mangostin or pharmaceutically acceptable salt thereof in preparation of a pseudomonas aeruginosa quorum sensing system inhibitor. The invention discloses alpha-mangostin which can be used as a PQS quorum sensing system inhibitor of pseudomonas aeruginosa. The Pseudomonas aeruginosa strain can inhibit the expression of a Pseudomonas aeruginosa pqsA operon, reduce the generation of pyocyanine, inhibit the formation of a biological membrane and reduce the athletic ability of the Pseudomonas aeruginosa, and also can reduce the expression of hydrogen cyanide, elastase, lectin and rhamnolipid synthetic genes, thereby weakening the toxicity and pathogenicity of the Pseudomonas aeruginosa. In-depth study finds that the action target of the alpha-mangostin is the transcription activator protein PqsR of a PQS quorum sensing system. The research proves that the natural active ingredient alpha-mangostin can obviously reduce the pathogenicity of the pseudomonas aeruginosa to crop and human lung cancer cells A549, and more thoughts are provided for prevention and control of pseudomonas aeruginosa diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of α-dextrin or its pharmaceutically acceptable salt in the preparation of Pseudomonas aeruginosa quorum sensing system inhibitors. Background Technology

[0002] Pseudomonas aeruginosa ( Pseudomonas aeruginosa *Pseudomonas aeruginosa* is a Gram-negative pathogen widely distributed in soil, water, and medical environments. It has been confirmed that *Pseudomonas aeruginosa* is involved in various human conditional pathogens and is also a plant pathogen, causing diseases in many economically important plants, including soybeans, lettuce, pumpkins, ginseng, and poplars. Quorum sensing (QS) is a cell-to-cell communication system in bacteria. Bacteria sense the density of their colony by secreting signaling molecules, thereby coordinating and regulating pathogenic behavior. Because the quorum sensing systems of different bacterial species are specific, developing quorum sensing inhibitors (QSIs) targeting specific pathogens allows for precise intervention. Unlike traditional antibiotics, quorum sensing inhibitors weaken virulence by blocking bacterial signal transduction rather than directly killing the bacteria. This "de-weaponization" strategy significantly reduces the selective pressure of drug resistance.

[0003] The pathogenicity and drug resistance of *Pseudomonas aeruginosa* are closely related to its quorum sensing system. *P. aeruginosa* has three quorum sensing systems: LAS, RHL, and PQS. These systems synergistically regulate the expression of multiple virulence factors through specific signaling molecules and their receptors. The LAS system consists of LasI and LasR. LasI is the synthase for the signaling molecule, synthesizing N-(3-oxododecanoyl)-L-homoserine lactone, and LasR is the receptor for this signaling molecule. Similar to the LAS system, the RHL system consists of RhlI and RhlR. RhlI is the synthase for the signaling molecule, synthesizing N-butyryl-L-homoserine lactone, and RhIR is the receptor for this signaling molecule. In the PQS system, PhnA, PhnB, PqsA, PqsB, PqsC, PqsD, PqsE, and PqsH co-catalyze the synthesis of two signaling molecules: 2-heptayl-4-hydroxyquinoline (HHQ) and 2-heptayl-3-hydroxy-4-quinolone (PQS), with PqsR acting as the receptor for these signaling molecules. The three quorum sensing systems—LAS, RHL, and PQS—jointly regulate the production of virulence factors in *Pseudomonas aeruginosa*, including the extracellular protease LasA, elastase LasB, alkaline protease, hydrogen cyanide, exotoxin A, rhamnolipids, pyocyanin, and biofilms. Therefore, in-depth research into the mechanisms of the quorum sensing system will provide new insights for developing novel therapeutic strategies against *Pseudomonas aeruginosa* infections.

[0004] α-Mangostin, CAS number 6147-11-1, is a natural xanthonone compound. Current research indicates that α-Mangostin possesses antibacterial, anti-inflammatory, antioxidant, and antitumor effects. Studies have confirmed that α-Mangostin has strong inhibitory effects on Gram-positive bacteria (such as Staphylococcus aureus) and plant pathogens (such as Bacterium tumefaciens, the causal agent of rice bacterial blight). Furthermore, studies have shown that α-Mangostin can inhibit the migration and invasion of lung cancer cells, thereby inhibiting their survival, suggesting that α-Mangostin is a potential cancer therapeutic. However, there are currently no reports on the inhibition of the PQS quorum sensing system of Pseudomonas aeruginosa by α-Mangostin. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a natural small-molecule bacterial quorum sensing inhibitor for the prevention and control of diseases caused by the Gram-negative bacterium Pseudomonas aeruginosa, thus offering more options for solving the problem of Pseudomonas aeruginosa disease control.

[0006] On the one hand, the present invention provides the use of α-dextrin or a pharmaceutically acceptable salt thereof in the preparation of Pseudomonas aeruginosa quorum sensing system inhibitors.

[0007] Furthermore, in the aforementioned applications, the Pseudomonas aeruginosa quorum sensing system inhibitor is used for the prevention and / or treatment of Pseudomonas aeruginosa diseases.

[0008] Furthermore, in the aforementioned application, α-dextrin or a pharmaceutically acceptable salt thereof acts on the PQS system in the Pseudomonas aeruginosa quorum sensing system.

[0009] Furthermore, in the aforementioned application, the α-dextrin or a pharmaceutically acceptable salt thereof inhibits the gene of the PQS system in the Pseudomonas aeruginosa quorum sensing system. pqsA The expression.

[0010] Furthermore, in the aforementioned application, the target of action of the α-dextrin or a pharmaceutically acceptable salt thereof is the PqsR protein.

[0011] Furthermore, in the aforementioned applications, the α-dextrin and its pharmaceutically acceptable salts inhibit the virulence and / or pathogenicity of Pseudomonas aeruginosa.

[0012] Furthermore, in the application, the virulence is the ability of Pseudomonas aeruginosa to produce pyocyanin.

[0013] Furthermore, in the application, the virulence refers to the motility of Pseudomonas aeruginosa, which includes swarming ability, swimming ability, and shuffling ability.

[0014] Furthermore, in the application, the virulence refers to the ability of *Pseudomonas aeruginosa* to produce LasA protease, and the expression capacity of genes synthesizing hydrogen cyanide, elastase, lectin, and rhamnolipid in *Pseudomonas aeruginosa*.

[0015] Furthermore, in the application, the virulence is the ability of *Pseudomonas aeruginosa* to form a biofilm.

[0016] Furthermore, in the aforementioned application, the pathogenicity refers to the ability of *Pseudomonas aeruginosa* to infect plants.

[0017] Furthermore, in the application described, pathogenicity refers to the ability of *Pseudomonas aeruginosa* to infect animals.

[0018] The "prevention and treatment" described in this invention refers to the use of this product to prevent or reduce the accumulation of Pseudomonas aeruginosa, and to cure or reduce the virulence and / or pathogenicity of Pseudomonas aeruginosa in the presence of possible Pseudomonas aeruginosa or factors that lead to Pseudomonas aeruginosa infection.

[0019] In this invention, the term "pharmaceutical acceptable" means that it has no long-term harmful effects on the general health of the subject receiving the treatment.

[0020] In this invention, the term "pharmaceuticalally acceptable salt" refers to a salt that retains the biological efficacy of α-dextrin and has no adverse effects in biological or other aspects. A pharmaceutically acceptable salt is an inorganic or organic acid salt formed by converting a base group in a parent compound into a salt form, such as an amino acid group. It is generally prepared by reacting the parent compound with a common type of acid in a solvent system. Inorganic acids generally include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid; organic acids generally include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, or salicylic acid.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention provides a natural small-molecule bacterial quorum sensing inhibitor—α-dextrin—for the preparation of formulations that inhibit the quorum sensing system of *Pseudomonas aeruginosa*. This discovery offers more options for addressing the problem of *P. aeruginosa* drug resistance. α-dextrin can effectively interfere with the quorum sensing system of *P. aeruginosa*, particularly the PQS system, and inhibit gene... pqsAThe expression of α-dextrin is inhibited, thereby weakening the virulence and pathogenicity of *Pseudomonas aeruginosa*. This inhibitory effect is not only reflected in the production of pyocyanin by *P. aeruginosa*, but also affects its motility. Furthermore, the application of α-dextrin significantly reduces the pathogenicity of *P. aeruginosa* in Chinese cabbage and the human alveolar epithelial cell A549 host model, demonstrating its potential in plant and animal protection. Compared with chemical antibacterial agents, the plant natural product α-dextrin has the advantages of wide availability, high safety, and is less likely to induce bacterial resistance. Moreover, this invention, through in-depth research, reveals that the target of α-dextrin is the transcriptional activator protein PqsR of the PQS quorum sensing system, providing a solid theoretical basis for its further development and application, and offering new ideas for the control of *P. aeruginosa* diseases. Attached Figure Description

[0022] Figure 1 This diagram illustrates the effects of α-pachydextrin on gene expression in the PQS, RHL, and LAS systems of *Pseudomonas aeruginosa*. In the diagram, A represents the effect of 100 μg / mL α-pachydextrin on the QS gene expression. pqsA Effects on expression; B at 100 μg / mL α-trachytin on QS gene pqsA Effects on expression; C = 100 μg / mL α-trachytin on QS gene lasI The impact of expression.

