Introduction
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic, Gram-negative microorganism causing acute or chronic infection in immunocompromised individuals and is a major cause of morbidity and mortality in hospital-acquired infections.1 Patients suffering from ventilator-associated pneumonia (VAP), bronchiectasis, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD) frequently develop chronic respiratory infections due to P. aeruginosa.2 The treatments of P. aeruginosa infection are extremely challenging due to its rapid mutations and resistant to multiple antibiotics. Carbapenem-resistant P. aeruginosa has been classified as a “critical” pathogen by World Health Organization (WHO),3 which urgently need to develop innovative effective medicines that can successfully and safely prevent P. aeruginosa resistance.
Quorum-sensing (QS) systems can generate signal molecule N-acyl homoserine lactones (AHLs) linked to extracellular virulence and internal communication.4 QS modulates various biological processes, including biofilm formation, virulence factor production, and microbe-host interactions.5 QS-inhibitory (QSI) agents, which can reduce bacterial virulence rather than kill it, may offer an effective therapeutic strategy for controlling P. aeruginosa infection.4 Researchers have gradually realized that plant-derived compounds can provide abundant materials for preventing and treating various diseases.6 Natural plants are one of the best potential sources of new drugs, as a World Health Organization (WHO) report mentioned.7 Extracts from various natural plants, such as Baicaetin,8 Naringenin,9 polyphenols from Salix tetrasperma,10 Falcarindiol from Notopterygium incisum,11 have been regarded as potential therapeutic agents due to their ability to inhibit the QS of P. aeruginosa.
Viola philippica Cav. (Violaceae), the traditional Chinese medicine Zǐ Huā Dì Dīng (ZHDD, in pinyin Chinese), also synonymized as Viola yedoensis Makino, is a perennial flowering plant native in China, Japan and Korea. To date, approximately 162 compounds have been identified in V. philippica, covering a wide range of chemical categories including coumarins, flavonoids, terpenoids, phenolic acids, alkaloids, and cyclopeptides.12,13 Recent studies have shown that extracts of V. philippica and its compounds exhibit a range of pharmacological effects, including anti-viral,13 anti-inflammatory,14,15 anti-coagulant16 and antimicrobial effects.14 Specifically, these extracts have shown antimicrobial efficacy against several bacterial strains, such as Streptococcus mutans,15 Staphylococcus aureus, Escherichia coli, Streptococcus lactis, Streptococcus agalactiae, Streptococcus dysgalactiae and Salmonella.17 Xie et al illustrated that the petroleum ether and ethyl acetate extracts of V. philippica effectively inhibited Bacillus subtilis and Pseudomonas syringae, while the methanol and methanol-water extracts failed to exhibit antibacterial activity.18 Given the rich content of bioactive compounds in V. philippica and its diverse antibacterial activities, its potential role in combating P. aeruginosa infections has attracted the attention of researchers. However, the effects of V. philippica on QS-regulated virulence against P. aeruginosa are poorly understood.
To the best of our knowledge, despite the diverse antibacterial activities of V. philippica, no study has yet investigated its anti-virulence effects against P. aeruginosa PAO1 via QS regulation. This study aims to fill this gap by investigating the effects of V. philippica and its compounds on QS-regulated virulence and genes in PAO1. We found that V. philippica granule herbal extract (VPE) acts as a QSI, inhibiting the production of QS-linked virulence traits in P. aeruginosa PAO1. Scopoletin and scoparone, two previously uncharacterized coumarin derivatives from VPE, were identified as novel QSIs. These compounds reduced pyocyanin, rhamnolipid, and hemolysin production, elastase activity, biofilm formation and swarming motility by inhibiting QS-associated genes (lasI, rhlI and pqsA), suggesting that scopoletin and scoparone were responsible for VPE’s effect on weakening QS-controlled virulence of PAO1. This study provides a laboratory basis for using scopoletin and scoparone as promising anti-virulence candidates for adjuvant therapy against P. aeruginosa infection.
Materials and Methods
Bacterial Strains and Reagents
The wild-type P. aeruginosa PAO1 strain (Strain No.: ATCC 15692) and Chromobacterium violaceum (C. violaceum) CV026 strain, both preserved in our laboratory, which had been used in our previous studies,19,20 were originally obtained from the American Type Culture Collection (ATCC) and the China General Microbiological Culture Collection Center (CGMCC), respectively. All strains were authenticated by 16S rDNA sequencing prior to use. The ATCC 15692 cell line was authenticated by short-tandem-repeat (STR) profiling, which confirmed 100% match with the ATCC reference profile. Luria-Bertani (LB) broth (0.5% yeast extract, 1.0% tryptone, 0.5% NaCl, pH 7.4) was used to cultivate both strains, with or without antibiotics. Unless otherwise noted, P. aeruginosa PAO1 and C. violaceum CV026 were cultured at 37°C and 28°C, respectively, with 200 rpm, in a shaking incubator for 16 h (overnight). Overnight cultured P. aeruginosa was inoculated into LB broth and cultured at 37°C with 260 rpm for 2 h to obtain mid-exponential phase P. aeruginosa PAO1. Bacterial density was recorded at OD600 using BioTek Synergy H1 microplate reader.
V. philippica granule herbal extract (VPE, Tianjiang Pharmaceutical Co., Ltd., Jiangsu, China) was dissolved in autoclaved double-distilled water to prepare a 1000 mg/mL solution, stored at −80°C until use. The extract was diluted with the corresponding solution (eg, sterilized water or LB medium) and sterilized through 0.22 μm syringe filters (Millipore) before use. Scopoletin standards (also known as 7-Hydroxy-5-methoxycoumarin, CAS: 92–61-5) and scoparone standards (also known as 6,7-dimethoxycoumarin/6,7-dimethylesculetin, CAS: 120–08-1) were purchased from Sigma-Aldrich (St Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions at 81.92 mg/mL and 102.4 mg/mL, respectively, and stored at −80°C until use.