[0023] rhlI This is a schematic diagram illustrating the effect of α-dextrin on the growth of Pseudomonas aeruginosa.

[0024] Figure 2 It is α-dextrin with bacterial OD 600 Value changes for Pseudomonas aeruginosa Figure 3 A schematic diagram illustrating the effects of gene expression.

[0025] pqsA Different concentrations of α-dextrin affect the PQS system genes of Pseudomonas aeruginosa. Figure 4 Schematic diagram illustrating the effect of α-retropine concentration on expression. Where A represents the effect of α-retropine concentration on expression in wild-type strains of *Pseudomonas aeruginosa*. pqsA Changes in expression levels; B represents the effect of α-retropine on wild-type strains of Pseudomonas aeruginosa. pqsA Half-inhibitory concentration (IC50) of expression level 50 ).

[0026] pqsA Different concentrations of α-dextrin affect the PQS system gene in strains overexpressing PqsR protein. Figure 5 Schematic diagram of the effect of expression. Where A represents the effect of increasing α-retropine concentration on wild-type and... pqsA Overexpression strainspqsR Changes in expression levels. B represents the effect of α-retropine on wild-type strains of Pseudomonas aeruginosa. pqsA Half-inhibitory concentration (IC50) of expression level 50 C represents α-dextrin against Pseudomonas aeruginosa. pqsA In overexpression strains pqsR Half-inhibitory concentration (IC50) of expression level 50 ).

[0027] pqsA This involves the molecular docking of α-retropine with PqsR protein and the quorum sensing system gene in *Pseudomonas aeruginosa* strains with point-mutated PqsR protein complementation by α-retropine. Figure 6 A diagram illustrating the impact.

[0028] pqsA This is a schematic diagram showing the changes in secondary structure and binding ability caused by the binding of α-dextrin to Pseudomonas aeruginosa PqsR protein and its mutants.

[0029] Figure 7 This is a schematic diagram illustrating the effect of α-retropine on pyocyanin synthesis in *Pseudomonas aeruginosa*. In the diagram, A represents the operon of the pyocyanin synthesis gene in *Pseudomonas aeruginosa*. Figure 8 1 B 1 C 1 D 1 E 1 F 1 G 1( phzA 1) Results of β-galactosidase activity assay in promoter-transcribed fusion strains; B represents the operon of the pyocyanin synthesis gene. phzA 2 B 2 C 2 D 2 E 2 F 2 G 2( phzA 2) The results of β-galactosidase activity detection of promoter transcription fusion reporter strains; C represents the quantitative results of Pseudomonas aeruginosa under different drug treatments.

[0030] phzA This is a schematic diagram illustrating the effect of α-dextrin on the motility of Pseudomonas aeruginosa. Specifically, A is a schematic diagram and statistical graph illustrating the effect of different drug treatments on the clustering motility of Pseudomonas aeruginosa; B is a schematic diagram and statistical graph illustrating the effect of different drug treatments on the swimming motility of Pseudomonas aeruginosa; and C is a schematic diagram and statistical graph illustrating the effect of different drug treatments on the scooting motility of Pseudomonas aeruginosa.

[0031] Figure 9This is a schematic diagram illustrating the effect of α-dextrin on LasA protease in Pseudomonas aeruginosa.

[0032] Figure 10 This is a schematic diagram illustrating the effects of α-retropine on the genes involved in the synthesis of hydrogen cyanide, elastase, lectin, and rhamnolipid in Pseudomonas aeruginosa.

[0033] Figure 11 This is a schematic diagram illustrating the effect of α-dextrin on biofilm formation in *Pseudomonas aeruginosa*. A and B represent the operons for extracellular polysaccharide synthesis in *Pseudomonas aeruginosa* under different drug treatments. Figure 12 and pel A schematic diagram showing the effect of expression; C is a schematic diagram showing the original integrated density statistical results of biofilm images under laser scanning confocal microscopy after different drug treatments of Pseudomonas aeruginosa; D is a biofilm image formed under different drug treatment conditions under laser scanning confocal microscopy.

[0034] psl This is a schematic diagram illustrating the effect of α-dextrin on the pathogenicity of *Pseudomonas aeruginosa*. Specifically, A is a schematic diagram and statistical graph showing the infectivity of *P. aeruginosa* on Chinese cabbage under different drug treatments; B is a graph showing the survival rate analysis of A549 cells under *P. aeruginosa* infection after different drug treatments; C is a graph showing the invasion rate of *P. aeruginosa* on A549 cells after different drug treatments; and D is a graph showing the invasion rate of *P. aeruginosa* on A549 cells after different drug treatments. Detailed Implementation

[0035] The technical solution of the present invention will be described below with reference to embodiments; however, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0036] The test materials in the following examples were sourced from the following sources: The Pseudomonas aeruginosa PAO1 used in this invention was obtained from the Pathogenic Microbiology Laboratory of Yan'an University. Other related strains were constructed based on PAO1 and used to explain this invention.

[0037] The α-mangostin and formononetin used in this invention were purchased from Chengdu Efa Biotechnology Co., Ltd., with the purity of α-mangostin and formononetin being 98%.

[0038] Using dimethyl sulfoxide (DMSO) as a solvent, α-dextrin and gentianin were prepared into a stock solution of traditional Chinese medicine monomers with a concentration of 10 mg / mL. MIC (minimum inhibitory concentration) analysis revealed that both α-dextrin and gentianin had MICs greater than 256 μg / mL against *Pseudomonas aeruginosa*, indicating that these two traditional Chinese medicine monomers do not affect the growth of *P. aeruginosa* or exert selective pressure on it within a specific concentration range (<256 μg / mL).

[0039] The primers and related restriction enzyme sites used in this invention are shown in Table 1 below: Table 1 Primer and related restriction enzyme site information

[0040] The underlined sequence represents the enzyme cleavage site.

[0041] Example 1 This embodiment describes the construction of a gene expression screening model for the Pseudomonas aeruginosa QS system.

[0042] (1) Construction of transcriptional fusion strains Step 1: Design primers using Pseudomonas aeruginosa genomic DNA as a template Figure 13 F / pqsA R, using this primer pair for PCR amplification. pqsA promoter sequence P pqsA The DNA fragment size is 1105 bp; Step 2, using pqsA I / Sal I to P Pst and plasmid mini-CTX- pqsA Perform double enzyme digestion; Step 3: Digest the P enzyme-digested P lacZ After purification, it was ligated into the plasmid mini-CTX- pqsA The cells were transformed into *E. coli* TG1 competent cells using a heat shock transformation method and screened on LB plates containing tetracycline (200 µg / mL, abbreviated as Tc200). In step 4, after PCR testing, the obtained colonies were used to extract plasmids to obtain recombinant vectors. lacZ I / Sal I performed enzyme digestion verification on the recombinant vector, obtaining the recombinant vector mini-CTX-P that showed a positive result. Pst - pqsA Step 5: Place the mini-CTX-P lacZ - pqsATransformed into S17-1 competent cells, yielding S17-1 mini-CTX-P lacZ - pqsA Step 6: The obtained recombinant strain S17-1 mini-CTX-P lacZ - pqsA Pseudomonas aeruginosa PAO1 was conjugated on LB agar plates at 37°C for 48 h. Subsequently, bacterial colonies were scraped from the plates, suspended in liquid LB, diluted, and spread onto LB agar plates containing kanamycin (Km, 30 µg / mL, abbreviated as Km30) and Tc200. The obtained colonies were streaked again on the LB agar plates to yield the transcriptional fusion strain: PAO1. lacZ ::mini-CTX-P attB - pqsA Using the same method, gene expression screening models for the other two quorum sensing systems of Pseudomonas aeruginosa (Rhl quorum sensing system and Las quorum sensing system) were obtained: PAO1 lacZ ::mini-CTX-P attB - rhlI and PAO1 lacZ ::mini-CTX-P attB - lasI .

[0043] (2) Detection of gene expression in quorum sensing system under the action of α-trachytin Step 1: Using argentin and DMSO as controls, α-argentin solution with a final concentration of 100 μg / mL was mixed with the transcriptional fusion reporter strain PAO1. lacZ ::mini-CTX-P attB - pqsA PAO1 lacZ ::mini-CTX-P attB - rhlI and PAO1 lacZ ::mini-CTX-P attB - lasI Co-incubate at 37°C and 200 rpm until OD. 600The value was 1.6; Step 2: Detection of β-galactosidase activity in the transcription fusion reporter strain. The method for detecting β-galactosidase activity in the transcription fusion reporter strain was to use o-nitrophenyl-β-D-galactosidase (ONPG) as a substrate: Take 20-100 µL of bacterial culture in the logarithmic phase, and add 420 µL of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH=7.0, 0.2% β-mercaptoethanol), 20 µL of chloroform, and 10 µL of 0.1% sodium dodecyl sulfate (SDS) sequentially to the bacterial culture. After rapid mixing for 20 s, incubate at 30℃ for 1 h. After incubation, add 100 µL of ONPG at a concentration of 4 mg / mL to the mixture to initiate the reaction. Terminate the reaction by adding 250 µL of 1 M Na2CO3, and record the reaction time. Finally, after centrifuging the mixture at 14000×g for 3 min, the supernatant was collected, and the OD of the mixture was measured. 420 and OD 550 Then, β-galactosidase activity is calculated in Miller units (MU) according to the following equation: MU = 1,000 × (OD) 420 -1.75 × OD 550 ) / [OD 600 [× Volume (mL) × Reaction Time (min)]; The measurement results are as follows lacZ As shown, compared with the DMSO solvent and mangiferin control groups, α-mangiferin at a concentration of 100 μg / mL significantly inhibited [the treatment]. Figure 1 The promoter activity was significantly increased, and the inhibitory effect exceeded 50%, a difference that was statistically significant (***). p <0.001). Furthermore, compared to pqsA α-Typocytosine pqsA and rhlI The expression of α-dextrin was not significantly affected, indicating that α-dextrin only had a significant inhibitory effect on the PQS quorum sensing system of Pseudomonas aeruginosa.

[0044] Example 2 This example is a test experiment to test the effect of α-dextrin on the growth of Pseudomonas aeruginosa.

[0045] Using the wild-type Pseudomonas aeruginosa strain PAO1 as the test strain, three experimental treatments were set up. The first group was treated with the same volume of DMSO solvent as the subsequent drugs. The second group was treated with α-dextrin at a final concentration of 100 μg / mL in the culture medium. The third group was treated with dextrin at a final concentration of 100 μg / mL in the culture medium. Each group was set up with three biological replicates. The specific test steps are as follows: Step 1: Pick a single colony of *Pseudomonas aeruginosa* PAO1 and place it in 5 mL of LB broth containing Km30. Incubate at 37°C and 200 rpm for 16-20 h until the stationary phase. Step 2: Add the *Pseudomonas aeruginosa* bacterial suspension at a 1:100 ratio to 5 mL of LB broth containing Km30 and 100 μg / mL α-dextrin (or gentianin and an equal volume of DMSO). Detect the initial OD. 600 DMSO solvent and gentianin were used as controls; Step 3: The transferred bacterial culture was cultured at 37℃ and 200 rpm with shaking, and the OD of the bacterial culture was measured and recorded every 2 hours. 600 The culture lasted for 24 hours.

[0046] Test results are as follows lasI As shown, α-dextrin had no effect on the growth of Pseudomonas aeruginosa compared with the DMSO and dextrin control groups.

[0047] Example 3 This embodiment tests the QS system for α-retropine against Pseudomonas aeruginosa. Figure 2 The impact on gene expression.

[0048] α-Retropine's QS system for Pseudomonas aeruginosa at different growth stages pqsA The specific steps of the experiment to detect the effects of gene expression are as follows: Step 1: Add α-dextrin to a final concentration of 100 μg / mL and the transcriptional fusion strain PAO1. pqsA ::mini-CTX-P attB - pqsA Co-incubated, with DMSO solvent and gentianin as negative controls, at 37℃ and 200 rpm until the decline phase, and then separately incubated at OD... 600 Sampling was performed at values ​​of 0.5, 0.8, 1.6, 1.9, 2.2, 2.6, and 3 to detect different OD values ​​under α-twistingin treatment. 600 The β-galactosidase activity of the transcribed fusion strain was detected according to the detection method in step 2 of the gene expression detection of the quorum sensing system under the action of α-retropine in Example 1 (2).

[0049] OD was detected separately 600When the values ​​are 0.5, 0.8, 1.6, 1.9, 2.2, 2.6, 3, lacZ The expression of [the information]. The detection results are as follows: pqsA As shown, compared with the DMSO solvent and mangiferin treatment groups, the treatment with 100 μg / mL α-dextrin resulted in a longer degradation period (OD). 600 The value is 3) Previously, the PAO1 quorum sensing system gene of Pseudomonas aeruginosa Figure 3 The promoter activity of all of them was inhibited. Compared with the DMSO control, the activity of the control herb, gentianin, was significantly reduced. pqsA The promoter activity remained similar to that of the DMSO-treated group, but α-twistingin significantly inhibited activity throughout the growth cycle. pqsA The expression. When OD 600 When it reaches 1.6, α-twistingin pairs pqsA When the promoter enzyme activity is inhibited to the maximum, OD 600 After reaching 2.2, the DMSO and α-dextrin treatment groups pqsA The expressions tend to stabilize when OD 600 When the concentration reaches 3, *Pseudomonas aeruginosa* enters the decline phase. α-Retropine is effective against *Pseudomonas aeruginosa*. pqsA The promoter inhibition disappeared. This result may be because, with bacterial growth and reproduction, the target site or corresponding competitive ligand of α-retropine in *Pseudomonas aeruginosa* is synthesized in large quantities, interfering with its effect.

[0050] Example 4 This embodiment tests the effects of different concentrations of α-retropine on the gene expression of the PQS quorum sensing system of *Pseudomonas aeruginosa*. pqsA The impact of expression PQS quorum sensing system genes under different concentrations of α-twistingin pqsA In the expression detection experiment, to detect the effect of different concentrations of α-trachytin on the PQS system, this invention followed the steps of reacting different concentrations of α-trachytin with PAO1::mini-CTX-PpqsA- pqsA The strains were co-incubated, and the β-galactosidase activity was measured to analyze the results. lacZ The specific steps for testing promoter expression are as follows: Step 1: Using argentin and DMSO solvent as negative controls, different concentrations of α-argentin solutions and the transcriptional fusion strain PAO1 were tested. pqsA ::mini-CTX-P attB - pqsA Co-incubate at 37°C and 200 rpm until OD. 600The value is 1.6; Step 2, the β-galactosidase activity of the transcribed fusion strain was detected according to the detection method in Step 2 of the gene expression detection of the quorum sensing system under the action of α-retropine in Example 1 (2).

[0051] The concentrations of α-dextrin were measured at 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100, respectively. lacZ The expression of [the information]. The detection results are as follows: pqsA As shown, Figure 4 Promoter activity decreased with increasing α-twistingin concentration, exhibiting concentration-dependent inhibition, and IC50... 50 It was 12.39 μg / mL. α-Dextrin pqsA The inhibitory efficiency of expression reached its maximum at 60 μg / mL. Therefore, to ensure the inhibitory activity of α-dextrin against the PQS system of Pseudomonas aeruginosa, the following studies all used a concentration of α-dextrin of 60 μg / mL and an OD value of [missing value]. 600 Further research will be conducted on 1.6.

[0052] Example 5: This example tests the effects of different concentrations of α-twistingin on the PQS quorum sensing system gene in PqsR protein-overexpressing strains. pqsA The impact of expression.