Determination of Minimum Inhibitory Concentration (MIC)
The agar dilution method was used to evaluate the minimum inhibitory concentration (MIC) of VPE against P. aeruginosa PAO1 in Müeller-Hinton agar containing VPE (VPE-MH, in serial two-fold dilution), in accordance with the M7-A11 protocol published by the Clinical and Laboratory Standards Institute Standards (CLSI), as stated19 with slight modifications. The final concentration of VPE ranged from 1 mg/mL to 128 mg/mL. MH agar without VPE served as control. The mid-exponential phase P. aeruginosa PAO1 was diluted in LB broth, and 2 μL of the diluted suspension was spotted onto the plates to achieve a final concentration of 104 CFU/spot. The plates were incubated statically at 37°C for 20 h. MIC was defined as the lowest concentration of VPE preventing visible growth.
Growth Curve
Growth curve of P. aeruginosa PAO1 with VPE treatment was constructed as previously reported.20 Briefly, 10 μL of mid-exponential phase P. aeruginosa PAO1 was added to 1 mL of LB broth with or without sub-MICs of VPE, and incubated at 37°C 200 rpm. Every 3 h, cells were washed twice with sterile saline, resuspended in 1 mL of sterile saline, and 200 μL of the suspension was withdrawn into a 96-well plate, and the OD600 was measured using BioTek Synergy H1 microplate reader to generate growth curves.
Growth curves of P. aeruginosa PAO1 with scopoletin/scoparone treatment were performed in another way as previously described.21 Mid-exponential phase P. aeruginosa PAO1 were diluted 1:200 final concentration in a 96-well plate with or without scopoletin/scoparone, incubated at 37°C with gentle shaking for 24 h. OD600 was continuously monitored using BioTek Synergy H1 microplate reader. Sub-doses lower than 1024 μg/mL scopoletin/scoparone were used in all assays in the present study.
Bioassay for QSI Activity Using C.violaceum CV026
C. violaceum bioassay was performed with several modifications as previously described.22 The overnight culture of C. violaceum CV026 was mixed with warm LB agar supplemented with N-Butanoyl-L-homoserine lactone (C4-HSL) to a final concentration of 20 μmol/L. The mixture was then poured onto LB plates with a solidified layer. Wells (5 mm in diameter) were punched into the agar, and 40 μL sub-MICs of VPE or scopoletin/scoparone were added to the wells. The plates were incubated at 28°C for 20 h. The absence of purple violacein pigment in the bacterial lawn surrounding the wells indicated QS inhibition.
Pyocyanin Production Assay
The pyocyanin assay was carried out as previously described.20 Mid-exponential phase P. aeruginosa PAO1 (30 μL) was added to 3 mL of LB broth with or without sub-MICs of VPE or scopoletin/scoparone, and incubated at 37°C with 200 rpm for 20 h. Pyocyanin was extracted from 2.5 mL of P. aeruginosa PAO1 culture supernatant using 1.5 mL of chloroform and 0.5 mL of 0.2 mol/L HCl. The absorbance of the extract was measured at OD520 nm using BioTek Synergy H1 microplate reader.
Rhamnolipid Assay
The rhamnolipid assay was performed as described with minor modifications.23 Mid-exponential phase P. aeruginosa PAO1 (20 μL) was inoculated into 2 mL of M9 medium (containing 0.4% glucose, 2 mmol/L MgSO4, and 100 μmol/L CaCl2) with or without sub-MICs of VPE or scopoletin/scoparone, and incubated at 37°C 200 rpm for 16 h. The centrifuged supernatant (1mL) was treated with 40 μL of 1 mol/L HCl and 4 mL of chloroform, followed by vigorous shaking. The chloroform extract (3 mL) was then reacted with 100 μL of 1 g/L methylene blue and 2.45 mL of deionized water. Finally, 200 μL of the chloroform layer was transferred to a 96-well plate to measure the OD at 638 nm using BioTek Synergy H1 microplate reader.
Elastase Assays
Elastase activity was carried out by elastin-Congo red (ECR) assays.24 Mid-exponential phase P. aeruginosa PAO1 (20 μL) was inoculated into 2 mL of LB medium in the presence or absence of scopoletin/scoparone and incubated at 37°C 200 rpm for 8 h. Subsequently, 200 μL culture supernatant was inoculated with 800 μL ECR solution (Sigma-Aldrich, St. Louis, MO) containing 0.1 M Tris (pH 7.2), 1 mM CaCl2 and 3 mg/mL ECR, and incubated at 37°C 200 rpm for 4 h. The reaction was terminated by adding 100 μL of 0.12 M EDTA. The insoluble ECR was removed by centrifugation, and its OD495 was measured using BioTek Synergy H1 microplate reader.
Motility Assays
Swimming and swarming motility were assessed as previously described.20 Mid-exponential phase P. aeruginosa PAO1 (5 μL) was spotted onto the center of swarming medium (LB containing 0.5% agar, 5 g/L glucose) and swimming medium (tryptone broth containing 0.3% agar), both supplemented with various concentrations of VPE (16 mg/mL and 32 mg/mL), scopoletin (256 µg/mL and 128 µg/mL), or scoparone (256 µg/mL and 128 µg/mL). Media without VPE or scopoletin/scoparone served as controls. After the incubation at 37°C for 16 h, the motility zones were measured using a Vernier caliper.