[0053] (1) Construction and enzyme activity detection of Pseudomonas aeruginosa strain overexpressing PqsR protein Step 1: Design primers using Pseudomonas aeruginosa genomic DNA as a template pqsA F / pqsR R, the gene was amplified by PCR using this primer. pqsR The sequence is 1000 bp, that is pqsR Gene fragment; Step 2, using pqsR I / Xho I pair Pst The gene fragment and plasmid pBBR1MCS-5 were double-digested; Step 3: The digested... pqsR After purification, the gene fragment was ligated into plasmid pBBR1MCS-5, transformed into E. coli TG1 competent cells, and plated on LB plates containing Gm100 for selection; Step 4: Positive clones were obtained by colony PCR screening, and the recombinant vector was extracted from them. pqsR I / Xho I performed enzyme digestion verification on the recombinant vector to obtain the recombinant vector pBBR1MCS-5- Pst Step 5: Pick PAO1 pqsR ::mini-CTX-P attB - pqsASingle colonies were incubated overnight at 37°C and 200 rpm in 5 mL of LB liquid medium. Step 6: The bacterial culture obtained in step 5 was transferred 1:100 to 50 mL of fresh LB liquid medium and incubated at 37°C and 200 rpm until OD500 was reached. 600 =0.75-1.25, centrifuge at 4°C, 8100×g for 8 min, and collect the bacterial cells; Step 7, resuspend the bacterial cells in 50 mL of pre-chilled 300 mM sucrose solution, centrifuge at 4°C, 8100×g for 8 min, and collect the bacterial cells; Step 8, resuspend the bacterial cells in 25 mL of pre-chilled 300 mM sucrose solution, centrifuge at 4°C, 8100×g for 8 min, and collect the bacterial cells; Step 9, resuspend the bacterial cells in 0.5 mL of pre-chilled 300 mM sucrose solution, incubate on ice for 30 min to obtain electrocompetent cells, aliquot 100 μL into each tube for later use, and store the remainder at 4°C; Step 10, add the recombinant vector pBBR1MCS-5- lacZ Add PAO1 pqsR ::mini-CTX-P attB - pqsA In the electrotransformation of competent cells, after mixing, the mixture was transferred to a sterile and pre-cooled electrotransformation cuvette for electrotransformation. The electrotransformation conditions were set as follows: 25 μF, 200 Ω, 1900 V, 3-5 ms. Step 11: The electrotransformed cells were transferred to 5 mL of fresh LB liquid medium and cultured at 37°C and 200 rpm for 3-5 h. Step 12: An appropriate amount of bacterial culture was spread onto TSB medium containing Km30, Tc200, and Gm100 for resistance selection. After culturing at 37°C for 2 days, the obtained colonies were streaked onto fresh TSB medium containing Km30, Tc200, and Gm100 to obtain... lacZ Gene overexpression strain PAO1 pqsR ::mini-CTX-P attB - pqsA (pBBR1MCS-5- lacZ Step 13: Obtain the empty vector control strain PAO1 using the same method. pqsR ::mini-CTX-P attB - pqsA (pBBR1MCS-5); Step 14: To detect the effect of different concentrations of α-dextrin on the expression of PQS quorum sensing system genes in PqsR protein overexpressing strains, dextrin and DMSO solvent, which have no effect on the PQS quorum sensing system of Pseudomonas aeruginosa, were used as negative controls. Different concentrations of α-dextrin were reacted with PAO1... lacZ ::mini-CTX-P attB - pqsA (pBBR1MCS-5- lacZ) and its empty vector control strain pqsR ::mini-CTX-P attB - pqsA (pBBR1MCS-5) were co-incubated at 37℃ and 200 rpm until OD. 600 The value is 1.6; Step 15: The β-galactosidase activity of the report strain was detected according to the detection method in step 2 of the quorum sensing system gene expression detection under the action of α-retropine in Example 1 (2).

[0054] Test results are as follows lacZ As shown, in Figure 5 In normally expressing strains, as the concentration of α-dextrin gradually increases, pqsR Promoter activity gradually decreased, reaching maximum inhibition efficiency at a concentration of 60 μg / mL. When overexpression... pqsA Subsequently, as the concentration of α-dextrin increased, pqsR If the promoter activity remains unchanged, IC 50 The concentration increased from 14.14 μg / mL to 518954 μg / mL. However, this did not apply to either normal or overexpression. pqsA In the control group pqsR Promoter activity remained largely unchanged. These results indicate that α-twistingin reduces PqsR protein activity through targeted inhibition. pqsA Gene expression.

[0055] Example 6 This embodiment demonstrates the molecular docking experiment between α-retropine and PqsR protein, and the effect of α-retropine on the quorum sensing system gene in a Pseudomonas aeruginosa point-mutant PqsR protein complementation strain. pqsA Tests to assess the impact of expression.

[0056] (1) Molecular docking experiment between α-twistingin and PqsR protein Step 1: The PqsR protein crystal structure was downloaded from the PDB database (PDB ID: 4jvd). The 3D structure of the small molecule α-taurin was constructed using Chem3D 14.1 and energy minimization was performed under the MMFF94 force field. Step 2: Molecular docking was performed using AutoDock Vina 1.1.2 software. Before docking, the receptor protein was processed using PyMol 2.5.4, including the removal of water molecules, salt ions, and small molecules. The docking box was then set to enclose the entire protein structure. Furthermore, ADFRsuite 1.0 was used to convert all processed small molecules and the receptor protein into the PDBQT format required for AutoDock Vina 1.2.3 docking. During docking, the parameters were kept at default settings. The docking conformation with the highest score was considered the binding conformation. Finally, the docking results were visualized and analyzed using PyMol 2.5.4 and Discovery Studio.

[0057] The docking results are as follows pqsA As shown, α-trachytin molecules can interact with the PqsR protein. Figure A shows α-trachytin binding in the central groove of the PqsR protein. Detailed interaction diagrams in Figures B and C reveal that the small molecule interacts hydrophobically with VAL-211, LEU-208, LEU-197, PHE-221, PRO-238, ILE-236, ILE-263, LEU-207, ALA-168, and ILE-149 on the PqsR protein, and undergoes a Pi-Sigma interaction with TYR-258. These interactions form the basis for the binding of α-trachytin to the PqsR protein. Furthermore, the affinity score for α-trachytin to PqsR is -7.755 kcal / mol, indicating a high binding potential between α-trachytin and the PqsR protein.

[0058] (2) α-Retropine's effect on the quorum sensing system gene in Pseudomonas aeruginosa point mutant PqsR protein complement strains Figure 6 Expression Influence Experiment Step 1: Using *Pseudomonas aeruginosa* genomic DNA as a template, primers were designed targeting amino acid residues 236, 263, 168, 207, 149, 197, 208, 238, 221, 258, and 211 of the PqsR protein to construct site-directed mutagenesis complementation vectors, such as the site-directed mutagenesis complementation vector pUC18T-mini-Tn7T-PqsR. A168L -Gm construction: We replaced the conserved amino acid residue alanine at position 168 of PqsR with leucine. Using primer PqsR... A168L upF / PqsR A168L upR / PqsRA168L lowF / PqsR A168L lowR amplification pqsA The upstream and downstream DNA sequences of the gene were ligated using overlap PCR to form the gene sequence. The product of the overlap PCR was then inserted into the pUC18T-mini-Tn7T-Gm plasmid. pqsR dIII / Hin At site I, the site-directed mutagenesis recombinant plasmid pUC18T-mini-Tn7T-PqsR was obtained. A168L -Gm. Site-directed mutagenesis vectors for other sites were obtained using the same method. Step 2: To further verify the interaction between α-dextrin and PqsR protein, this invention constructed site-directed mutagenesis complementation gene transcription fusion reporter strains targeting these amino acid sites (VAL-211, LEU-208, LEU-197, PHE-221, PRO-238, ILE-236, ILE-263, LEU-207, ALA-168, ILE-149, TYR-258). These vectors were transformed into ∆ Sac ::pMini-CTX-PpqsA pqsR In the middle, testing -lacZ Gene expression was assessed, and the effect of adding 60 μg / mL α-dextrin on the β-galactosidase activity of these reporter strains was examined according to Example 1. The test results are as follows: pqsA As shown in D, the results indicate that, compared with the reintroduced wild type Figure 6 In comparison, at ∆ pqsR ::pMini-CTX-P pqsR China and the United States complement each other. pqsA-lacZ L207A , pqsR L197A , pqsR I149A , pqsR L208A , pqsR F221A , pqsR V221A and pqsR P238A Almost undetectable pqsR Gene expression indicates that these sites are key active sites associated with PqsR-activated transcription. (Compared to wild-type replacement) pqsA Compared to transcriptional fusion reporter strains, PqsR I263A The mutation enhanced pqsR The expression suggests that changing the ILE residue to a LEU residue alters the structure of the PqsR protein, making its active site conformation more readily adaptable to [the target protein]. pqsA Sequence binding, and with complementary wild type pqsAIn comparison, although complementary pqsR Y258L , pqsR I236L back pqsR The expression of [a substance] was reduced; however, after treatment with α-twistingin, the expression of [a substance] in the transcriptional fusion reporter strain was reduced. pqsA Expression activity was still inhibited by α-trachytin, suggesting that ILE263, ILE-236, and TYR-258 may not be direct targets of α-trachytin. In contrast, complementary... pqsA A168L strains pqsR Although expression was weakened, it was not affected by α-trachytin, indicating that ALA-168 is a key site for the interaction between α-trachytin and PqsR.

[0059] Example 7 This example illustrates α-retropine and PqsR protein, as well as α-retropine and point-mutated PqsR protein. pqsA A168L Tests to detect protein binding capacity.

[0060] (1) PqsR and pqsR A168L Protein preparation Step 1: Using Pseudomonas aeruginosa genomic DNA as a template, design primers targeting the soluble portion of the PqsR protein, for example... pqsR F / pqsR R, pqsR A168L F / pqsR A168L R, the sequence was amplified by PCR using this primer; Step 2, using... pqsR I / Nde I amplified Sal Step 3: The gene fragment and plasmid pET28b were double-digested; Step 4: The digested fragment was purified and ligated into the plasmid, transformed into E. coli TG1 competent cells, and screened on LB plates containing Km30; Step 5: After PCR testing, the obtained colonies were used to extract the plasmid to obtain the recombinant vector. pqsR I / Nde I performed enzyme digestion verification on the recombinant vector, obtaining the recombinant vector pET28b- which showed a positive result. Sal pET28b- pqsR A168L Step 5: Transform the obtained recombinant vector into *E. coli* Transetta to obtain the His-tagged expression vector *Transetta pET28b-* pqsR TransettapET28b- pqsR A168LStep 6: Use sonication to lyse bacterial cells that have successfully expressed protein after induction with 0.5 mM IPTG at 16°C, and obtain cell lysis supernatant containing the target protein. Step 7: Take the His purification column, wash it with dd H2O water, and then equilibrate the column with low flow rate lysis buffer. Step 8: Filter the cell lysis supernatant from Step 6 and pass it through the column to adsorb the His fusion protein into the packing material. Step 9: Add washing buffer until no protein is washed out. Step 10: Elute with elution buffer, collect the protein sample, and after SDS-PAGE detection, dialyze the protein sample overnight at 4°C to obtain PqsR protein.

[0061] The buffers used for His protein purification are as follows: Lysis buffer (pH 8.0): 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole. Wash buffer (pH 8.0): 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole. Elution buffer (pH 8.0): 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole. Dialysis buffer (pH 8.0): 50 mM NaH₂PO₄, 300 mM NaCl, 10% glycerol.

[0062] (2) Circular dichroism detection test Circular dichroism (CD) is an optically active spectroscopic technique that analyzes the conformational changes of molecules' secondary structures by measuring the difference in absorption of left-handed and right-handed circularly polarized light. By comparing the CD spectra of proteins before and after small molecule binding (far ultraviolet region 150–400 nm), it can be determined whether the small molecule induced changes in the protein's secondary structure (such as an increase or decrease in the content of α-helices and β-sheets). The specific steps are as follows: Prepare specific concentrations of the herbal monomer α-dextrin and PqsR protein / pqsR. A168L After mixing to ensure a complete reaction, a certain amount of the mixture was pipetted into a 1 cm quartz cuvette for circular dichroism spectroscopy (CD) analysis of the interaction. The CD spectra were obtained before and after the interaction of α-twistingin with PqsR protein, purged with nitrogen in the wavelength range of 150-400 nm at room temperature using a buffer solution as a reference. Each CD spectrum is the average of three scans.

[0063] (3) Isothermal Titration Calorimetry (ITC) Detection Test When studying the interaction between small molecules and proteins, the main purpose of ITC is to quantitatively determine the thermodynamic parameters during the binding process, thereby revealing the mechanism, affinity, and specificity of molecular recognition. The specific steps are as follows: Step 1: Prepare a 0.2 mM α-twistingin solution and a 0.022 mM PqsR protein solution; Step 2: Draw 50 μL of α-twistingin into a syringe; add 300 μL of PqsR protein to the sample cell; this is the experimental group. The control group is: α-twistingin titration blank solvent (10% DMSO buffer); Step 3: Titrate PqsR protein with α-twistingin, a total of 20 drops, 2 μL per drop, titration interval 120 s, temperature 25℃, stirring speed 350 r / min; α-twistingin and PqsR A168L The steps for protein-protein interaction are the same as above.

[0064] To further investigate the interaction between α-trachytin and PqsR protein, and the key binding sites of PqsR protein and α-trachytin, circular dichroism (CD) chromatography and isothermal titration calorimetry (ITC) were used to determine whether the protein and α-trachytin could bind and the magnitude of the binding affinity. First, the circular dichroism chromatograms of PqsR and the α-trachytin-PqsR system were compared. The results are shown below. pqsR As shown in Figure AB, the interaction between α-trachytin and PqsR protein resulted in a 0.5% decrease in the α-helix H (r, d%) content, a 2.7% decrease in the β-turn (Turn%) content, a 1.7% decrease in the β-sheet S (r, d%) content, and a 4.8% increase in the random coil (Unrd%) content. This indicates that the interaction between α-trachytin and PqsR alters the secondary structure of the PqsR protein. Previous studies have shown that ALA-168 is a key site for the interaction between α-trachytin and PqsR, therefore, PqsR was tested... A168L Protein and α-PqsR A168L The CD of the system revealed that, under the influence of α-twistingin, PqsR A168L Although the secondary structure of the protein underwent some changes, the magnitude of these changes was significantly reduced compared to the intact PqsR protein. This further demonstrates that ALA-168 is the key site for the interaction between α-trachytin and PqsR. To investigate the binding affinity between α-trachytin and PqsR protein, the α-trachytin-PqsR system was compared and tested. A168L The ITC results are as follows Figure 7 As shown in CD: In the ITC experiment of the α-trollinosin-PqsR system, Kd = (9.213 × 10⁻ 6 ±2.002 ×10⁻ 6At this point, Kd is at the micromolar level, indicating that α-dextrin has a high binding affinity to PqsR protein. Therefore, α-dextrin exhibits a strong micromolar binding ability to PqsR protein, and the binding process is an enthalpy-driven, exothermic reaction, further confirming that the target of α-dextrin is indeed PqsR protein. In the α-dextrin-PqsR... A168L In the ITC experiment of the system, the Kd value increased to (3.66 × 10⁻⁵ ± 4.341 × 10⁻⁵). 6 The presence of M indicates that the mutation significantly weakens the protein's binding affinity to small molecules, decreasing it by approximately 3.97-fold. This further confirms that ALA-168 is a key site for the interaction between α-trachytin and PqsR. Specific data from the CD and ITC experiments are shown in the table below:

[0065] Example 8 This example is a test experiment to test the effect of α-retropine on the synthesis of pyophyll aeruginosa by Pseudomonas aeruginosa.

[0066] (1) Detection of gene expression for pyocyanin synthesis under the action of α-retropine The expression of the pyocyanin synthesis gene under the action of α-retropine was detected to identify the operon of the pyocyanin synthesis gene. Figure 7 1 and phzA The expression of 2 is obtained by constructing PAO1. phzA ::mini-CTX-P attB 1- phzA PAO1 lacZ and ::mini-CTX-P attB 2- phzA Transcriptional fusion strains were used to detect β-galactosidase activity in the strains and to determine the operon of the pyocyanin synthesis gene. lacZ 1 and phzA The expression of 2 is as follows: The operon for the pyocyanin synthesis gene was constructed according to the method in Example 1. phzA 1 B 1 C 1 D 1 E 1 F 1 G 1( phzA 1) and phzA 2 B 2 C 2 D 2 E 2 F 2 G 2( phzA 2) Transcriptional fusion strain PAO1phzA ::mini-CTX-P attB 1- phzA PAO1 lacZ ::mini-CTX-P attB 2- phzA ; The β-galactosidase activity of the transcribed fusion strain was detected according to the detection method in step 2 of the gene expression detection of the quorum sensing system under the action of α-retropine in Example 1 (2).

[0067] (2) Detection of Pyrrosin Production under the Action of α-Retropine The specific experimental steps for detecting pyocyanin production under the action of α-dextrin are as follows: Step 1: Pick a single colony of PAO1 and place it in 5 mL of fresh LB liquid medium containing Km30. Incubate at 37°C and 200 rpm for 16-20 h with shaking. Step 2: Add Pseudomonas aeruginosa bacterial suspension at a ratio of 1:100 to 5 mL of PB liquid medium containing Km30 and α-dextrin, gentianin, or an equal volume of DMSO at a final concentration of 60 μg / mL. Incubate at 37°C and 200 rpm until the logarithmic growth phase. The preparation method of the PB liquid culture medium is as follows: Weigh 1.4 g of MgCl2, 10 g of K2SO4, and 20 g of bacterial peptone, dissolve them in distilled water, and then bring the volume to 1 L with distilled water. Sterilize at 121℃ for 20 min; Step 3: After the culture reaches the stationary phase, centrifuge and collect the supernatant; Step 4: Add 3 mL of chloroform to 5 mL of supernatant, stir vigorously for 2 min, and then centrifuge at 8000 rpm for 10 min; Step 5: Transfer the chloroform layer to a new test tube and mix with 1 mL of 0.2 M hydrochloric acid; Step 6: Centrifuge again at 8000 rpm for 10 min, aspirate the top red solution, and measure the OD. 520 .