Biofilm Formation Assay
Crystal violet staining was used to perform biofilm formation assay.8 Overnight cultured P. aeruginosa PAO1 (100 μL, 1 × 107 CFU/mL) was cultured in a 96-well plate with or without sub-MICs of VPE and scopoletin/scoparone for 24 h at 37°C statically. Planktonic/non-adherent cells were discarded, and the wells were gently washed with sterile saline. The remaining bacterial biomass was fixed with methanol for 5 min. After discarding the methanol, the wells were air-dried and stained with 1% crystal violet. After rinsing with sterile saline three times, the crystal violet-stained biofilms were eluted with 95% ethanol, and the OD595 was measured using BioTek Synergy H1 microplate reader.
Characterization of the Extract by Ultrahigh Performance Liquid Chromatography Coupled with Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometry (UHPLC-Q Exactive-Orbitrap MS)
Qualitative identification of V. philippica compounds was performed using a Thermo Scientific UltiMate 3000 UHPLC system and ultra-high-performance liquid chromatography coupled with a Q Exactive Orbitrap mass (Thermo Fisher Scientific, Waltham, MA, USA). A Waters ACQUITY UPLC HSS T3 Column (2.1 mm × 100 mm, 1.8 μm) with an ACQUITY UPLC HSS T3 (1.8 μm) precolumn was employed. Gradients of solvents: Acetonitrile (A) and 0.1% formic acid aqueous solution (B) were used as the mobile phase, with a flow rate of 0.2 mL/min. The gradient elution was as follows: 0–2 min, 5–10% of A; 2–5 min, 10–30% of A; 5–15 min, 30–35% of A; 15–18 min, 35–40% of A; 18–21 min, 40–100% of A; 100% – 21–24 min, 30% of A; 24–26 min, 30–5% of A. The injection volume was 5 μL and the total analysis time was 26 min. The main parameters for Q Exactive-Orbitrap MS were set as follows: heat electrospray ionization (ESI) positive and negative ion modes; capillary temperature, 350°C; sheath gas flow rate, 40; NCE, 20, 30, 45 eV; auxiliary gas heater temperature, 350°C; auxiliary gas flow rate, 15. In the full MS-dd MS2 experiment, the full MS scan range was set at m/z 100–2000 and the resolution was set at 70000. The resolution of the dd-MS2 was 17500. The parameters of the excluded isotopes were operated, and the dynamic exclusion time was 10s.
Blood Hemolysis Assays
Blood hemolysis assays were performed as previously described.20 Human whole blood was collected from healthy individuals and washed with phosphate-buffered saline (PBS, PH 7.4) to remove the leukocyte layer by centrifugation at 2,000 rpm for 5 min. The erythrocytes were then resuspended to a 4% concentration. A 300 μL aliquot of 4% washed erythrocytes was added to 300 µL of mid-exponential phase P. aeruginosa PAO1 resuspended in PBS (experimental group, EG), 300 µL of PBS (negative control, NC), and 300 µL of 2% Triton X-100 solution (positive control, PC), and scopoletin/scoparone was added. The mixtures were incubated at 37°C 6 h, and supernatant was measured at OD450 using BioTek Synergy H1 microplate reader. The percentage of hemolysis was determined using the following formula: (AEG − ANC)/(APC − ANC) × 100% = hemolysis%.
Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Overnight cultured P. aeruginosa PAO1 (30 μL) was added to 3 mL of LB broth with or without scopoletin/scoparone and incubated at 37°C 200 rpm. After collecting the cultures at 8 h intervals beginning at 4 h, total RNA was extracted using RNAiso Plus reagent (Takara, Liaoning, China), and reverse-transcribed using the PrimeScript RT Reagent Kit (Takara, Liaoning, China). PCR was performed using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China) on a ViiATM7 Dx system (Applied Biosystems, Foster, CA, United States), with the primers listed in Table 1. The 2−ΔΔCt method was used to calculate target gene expression levels, with the rpoD gene serving as an internal control.
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Table 1 Primers Used for Real-Time RT-PCR Analysis
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Statistical Analysis
All data are presented as the mean ± standard deviation (SD) of three independent experiments unless otherwise indicated. One-way ANOVA was performed using SPSS Statistics (version 23.0) to compare the differences between groups. Graphs were generated using the GraphPad Prism software (version 5.0; GraphPad Software, United States). Each experiment was repeated at least thrice to yield comparable results.P < 0.05. P values are represented as *P < 0.05; **P < 0.01; and ***P < 0.001.
Results
VPE Inhibits the Production of QS-Related Virulence Factors in P.aeruginosa PAO1
First, the growth of P. aeruginosa PAO1 was evaluated after VPE treatment. Our results showed that the MIC of VPE against PAO1 was 128 mg/mL, using the agar dilution method. At sub-MIC concentrations (1/2, 1/4, and 1/8 MIC, corresponding to 64, 32, and 16 mg/mL, respectively), PAO1 growth kinetics remained unaffected, indicating no inhibitory effect of VPE on P. aeruginosa growth (Figure S1). Sub-MICs of VPE exhibited dose-dependent anti-QS activity, evidenced by the notable halo zones around the disc against purple colonies (Figure S2), indicating the extremely promising anti-QS activity of VPE. As shown in Table 2, analysis of virulence factors revealed that sub-MICs of VPE significantly reduced the production of pyocyanin (1/8 MIC VPE treatment: 52% decrease; 1/4 MIC VPE treatment: 80% decrease) and rhamnolipids (1/8 MIC VPE treatment: 26% decrease; 1/4 MIC VPE treatment: 28% decrease). Correspondingly, swarming motility, related to rhamnolipid production, was also inhibited by VPE treatment, while swimming motility was unaffected (Table 2). Biofilm formation of P. aeruginosa was also inhibited by VPE treatment (1/8 MIC VPE treatment: 19% decrease; 1/4 MIC VPE treatment: 51% decrease) (Table 2). RT-qPCR assays showed that QS-regulatory genes (lasI, rhlI and pqsA) were downregulated in the presence of VPE, consistent with the suppression of virulence phenotypes including pyocyanin, rhamnolipids, motility, and biofilms (Figure S3). Overall, these results indicate that sub-MICs of VPE can inhibit QS-linked virulence traits in P. aeruginosa PAO1.