[0068] Calculate the content of pyocyanin according to the formula: OD 520 / OD 600 ×17.072.

[0069] The results of the detection of the effect of α-retropine on the synthesis of pyophyll in Pseudomonas aeruginosa are as follows: lacZ As shown, where Figure 8 A shows that, compared to the DMSO treatment group, the α-dextrin treatment group... Figure 8 The expression level decreased by 19.93%, and the difference reached a highly significant level (**). p <0.01); Compared with the control group treated with gentian root extract, the α-dextrin treatment group phzA1 The expression level decreased by 19.69%, and the difference reached a statistically significant level (**,p <0.01). phzA1 Results B showed that the α-dextrin treatment group Figure 8 The expression level was reduced by 41.99% compared to the DMSO treatment group, and the difference was statistically significant (**). p <0.01); compared with the control group treated with gentian root extract, the level decreased by 43.12%. The difference was also statistically significant (**, p <0.01). The results showed that 60 μg / mL α-dextrin could inhibit the expression of the Pseudomonas aeruginosa pyocyanin synthesis gene by inhibiting the PQS system.

[0070] The test results of the effect of α-retropine on the production of pyophyll aeruginosa by Pseudomonas aeruginosa PAO1 are as follows: phzA2 As shown in Figure C, the pyocyanin production in the α-dextrin-treated group was reduced by 65% ​​compared to the DMSO-treated group, and the difference was statistically significant (***). p <0.001), compared to the control group treated with gentian root extract, the level was reduced by 64.56%, and the difference was statistically significant (***, p <0.001). This indicates that α-dextrin inhibits the production of pyocyanin by Pseudomonas aeruginosa.

[0071] Example 9 This example is a test experiment to test the effect of α-retropine on the motility of Pseudomonas aeruginosa.

[0072] (1) Detection test of Pseudomonas aeruginosa swarming Step 1: Add α-dextrin or 60 μg / ml mangiferin or the same volume of DMSO to the Swarming medium to prepare different Swarming plates. The Swarming medium is prepared as follows: Weigh 3.0 g of agarose, 5 g of glucose, and 8 g of nutrient broth (NB), dissolve them in distilled water, and then bring the volume to 1 L with distilled water. Sterilize at 121℃ for 20 min. Step 2: Pick a single colony of PAO1 and place it in 5 mL of fresh LB liquid medium containing Km30. Incubate at 37℃ with shaking at 200 rpm for 16-20 h. Step 3: Measure the OD of the bacterial culture. 600 Dilute the bacterial solution until the OD of the diluted bacterial solution reaches the specified level. 600 =0.5; Step 4: Take 1 μL of the diluted bacterial solution and drop it into the center of the plate; Step 5: After the bacterial solution has penetrated the plate, place the plate in a 30℃ incubator and incubate for 24 h. Observe the diffusion of the bacterial solution and measure its diameter. All experiments were performed in triplicate.

[0073] (2) Swimming test of Pseudomonas aeruginosa Step 1: Add α-dextrin or 60 μg / mL mangiferin or the same volume of DMSO to the Swimming medium to prepare different Swimming plates. The preparation method of the Swimming medium is as follows: Weigh 2.5 g of agarose, 5 g of NaCl and 10 g of peptone, dissolve them in distilled water, and then make up to 1 L with distilled water. Sterilize at 121℃ for 20 min. Step 2: Pick a single colony of PAO1 and place it in 5 mL of fresh LB liquid medium containing Km30. Incubate at 37℃ with shaking at 200 rpm for 16-20 h. Step 3: Measure the OD of the bacterial culture. 600 Dilute the bacterial solution until the OD of the diluted bacterial solution reaches the specified level. 600 = 0.5; Step 4: Take 1 μL of the diluted bacterial solution and drop it into the center of the plate; Step 5: After the bacterial solution has penetrated the plate, place the plate in a 30℃ incubator and incubate for 24 h. Observe the diffusion of the bacterial solution and measure its diameter. All experiments were performed in triplicate.

[0074] (3) Test for twitching movement of Pseudomonas aeruginosa Step 1: Add α-dextrin or 60 μg / mL gentianin or the same volume of DMSO to Twitching medium to prepare different Twitching plates. Dry the plates in a clean bench before use. The Twitching medium is prepared as follows: Weigh 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g / L agar powder, dissolve in distilled water, and then bring the volume to 1 L with distilled water. Sterilize at 121℃ for 20 min. Step 2: Pick a single colony of PAO1 and place it in 5 mL of fresh LB liquid medium containing Km30. Incubate at 37℃ with shaking at 200 rpm for 16-20 h. Step 3: Use the tip of a toothpick to pierce the Twitching plate with fresh Pseudomonas aeruginosa bacterial suspension. Incubate upside down at 37℃ for 24 h. Step 4: After incubation, stain with Coomassie Brilliant Blue solution (0.05 g Coomassie Brilliant Blue R250, 40...). Mix 10 mL of methanol and 10 mL of acetic acid thoroughly, and bring the volume to 100 mL with deionized water to completely immerse the plate. Step 5: Decolorize with 95% ethanol until a diffusion zone is visible, and measure the diameter of the diffusion zone. All experiments were performed in triplicate.

[0075] Test results as follows Figure 8 As shown, Figure 9A showed that, compared with the DMSO treatment group, the swarming diameter of *Pseudomonas aeruginosa* in the α-dextrin treatment group was reduced by approximately 73.30%, a significant difference (***, p <0.001), compared with the control group treated with gentian root extract, the swarming diameter decreased by approximately 72.96%, and the difference reached a statistically significant level (***, p <0.001), indicating that 60 μg / mL α-dextrin can significantly inhibit the swarming movement of Pseudomonas aeruginosa; Figure 9 B showed that, compared with the DMSO treatment group, the swimming diameter of *Pseudomonas aeruginosa* in the α-dextrin treatment group was reduced by approximately 36.67%, a significant difference (**, p <0.01), compared with the control group treated with gentian root extract, the Swimming diameter decreased by approximately 34.48%, and the difference reached a statistically significant level (**, p <0.01), indicating that 60 μg / mL α-dextrin can significantly inhibit the swimming motility of Pseudomonas aeruginosa; Figure 9 C showed that, compared with the DMSO-treated group, the diameter of *Pseudomonas aeruginosa* twitching motility decreased by approximately 63.31% in the α-twistingin-treated group, a significant difference (***, p <0.001), compared with the control group treated with gentian root extract, the diameter of twitching was reduced by approximately 63.42%, and the difference reached a statistically significant level (***, p The result (<0.001) indicates that 60 μg / mL α-dextrin significantly inhibits the twitching motility of *Pseudomonas aeruginosa*. Swimming, swarming, and twitching motility in *P. aeruginosa* depend on the normal function of flagella, rhamnolipin, and pili, respectively. Swimming depends on flagellar activity, swarming requires the participation of rhamnolipin, and twitching is driven by pili. These three motility characteristics are closely related to the pathogenicity of *P. aeruginosa* and are all regulated by the PQS quorum sensing system. These results demonstrate that α-dextrin significantly reduces the motility of *P. aeruginosa* by inhibiting the PQS quorum sensing system, further validating the potential application value of α-dextrin in combating *P. aeruginosa* infection.

[0076] Example 10 This embodiment is an experiment to detect the effect of α-retropine on the activity of Pseudomonas aeruginosa LasA protease.

[0077] The activity of the LasA protease was detected by monitoring the decrease in absorbance of the Staphylococcus aureus suspension at 595 nm, thus determining its staphylococcal lysis activity in the Pseudomonas aeruginosa supernatant. LasA is a lysosomal protease, an enzyme that dissolves Staphylococcus aureus by cleaving peptidoglycan-5-glycine metapeptide.

[0078] Step 1: Pick a single colony of *Pseudomonas aeruginosa* PAO1 and place it in 5 mL of LB broth containing Km30. Incubate at 37°C and 200 rpm with shaking for 16-20 h until the stationary phase. Step 2: Add the *Pseudomonas aeruginosa* bacterial suspension at a 1:100 ratio to 5 mL of LB broth containing Km30 and α-dextrin (or gentianin and an equal volume of DMSO) at a final concentration of 60 μg / mL. Incubate at 37°C and 200 rpm until the OD phase is reached. 600 The concentration was 1.6, and Staphylococcus aureus was picked and placed in 5 mL of LB liquid medium, and cultured at 37°C with shaking at 200 rpm until the stationary phase; Step 3: The Staphylococcus aureus cultured to the stationary phase was centrifuged to collect the cells, resuspended in 20 mM / pH=8.8 Tris-HCl, and the OD was measured. 595 The results were recorded, and the cells were killed by incubation at 100°C for 30 min. Step 4: 0.1 mL of Pseudomonas aeruginosa supernatant was added to 0.9 mL of heat-killed Staphylococcus aureus suspension. The absorbance was measured every 30 min at 595 nm using an ELISA reader, and the change in absorbance was measured over at least 6 h to determine the staphylococcal lysis activity of the sample.