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Table 2 Inhibitory Effect of VPE on QS-Regulated Virulence
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VPE Components Assay by UHPLC-Q Exactive-Orbitrap MS
The total ion chromatograms (TIC) of V. philippica granule herbal extract, analyzed by UHPLC-Q Exactive-Orbitrap MS in both positive (ESI+) and negative (ESI−) ion mode, are presented in Figure 1. A total of 16 compounds were identified based on published literature25 (Table 3), comprising six coumarins, five flavonoids, and five organic acids. The coumarins identified are esculin (2), esculetin (3), 7-hydroxy-8-methoxyecoumarin (6), scopoletin (8, known as 6-methoxy-7-hydroxycoumarin), scoparone (9, known as 6,7-dimethoxycoumarin), and prionanthoside (11). Given that coumarins are widely found in various plants and some have shown potential antibacterial and antifungal effects, we are motivated to explore the coumarin derivatives scopoletin and scoparone in VPE. However, the role of these derivatives in the QS-related virulence of P. aeruginosa PAO1 has not been previously studied.
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Table 3 Compounds Identified from V.philippica Granule Herbal Extract
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Figure 1 The total ion chromatograms (TIC) of V. philippica granule herbal extract detected by UHPLC-Q Exactive-Orbitrap MS in positive ion mode (A) and negative ion mode (B).
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Scopoletin and Scoparone Influence the Production of QS-Related Virulence Factors in P.aeruginosa PAO1
We further studied the effects of scopoletin and scoparone on QS-related virulence factors of P. aeruginosa PAO1. At 1024 μg/mL, PAO1 growth kinetics were inhibited, while no significant effects were observed at sub-doses of 512, 256, and 128 μg/mL of scopoletin/scoparone treatment (Figure 2A). C. violaceum bioassay revealed dose-dependent increases in halo zones under scopoletin/scoparone treatment, with more pronounced zones under scopoletin treatment, suggesting its stronger QSI activity (Figure 2B). Pyocyanin synthesis decreased dose-dependently with scopoletin/scoparone exposure (128, 256, and 512 μg/mL), though the decrease was not significant at 128 μg/mL scoparone treatment (Figure 2C). Scopoletin significantly repressed rhamnolipid production, elastase activity, and biofilm formation (Figure 2D–F), while scoparone only significantly attenuated rhamnolipid production (P < 0.05) and biofilm formation (P < 0.01) at 512 μg/mL. Elastase activity was significantly inhibited by 256 and 512 μg/mL scoparone treatment, with a slight but insignificant downregulation at 128 μg/mL (Figure 2E). Consistent with the effects of VPE on PAO1 motility, swarming motility was also dose-dependently inhibited by scopoletin/scoparone treatment (Figure 3A), whereas swimming motility remained unaffected (Figure 3B). Overall, these results indicate that scopoletin and scoparone negatively regulated pyocyanin synthesis, rhamnolipid production, elastase activity, biofilm formation, and swarming motility, with scopoletin being a more potent inhibitor than scoparone.
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Figure 2 Scopoletin and scoparone influenced the production of QS-related virulence factors in P. aeruginosa PAO1. (A) Growth curve analysis of P. aeruginosa PAO1 under different concentrations of scopoletin/scoparone treatment. LB (DMSO), compound-free controls which added equal amount of DMSO for dissolving the compounds. (B) Different concentrations of scopoletin/scoparone were added into the well of LB agar plates containing C. violaceum CV026 which supplemented with C4-HSL and cultured at 28°C for 20 h. The violacein halo production was observed (top) and the diameter of the halo was measured (bottom). (C) Pyocyanin in the supernatant of PAO1 cultured with different concentrations of scopoletin/scoparone in LB medium for 20 h was measured. The pyocyanin was observed (top) and the extracted pyocyanin was measured at OD520 (bottom). (D) Rhamnolipid in the supernatant of PAO1 cultured with different concentrations of scopoletin/scoparone in M9 medium for 16 h was measured. The extracted rhamnolipid was observed (top) and measured at OD638 (bottom). (E) Elastase in the supernatant of PAO1 cultured with different concentrations of scopoletin/scoparone in LB medium for 8 h was measured. The elastase incubated with ECR for 4 h was observed (top) and measured at OD495 (bottom). (F) Biofilm formation of PAO1 cultured with different concentrations of scopoletin/scoparone in LB medium in the 96-well plate for 24 h was determined at OD595. SPL, scopoletin; SPR: scoparone; Ctrl, compound-free treatment. 128, 256 and 512: 128 μg/mL, 256 μg/mL and 512 μg/mL of scopoletin or scoparone. Data are shown as mean ± SD of at least three independent experiments. ns, nonsignificant. *P < 0.05; **P < 0.01; and ***P < 0.001.
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Figure 3 Scopoletin and scoparone inhibited swarming motility of PAO1, but had no impact on swimming. (A) PAO1 (5 μL) was spotted onto the middle of pre-prepared swarming plates with or without scopoletin or scoparone at 37°C for 16 h, and the diameters of the culture were measured. (B) PAO1 (5 μL) was spotted onto the middle of pre-prepared swimming plates with or without scopoletin or scoparone at 37°C for 16 h, and the diameters of the culture were measured. SPL, scopoletin; SPR: scoparone; Ctrl, compound-free plate. 128, 256 and 512: 128 μg/mL, 256 μg/mL and 512 μg/mL of scopoletin or scoparone. Data are shown as mean ± SD of at least three independent experiments. ns, nonsignificant. **P < 0.01; ***P < 0.001.
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Scopoletin and Scoparone Inhibit Hemolytic Activity of P.aeruginosa PAO1
As hemolysin is another important QS-related virulence factor, hemolysin production by P. aeruginosa PAO1 was evaluated after scopoletin and scoparone treatment. As shown in Figure 4, scopoletin significantly downregulated the hemolysis rate of PAO1 at doses of 256 and 512 μg/mL. Low doses of scoparone had no impact on the hemolysis rate, whereas a high dose of scoparone (512 μg/mL) inhibited hemolysin production of P. aeruginosa PAO1. Thus, these data indicate that scopoletin and scoparone decreased hemolytic activity.
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Figure 4 Scopoletin and scoparone inhibited hemolytic activity P.aeruginosa PAO1. 300 μL of 4% washed erythrocytes were added with 300 μL of mid-exponential phase P.aeruginosa PAO1 resuspended with PBS (experimental group, EG), 300 μL of PBS (negative control, NC) and 300μL of 2% Triton X-100 solution (positive control, PC) respectively, then scopoletin/scoparone was added. The cells were incubated at 37°C 6 h and supernatant was measured at OD450. SPL, scopoletin; SPR: scoparone; 128, 256 and 512: 128 μg/mL, 256 μg/mL and 512 μg/mL of scopoletin or scoparone. Data are shown as mean ± SD of at least three independent experiments. ns, nonsignificant. *P < 0.05; ***P < 0.001.
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Mechanism of the Anti-QS Activity of Scopoletin and Scoparone
As QS systems in P. aeruginosa govern the production of virulence factors, we investigated the effects of the compounds on QS-associated genes. P. aeruginosa has three canonical QS systems: two LuxI/LuxR QS circuits (LasI/LasR and RhlI/RhlR), and a quinolone-based signaling (pqs) system. Therefore, QS regulatory genes such as lasI, rhlI and pqsA were tested. P. aeruginosa PAO1 was co-cultured with various concentrations of scopoletin or scoparone for 4, 12, and 20 h, corresponding to the early, middle, and late stages of bacterial growth, respectively. qRT-PCR was used to detect the expression levels of lasI, rhlI and pqsA. As shown in Figure 5, scopoletin at 256 and 512 μg/mL extensively inhibited lasI, rhlI and pqsA expression, with more pronounced effects at 4 and 12 h. Scopoletin at 128 μg/mL also downregulated rhlI and pqsA at 4 and 12 h, lasI was downregulated, though not significantly at all time points. Scoparone at 256 and 512 μg/mL also had inhibitory effects on lasI, rhlI and pqsA, but the inhibitory potential was weaker than that of scopoletin treatment. Scoparone at 128 μg/mL had no significant impact. Our findings suggest that scopoletin and scoparone may universally suppress QS-associated genes in P. aeruginosa, mainly in the early and middle growth stages, thereby downregulating virulence factors synthesis.
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Figure 5 Effects of scopoletin/scoparone on the expression of QS regulator genes in PAO1. PAO1 was incubated with different concentrations of scopoletin/scoparone at 37°C 200 rpm for 4 h, 12 h and 20 h, then the expression levels of lasI (A), rhIl (B), and pqsA (C) were measured by qRT-PCR. PAO1 incubation without scopoletin/scoparone was used as a control. SPL: scopoletin; SPR: scoparone; 128, 256 and 512: 128 μg/mL, 256 μg/mL and 512 μg/mL of scopoletin or scoparone. Data are shown as mean ± SD of at least three independent experiments. ns, nonsignificant. *P < 0.05; **P < 0.01; and ***P < 0.001.
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Discussion
In this study, for the first time, we focused on P. aeruginosa PAO1 as a model to investigate mode of action of traditional Chinese medicinal V. philippica and its compounds against bacterial infections. Consistent with Zhang’s report on the virulence attenuation of P. aeruginosa by falcarindiol from an antipyretic drug Notopterygium incisum,26 we report for the first time that V. philippica, also an antipyretic herb, as a novel QSI inhibits QS-controlled virulence in P. aeruginosa PAO1. Further component analysis of VPE based on UHPLC-Q Exactive-Orbitrap MS reveal that two previously uncharacterized coumarin derivatives scopoletin and scoparone in VPE are identified as novel QSIs using C. violaceum CV026 bioassay. Scopoletin and scoparone significantly downregulate QS-related virulence of P. aeruginosa PAO1, such as pyocyanin, rhamnolipid, elastase, hemolysin, biofilm formation and swarming motility by inhibiting QS-related genes. This research fills the gap in the study of anti-virulence properties of V. philippica and its compounds against P. aeruginosa. Our results position V. philippica and its compounds as promising candidates for adjuvant therapy against drug-resistant P. aeruginosa infections.