[0079] The results of the test on the effect of α-retropine on the LasA protease activity of Pseudomonas aeruginosa are as follows: Figure 9 As shown, compared with the control traditional Chinese medicines gentianin and DMSO, the activity of LasA protease in the supernatant of Pseudomonas aeruginosa was significantly reduced under α-gentianin treatment, with the difference reaching a highly significant level (***). p <0.001). This indicates that α-dextrin inhibits the production of LasA protease in Pseudomonas aeruginosa.

[0080] Example 11 This embodiment aims to investigate the effect of α-retropine on the gene expression of virulence factors in *Pseudomonas aeruginosa*. Step 1: Hydrogen cyanide (…) was constructed according to the method in Example 1. Figure 10 ), elastase ( hcnA ) and lectins ( lasB ), rhamnolipid ( lecA Transcriptional fusion strain PAO1 rhlA ::mini-CTX-P attB - hcnAPAO1 lacZ ::mini-CTX-P attB - lasB PAO1 lacZ ::mini-CTX-P attB - lecA PAO1 lacZ ::mini-CTX-P attB - rhlA Step 2: Co-culture α-dextrin (or gentianin and the same volume of DMSO) at a final concentration of 60 μg / mL with the transcriptional fusion strains, with three biological replicates for each group, and incubate at 37℃ and 200 rpm with shaking until OD. 600 The value is 1.6; Step 3: Detect the β-galactosidase activity of the transcriptional fusion strain according to the gene expression detection method of quorum sensing system under the action of α-retropine in Example 1 (2).

[0081] The results are as follows lacZ As shown in Figure A, α-retropine significantly reduced the levels of Pseudomonas aeruginosa. Figure 11 The expression level of [the substance] was reduced by 35.85% compared to the control group (DMSO), a difference that was statistically significant (**, [missing information]). p <0.01); such as hcnA As shown in B, α-retropine significantly reduced the levels of Pseudomonas aeruginosa. Figure 11 The expression level of [the substance] was reduced by 39.16% compared to the control group (DMSO), and the difference was statistically significant (**, [missing information]). p <0.01); such as lasB As shown in C, α-retropine significantly reduced the levels of Pseudomonas aeruginosa. Figure 11 The expression level of [the substance] was reduced by 45.09% compared to the control group (DMSO), and the difference was statistically significant (**, [missing information]). p <0.01); such as lecA As shown in Figure D, α-retropine significantly reduced the levels of Pseudomonas aeruginosa. Figure 11 The expression level of [the substance] was reduced by 33.72% compared to the control group (DMSO), and the difference was statistically significant (*). p <0.05). These results indicate that α-thujone can inhibit the synthesis of hydrogen cyanide, lectin, elastase, and rhamnolipid by inhibiting the PQS system.

[0082] Example 12 This example is a test experiment to test the effect of α-retropine on biofilm formation in Pseudomonas aeruginosa.

[0083] Construct a planktonic biofilm synthesis gene cluster according to the method in Example 1.rhlA and pel Transcriptional fusion strain PAO1 psl ::mini-CTX-P attB - pel PAO1 lacZ ::mini-CTX-P attB - psl ; The β-galactosidase activity of the transcriptional fusion strain was detected according to the detection method in the quorum sensing system gene expression detection under the action of α-retropine in Example 1.

[0084] The planktonic biofilm was observed using laser confocal scanning microscopy (LSCM). The specific operating steps are as follows: Step 1, pBBR- lacZ -mut3 vector (green fluorescent protein expression vector) was transformed into E. coli S17-1 and conjugated with PAO1 on TSB plates at 37 ℃ for 48 h; Step 2, the conjugated bacterial colony was suspended in TSB medium and appropriately diluted, and then spread on TSB triple antibody plates containing Km30, Tc200, and gentamicin (Gm, 100 μg / mL, abbreviated as Gm100) to screen for strains labeled with green fluorescent protein (GFP); Step 3, sterile glass coverslips were immersed in Petri dishes containing 5 mL of TSB medium. At a 1:100 inoculation rate, each strain labeled with GFP (green fluorescent protein) was transferred to a petri dish and cultured to the stationary phase; the culture was carried out at 37 ℃ with shaking at 100 r / min for 72 h; step 4: the coverslip was removed, rinsed twice with PBS to remove non-adherent cells and culture medium, and the coverslip was air-dried to obtain the biofilm of each strain labeled with GFP; step 5: the formation of the biofilm was observed under a laser confocal scanning microscope. The non-GFP-labeled PAO1 strain was used as a blank control.

[0085] The results are as follows gfp As shown, Figure 12 A shows that, compared to the dimethyl sulfoxide treatment group, the α-twistingin treatment group... Figure 12 There was no significant difference in expression levels. pel B shows that, compared to the dimethyl sulfoxide treatment group, the α-twistingin treatment group Figure 12 The expression level decreased by 23.42%, and the difference reached a statistically significant level (**). p <0.01), compared with the control group treated with gentian root extract. pslThe expression level decreased by 21.19%, and the difference reached a statistically significant level (**, p <0.01). This indicates that 60 μg / mL α-dextrin can inhibit the expression of Pseudomonas aeruginosa biofilm synthesis genes by inhibiting the PQS system; subsequently, the effect of α-dextrin on Pseudomonas aeruginosa PAO1 biofilm formation was investigated. pelA Figure 12 D shows that the biofilms in the dimethyl sulfoxide (DMSO) and mangiferin (MnS) treated groups were relatively thick and continuous, while the biofilm thickness and density decreased and the distribution became sparse in the α-mangiferin (MnS) treated group. Analysis of the raw integrated density (Rawintden) of the biofilm on the coverslips of different treatment groups yielded the following results: Figure 12 As shown in C, the Rawintden concentration in the α-twistingin-treated group was significantly lower than that in the dimethyl sulfoxide-treated group, approximately twice as low (***). p <0.001); Compared with the gentianin-treated group, the surface area to volume ratio was approximately 1.8 times lower (***, p The result (<0.001) indicates that the biofilm structure of *Pseudomonas aeruginosa* was more dispersed and weaker after treatment with α-dextrin. This suggests that α-dextrin has a significant effect on inhibiting the formation of planktonic biofilms in *P. aeruginosa*, further supporting the potential of α-dextrin to inhibit biofilm formation in *P. aeruginosa*.

[0086] Example 13 This example is a test experiment to determine the effect of α-retropine on the pathogenicity of Pseudomonas aeruginosa. (1) Effect of α-dextrin on pathogenicity of Pseudomonas aeruginosa in Chinese cabbage Using DMSO as a solvent control and gentianin as a reference herb, the infection ability of *Pseudomonas aeruginosa* on Chinese cabbage leaves after the addition of α-dextrin was tested. The specific experimental steps are as follows: Step 1: Pick a single colony of Pseudomonas aeruginosa PAO1 and place it in 5 mL of fresh LB liquid medium containing Km30, 60 μg / mL α-dextrin, 60 μg / mL gentianin, and the same volume of DMSO. Incubate at 37°C with shaking at 200 rpm until OD200. 600 =1.6; Step 2: Take the bacterial suspension, centrifuge at 5500 rpm for 5 min, discard the supernatant, then add an equal volume of 10 mM MgSO4 to wash the bacteria, repeat twice; Step 3: Dilute the bacterial suspension to OD with 10 mM MgSO4. 600 =1.5; Step 4: Select fresh Chinese cabbage leaves, disinfect the leaves with 0.2% sodium hypochlorite, and air dry; Step 5: Inoculate 10 μL of the prepared bacterial solution at the same position on the stem of the Chinese cabbage, incubate at 30℃ for 3 days, observe the area of ​​rot of the Chinese cabbage and calculate it using ImageJ software; Each experiment is repeated 3 times.

[0087] (2) Cell infection assay of Pseudomonas aeruginosa A549 cells by α-dextrin Using DMSO as a solvent control and gentianin as a reference traditional Chinese medicine, the effect of Pseudomonas aeruginosa on the survival rate of A549 cells was investigated after treatment with α-gentianin. The specific steps are as follows: Step 1: Remove the frozen cells from liquid nitrogen, suspend them in culture medium, and incubate them in a CO2 incubator until the cells adhere. Step 2: Remove the adhered and healthy cells from the CO2 incubator. In a clean bench, aspirate the culture medium from the flask, digest the cells with trypsin, and prepare a cell suspension. Step 3: Transfer the cell suspension to 96-well culture dishes. After the cells have completely adhered, remove the cell culture and gently rinse each well with 1×PBS. Step 4: Add a certain amount of bacteria to each well for infection and incubate in a CO2 incubator for 6 hours. Step 5: Remove the culture from each well, rinse with 1×PBS, add CCK8 solution, and incubate in a CO2 incubator for 2-4 hours. Step 6: Remove the 96-well culture dish, measure the absorbance at 450 nm using a microplate reader, and calculate the cell viability (%). Each experiment is repeated in triplicate. Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100% As: Absorbance of experimental wells (cells infected by bacteria treated with drugs, culture medium, CCK-8 solution); Ac: Absorbance of control wells (cells, culture medium, and CCK-8 solution not infected with bacteria) Ab: Absorbance of blank wells (including culture medium and CCK-8 solution, but without cells) (3) Cell invasion assay of Pseudomonas aeruginosa A549 cells by α-dextrin Using DMSO as a solvent control and gentianin as a reference traditional Chinese medicine, the effect of Pseudomonas aeruginosa on A549 cell invasion was investigated after treatment with α-gentianin. The specific steps are as follows: Step 1: Prepare the cells in advance according to the operation steps of Example 13 (2); Step 2: Transfer the cell suspension to a 96-well culture dish. After the cells are completely attached to the wall, remove the cell culture and gently rinse each well with 1×PBS; Step 3: Add a certain amount of bacteria to each well for infection and incubate in a carbon dioxide incubator for 6 h; Step 4: Discard the culture, rinse with 1×PBS, add Gm100 to each well and incubate in a carbon dioxide incubator for 1 h; Step 5: Discard the culture, rinse with 1×PBS, add pre-cooled Triton-x 100 and incubate for 15 min; Step 6: Resuspend the bacteria in each well using fresh LB medium, dilute and spread on Km30 plates for counting. Set up 3 replicates for each experiment.

[0088] (4) Cell adhesion assay of α-dextrin to Pseudomonas aeruginosa-invaded A549 cells Step 1: Prepare the cells in advance according to the operating steps in Example 13 (2); Step 2: Transfer the cell suspension to a 96-well culture dish. After the cells are completely attached to the wall, remove the cell culture and gently rinse each well with 1×PBS; Step 3: Add a certain amount of bacteria to each well for infection and incubate in a carbon dioxide incubator for 6 h; Step 4: Remove the culture, rinse with 1×PBS, and incubate with pre-cooled Triton-x 100 for 15 min; Step 5: Resuspend the bacteria in each well using fresh LB medium, dilute and spread on Km30 plates for counting. Set up 3 replicates for each experiment.

[0089] The results of the test on the infectivity of Pseudomonas aeruginosa to Chinese cabbage leaves are as follows: Figure 12 As shown in Figure A, compared with the DMSO treatment group, the area of ​​decay caused by *Pseudomonas aeruginosa* infection in Chinese cabbage was reduced by approximately 76.08% after α-dextrin treatment, and the difference reached a statistically significant level (***). p <0.001). Compared with the control group treated with the traditional Chinese medicine gentian root extract, the rotten area in the α-dextrin treatment group was reduced by approximately 77.55%, and the difference reached a statistically significant level (***, p <0.001), indicating that α-dextrin can reduce the pathogenicity of Pseudomonas aeruginosa to Chinese cabbage.

[0090] The results of the Pseudomonas aeruginosa survival test on A549 cells after treatment with α-dextrin are as follows: Figure 12 As shown in Figure B, compared with the DMSO and control group treated with the traditional Chinese medicine gentianin, the survival rate of Pseudomonas aeruginosa-infected A549 cells treated with α-gentianin was significantly increased by approximately 1.5 times, and the difference reached a statistically significant level (***). p <0.001), the relative invasion rate of Pseudomonas aeruginosa against cell A549 under α-dextrin treatment, the results are as follows: Figure 12As shown in Figure C, compared to the DMSO treatment group and the control group treated with the traditional Chinese medicine gentian root extract, the invasive ability of *Pseudomonas aeruginosa* treated with α-gentian root extract was significantly reduced, decreasing by approximately 1.4 times, with the difference reaching a statistically significant level (**, p <0.01). Furthermore, compared to the DMSO treatment group and the control group treated with the traditional Chinese medicine gentian root extract, the adhesion ability of *Pseudomonas aeruginosa* treated with α-gentian root extract decreased by approximately 1.3 times, as shown in the results. Figure 12 As shown in D, the difference reached a significant level (**, p <0.01). This indicates that α-dextrin can significantly inhibit the invasive ability of Pseudomonas aeruginosa on A549 cells. The above results show that α-dextrin can reduce the pathogenicity of Pseudomonas aeruginosa on A549 cells.

[0091] In summary, while the α-dextrin monomer screened in this invention has no significant effect on the normal growth of *Pseudomonas aeruginosa*, it can significantly inhibit the PQS quorum sensing system of *P. aeruginosa* in a concentration-dependent manner, with the most significant inhibitory effect at 60 μg / mL. Gene overexpression and molecular docking experiments verified that α-dextrin reduces PQS signal generation and inhibits the expression of PQS-related genes by binding to the transcriptional activator protein PqsR of the PQS quorum sensing system. Further circular dichroism (CD) and isothermal titration calorimetry (ITC) experiments using in vitro purified PqsR protein confirmed that PqsR is the direct target protein of α-dextrin, and its binding site is the key site of alanine residue 168. Further research shows that α-dextrin not only inhibits the expression of virulence factors such as pyocyanin, lectins, and hydrogen cyanide, but also significantly weakens the motility of *P. aeruginosa*, including its swarming, swimming, and rubbing abilities. Furthermore, the protective effect of α-dextrin on the host was also verified. The results showed that α-dextrin could reduce the pathogenicity of *Pseudomonas aeruginosa* in plants (such as Chinese cabbage) and animal cell models (such as human alveolar epithelial cells A549), significantly alleviating the infection damage suffered by the host. These results strongly suggest that α-dextrin is an effective compound for inhibiting the pathogenicity of *Pseudomonas aeruginosa*.

[0092] In summary, α-dextrin effectively inhibits the PqsR quorum sensing system of *Pseudomonas aeruginosa* without affecting its normal growth, reducing its pathogenicity in plant and animal models and providing protection to the host. Chemical antibacterial drugs easily lead to bacterial resistance, while plant-derived natural products have attracted much attention due to their abundant resources and high safety. Currently, the number of quorum sensing inhibitors (QSIs) with clearly defined targets is limited. This invention, through a series of experiments, discovered that the target of α-dextrin is the PqsR protein. This discovery provides strong support for the development and application of therapeutic or adjuvant drugs against *Pseudomonas aeruginosa* infection by studying this target. Furthermore, α-dextrin, as a quorum sensing inhibitor, has broad application prospects in the development and preparation of therapeutic and adjuvant drugs against *Pseudomonas aeruginosa* infection.

[0093] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The use of α-dextrin or a pharmaceutically acceptable salt thereof in the preparation of inhibitors of the quorum sensing system of Pseudomonas aeruginosa.

2. The application according to claim 1, characterized in that, The Pseudomonas aeruginosa quorum sensing system inhibitor is used for the prevention and / or treatment of Pseudomonas aeruginosa diseases.

3. The application according to claim 1, characterized in that, The α-dextrin or its pharmaceutically acceptable salt acts on the PQS system in the Pseudomonas aeruginosa quorum sensing system.

4. The application according to claim 3, characterized in that, The α-retropine or a pharmaceutically acceptable salt thereof inhibits the gene of the PQS system in the Pseudomonas aeruginosa quorum sensing system. pqsA The expression.

5. The application according to claim 3, characterized in that, The target of the α-dextrin or its pharmaceutically acceptable salt is the PqsR protein of the PQS system in the Pseudomonas aeruginosa quorum sensing system.

6. The application according to any one of claims 1-5, characterized in that, The α-dextrin and its pharmaceutically acceptable salts inhibit the virulence and / or pathogenicity of Pseudomonas aeruginosa.

7. The application according to claim 6, characterized in that, The virulence refers to the ability of Pseudomonas aeruginosa to produce pyocyanin.

8. The application according to claim 6, characterized in that, The virulence refers to the motility of Pseudomonas aeruginosa.

9. The application according to claim 6, characterized in that, The virulence is defined as the ability of *Pseudomonas aeruginosa* to produce LasA protease, the expression of genes for the synthesis of hydrogen cyanide, elastase, lectin, and rhamnolipid in *Pseudomonas aeruginosa*, and the ability of *Pseudomonas aeruginosa* to form biofilms.

10. The application according to claim 6, characterized in that, The pathogenicity refers to the ability of *Pseudomonas aeruginosa* to infect plants or animals.

Images