The virulence factors produced by P. aeruginosa, including pyocyanin, rhamnolipid, elastases and proteases, are intricately regulated by a hierarchical QS regulatory network and are directly linked to the pathogenicity of the bacteria.27 Researchers have paid extensive attention to this elaborate regulatory strategy to develop therapeutics for infections caused by P. aeruginosa. Numerous bacteria use the QS cell–cell communication system to sense the density of their population. These bacteria create and recognize diffusible signaling molecules known as autoinducers, which regulate the synthesis of virulence factors, motility, and biofilm formation.28 The discovery of the P. aeruginosa QS system offers an alternative treatment to traditional chemotherapy. In particular, many plant-based natural products have been shown to possess anti-QS and anti-virulence properties.29 However, no reports have claimed V. philippica and its compounds to be anti-QS agents for the control of P. aeruginosa infection.
Currently, UHPLC-Q Exactive-Orbitrap MS system has been widely used to identify significantly different chemical compounds in various Chinese herb samples. In this study, we used UHPLC-Q Exactive-Orbitrap MS to qualitatively identify the V. philippica compounds. Previous research has shown that V. philippica contains flavonoids,30 coumarins,17 anthocyanins31 and cyclotides, etc.32 Though Wu et al reported 32 compounds within water extract of V. philippica using UHPLC-ESI-Q-TOF-MS/MS, including coumarin, flavonoids and organic acids,25 we identified 16 compounds (6 coumarins, 5 flavonoids and 5 organic acids) from VPE. Coumarins are a group of natural compounds containing a variety of molecules with similar basic structures. Coumarins have been found in various plants and some have been shown to have antibacterial, antifungal, and pharmacological potential.33 It has been identified that coumarins, aesculetin and aesculin, can interact with the signal-adhering domain of the TraR protein, thus inhibiting the biofilm formation of P. aeruginosa.34 R.E. D’Almeida et al compared the QS inhibitory effects of seven structurally related coumarins (dihydrocoumarin, coumarin, 3-hydroxycoumarin, 4-hydroxycoumarin, 6-hydroxycoumarin, 7-hydroxycoumarin and 6,7-dihydroxycoumarin) on P. aeruginosa and revealed the intensity of anti-biofilm and anti-QS activity to P. aeruginosa was structure-dependent.35 Therefore, we focused on five coumarin derivatives identified in VPE. Scopoletin, also known as 6-methoxy-7-hydroxycoumarin, is a phenolic coumarin isolated from many plants and has been shown to possess antibacterial activity in inhibiting the growth of certain pathogenic bacteria, such as S. aureus, Escherichia coli and Salmonella spp.36 Scoparone, also known as 6,7-dimethoxycoumarin/6,7-dimethylesculetin, is the major component of Artemisia capillaris Thunb. Scoparone is the main efficacious and active component of the Yin-Chen-Hao decoction (YCHD) and Yin-Zhi-Huang decoction (YZHD) for the treatment of hepatic and cholestatic disorders.37,38 Recent studies have shown that scoparone has extensive pharmacological activities such as anti-inflammatory, anti-fibrotic, anti-apoptotic, anti-coagulant, and anti-diabetic properties.39–41 Scopoletin is the demethylation product of scoparone, and we hypothesized that scoparone might also exert antibacterial effects. Altogether, we investigated whether the coumarin derivatives, scopoletin and scoparone, were responsible for the anti-QS activity of VPE (Table 2).
Considering the antibacterial activity of V. philippica18 and to prevent exerting excessive selective pressure on PAO1, the MIC of VPE was evaluated. Our findings revealed that the MIC of VPE against PAO1 was 128 mg/mL using the agar dilution method. Moreover, PAO1 growth kinetics were unaffected at different concentrations (1/2, 1/4, and 1/8 MIC, corresponding to 64 mg/mL, 32 mg/mL, and 16 mg/mL, respectively). Likewise, exposure of PAO1 to scopoletin/scoparone resulted in growth inhibition at a concentration of 1024 μg/mL, while no impact was observed at sub-doses of 512 μg/mL, 256 μg/mL, and 128 μg/mL. Consequently, sub-doses of VPE, scopoletin, and scoparone were employed in the subsequent phenotypic and genetic investigations, given that these doses did not exert any inhibitory effects on the growth of P. aeruginosa.
QS inhibitors (QSIs) are a promising alternative to antibacterial therapies, exerting less selective pressure for resistance than conventional antibiotics.42 The biosensor C. violaceum CV026 is a double mini-Tn5 mutant that is limited to producing the purple pigment violacein when given an exogenous short-chain AHL under the control of the CVi/R QS system.43 Approximately 200 studies have been published regarding its use as an indicator for QS activity.44 Here, we observed that the range of the nonviolacein halo around C. violaceum CV026 progressively expanded in tandem with the increased concentration of VPE, scopoletin, and scoparone, suggesting that VPE and its compounds impede C. violaceum CV026’s capacity to respond to exogenously given N-hexanoylhomoserine lactone. This finding is in line with the work of Tajani AS et al,45 who reported that a variety of coumarin derivatives exhibit anti-QS, and scopoletin and scoparone are two newly discovered coumarin derivatives with anti-QS functions.
P. aeruginosa produces many virulence factors including pyocyanin, rhamnolipids, elastases, and hemolysin. Interestingly, our findings revealed that VPE treatment decreased the production of pyocyanin and rhamnolipids without affecting cell viability (Table 2). Similarly, scopoletin and scoparone exhibited inhibitory effects against P. aeruginosa PAO1, resulting in reduced expression of virulence factors such as pyocyanin (Figure 2C), rhamnolipid (Figure 2D), elastase (Figure 2E), and hemolytic activity (Figure 4). Pyocyanin is synthesized from chorismate by the phzABCDEFG operons, along with phzM and phzS,46 and is responsible for the blue-green color characteristic of P. aeruginosa. A possible mechanism of pyocyanin inhibition is that VPE and scopoletin/scoparone affect quinolone biosynthesis, resulting in reduced pyocyanin synthesis. Rhamnolipid, a so-called “secondary metabolite”, is a biosurfactant regulated by rhlA and rhlB operons that cluster with rhlI and rhlR. Rhamnolipids also play multiple roles in biofilm formation by P. aeruginosa.47 Scopoletin significantly inhibited the production of rhamnoolipid, whereas scoparone only showed moderate attenuation at 512 μg/mL. Hence, VPE and scopoletin/scoparone might have operated by upsetting QS systems or resulting in dysregulation of rhamnolipid synthesis. Hemolysins are las-regulated by various virulence factors. Our results showed that scopoletin treatment significantly inhibited the hemolysis rate of PAO1, whereas scoparone treatment only inhibited the hemolysis rate at high doses (512 μg/mL). To sum up, our research has revealed that VPE, along with its bioactive components scopoletin/scoparone, effectively inhibit QS-regulated virulence factors in P. aeruginosa.
Pyocyanin and rhamnolipid are virulence phenotypes in liquid media, and motility is one of the virulence phenotypes manifested by surface-associated behaviors that are crucial for colonization, biofilm formation, and production of virulence factors. Swarming motility is an uncoordinated surface-associated motility of hyperflagellated and highly motile bacterial cells.48 Swarming motility necessitates the involvement of flagella, type IV pili, and rhamnolipid biosurfactants. In contrast to swarming, swimming motility refers to the movement of individual bacterial cells within liquid environments. Our research revealed that VPE and its compounds, scopoletin and scoparone, inhibited P. aeruginosa swarming motility, but not swimming motility, suggesting that VPE and its compounds may hinder flagellar functions. Considering that QS regulates bacterial motility, it is plausible that V. philippica inhibited the motility of P. aeruginosa either by either directly targeting flagella or by disrupting with the QS signaling pathway. Such interference would, in turn, hinder bacterial adhesion and colonization, biofilm formation, as well as the production of other virulence factors regulated by QS. The manifestation that anti-QS compounds only affect swarming but not swimming has likewise been reported in several other studies, such as those by Luo et al,8 Che et al49 and Thomas et al.50
Additionally, P. aeruginosa forms surface-attached communities known as biofilms, the so-called “bacterial cities”.51 Bacteria living in biofilms cause 80% of bacterial infections.52 Numerous studies have found that natural products, such as baicalin, hyperoside, allicin and garlicin, exhibit an inhibitory effect on biofilms.8,53,54 Similarly, we showed here that VPE and its compound scopoletin/scoparone can significantly inhibit the development of biofilm, suggesting their potential for treating chronic infections. The early stages of biofilm formation rely on bacterial motility and rhamnolipid production. Thus, changes in biofilm, motility, and rhamnolipid production are interrelated.
Many plant agents downregulate the P. aeruginosa virulence via unknown QS-inhibiting mechanisms.29 Studies have elucidated some anti-QS molecular pathways. In P. aeruginosa, the rhl and pqs systems are regulated by the las system, and all three QS systems are required for producing virulence factors such as pyocyanin, biofilm formation, drug resistance, and full pathogenicity.55 Our results showed that under VPE treatment, QS regulatory genes (lasI, rhlI and pqsA) were downregulated, correlating with the suppression of virulence phenotypes, including pyocyanin, rhamnolipids, motility, and biofilms. Consistent with the crude extract findings, high concentrations of compounds extensively inhibited lasI, rhlI and pqsA, with pqsA having a dramatic inhibitory effect, mainly in the early and middle stages of growth. These results suggest that scopoletin and scoparone may act as multi-target inhibitors of bacterial virulence, given the inhibition of all three hierarchical QS systems in P. aeruginosa. The inhibition mechanisms could be (i) top-to-bottom regulation: scopoletin/scoparone shutting down the las system, thereby downregulating the pqs and rhl systems; and (ii) scopoletin/scoparone inhibiting the pqs system, leading to the subsequent shutdown of the rhl system. Nevertheless, the inhibitory mechanisms of scopoletin and scoparone on QS-regulated virulence factors may be multifaceted, including: (a) inhibiting the synthesis of signaling molecules (eg, C4-HSL and 3-O-C12-HSL; (b) enzymatic degradation of signaling molecules (eg, acylase hydrolysis of AHLs); (c) competing with signaling molecules for binding to related receptors (eg, lasR, RhIR, PqsR); (d) interfering with the binding of signaling molecules to promoters to repress gene expression. Further in-depth studies, such as transcriptomics and proteomics research, are needed to reveal unknown targets.
Although this study demonstrates that scopoletin and scoparone exert anti-virulence effects by broadly inhibiting QS-regulated genes, the precise molecular mechanism remains unclear. Additionally, as this study is based on in vitro results, the in vivo effects of scopoletin and scoparone are yet to be determined. Future research should focus on two main aspects: (1) Elucidation of molecular mechanisms: using molecular docking, site-directed mutagenesis, signal synthesis and degradation assays, and protein–protein interaction studies to provide a deeper understanding of the molecular mechanisms underlying the anti-QS activities of scopoletin and scoparone, and further validate their potential as effective anti-virulence agents against P. aeruginosa infections; (2) In vivo experiments: establishing animal models of P. aeruginosa infection (eg, mouse lung infection models) to investigate the in vivo mechanisms of these compounds. In summary, our findings suggest that scopoletin and scoparone from V. philippica can be used as potential anti-virulence agents to control harmful infections caused by P. aeruginosa via QS inhibition.
Conclusion
P. aeruginosa is a common nosocomial pathogen causing various infections. There is an urgent need to search for innovative effective medicines that can prevent microbial resistance owing to the rise in carbapenem-resistant P. aeruginosa and antibiotic abuse. The virulence of P. aeruginosa is regulated by QS. Plant-based natural products are repositories of QS inhibitors (QSIs), which can block the generation or transmission of QS signals. Therefore, understanding the compounds from V. philippica that regulate the QS-related virulence of P. aeruginosa is important to combat infection. In this study, two previously uncharacterized coumarin derivatives, scopoletin and scoparone from V. philippica were identified as novel QSIs. They have been found to inhibit QS-mediated virulence factors, including pyocyanin, elastase, rhamnolipid, hemolysin, biofilm, and swarming by inhibiting the QS-related genes lasI, rhlI, and pqsA. This study provides a laboratory basis for using scopoletin and scoparone as promising anti-virulence candidates for adjuvant therapy against P. aeruginosa infection. Future research will focus on elucidating the molecular mechanisms and conducting in vivo investigations.
Data Sharing Statement
The datasets generated and/or analyzed during the current study are available from the corresponding author (email: [email protected]) upon reasonable request.
Ethics Approval and Informed Consent
For whole blood hemolysis assays, informed consent was obtained from each volunteer prior to study commencement. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (BE2024-311-01).
Author Contributions
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
Funding
This work was supported by the Guangzhou Basic and Applied Foundation (grant number 2023A03J0225), the the Traditional Chinese Medicine Bureau of Guangdong Province (grant numbers 20233022 and 20251151), Key Project of State Key Laboratory of Traditional Chinese Medicine Syndrome (grant number SKLKY2024B0003Z and QZ2023ZZ34), and the Guangdong Hospital of Traditional Chinese Medicine, China (grant number YN2022QN09).
Disclosure
The authors report no conflicts of interest in this work.
References
1. Qin S, Xiao W, Zhou C, et al. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther. 2022;7(1):199. doi:10.1038/s41392-022-01056-1
2. Rossi E, La Rosa R, Bartell JA, et al. Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis. Nat Rev Microbiol. 2021;19(5):331–342. doi:10.1038/s41579-020-00477-5
3. WHO. Who publishes list of bacteria for which new antibiotics are urgently needed. Available from: https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed.
4. Kachhadiya DK, Georrge JJ. Quorum Sensing and its Inhibition in Pseudomonas aeruginosa: molecular targets and mode of action. Curr Drug Targets. 2025;26(9):613–631. doi:10.2174/0113894501373124250410105111
5. Moradali MF, Ghods S, Rehm BH. Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol. 2017;7:39. doi:10.3389/fcimb.2017.00039
6. Woo S, Marquez L, Crandall WJ, Risener CJ, Quave CL. Recent advances in the discovery of plant-derived antimicrobial natural products to combat antimicrobial resistant pathogens: insights from 2018-2022. Nat Prod Rep. 2023;40(7):1271–1290. doi:10.1039/d2np00090c
7. Efferth T. From ancient herb to modern drug: Artemisia annua and artemisinin for cancer therapy. Semin Cancer Biol. 2017;46:65–83. doi:10.1016/j.semcancer.2017.02.009
8. Luo J, Kong JL, Dong BY, et al. Baicalein attenuates the quorum sensing-controlled virulence factors of Pseudomonas aeruginosa and relieves the inflammatory response in P. aeruginosa-infected macrophages by downregulating the MAPK and NFkappaB signal-transduction pathways. Drug Des Devel Ther. 2016;10:183–203. doi:10.2147/DDDT.S97221
9. Hernando-Amado S, Alcalde-Rico M, Gil-Gil T, Valverde JR, Martinez JL. Naringenin inhibition of the Pseudomonas aeruginosa quorum sensing response is based on its time-dependent competition with n-(3-oxo-dodecanoyl)-l-homoserine lactone for lasr binding. Front Mol Biosci. 2020;7:25. doi:10.3389/fmolb.2020.00025
10. Mostafa I, Abbas HA, Ashour ML, et al. Polyphenols from salix tetrasperma impair virulence and inhibit quorum sensing of Pseudomonas aeruginosa. Molecules. 2020;25(6):1341. doi:10.3390/molecules25061341
11. Zhao C, Zheng H, Zhou L, et al. Falcarindiol isolated from notopterygium incisum inhibits the quorum sensing of Pseudomonas aeruginosa. Molecules. 2021;26(19):5896. doi:10.3390/molecules26195896
12. Yu L, Pan H, Chen X, et al. Comprehensive mapping of cyclotides from Viola philippica by using mass spectrometry-based strategy. Molecules. 2024;29(18):4344. doi:10.3390/molecules29184344
13. Wang YL, Zhang L, Li MY, Wang LW, Ma CM. Lignans, flavonoids and coumarins from Viola philippica and their alpha-glucosidase and hcv protease inhibitory activities. Nat Prod Res. 2019;33(11):1550–1555. doi:10.1080/14786419.2017.1423305
14. Muluye RA, Bian Y, Alemu PN. Anti-inflammatory and antimicrobial effects of heat-clearing Chinese herbs: a current review. J Tradit Complement Med. 2014;4(2):93–98. doi:10.4103/2225-4110.126635
15. Jeong YH, Oh YC, Cho WK, Shin H, Lee KY, Ma JY. Anti-inflammatory effects of Viola yedoensis and the application of cell extraction methods for investigating bioactive constituents in macrophages. BMC Complement Altern Med. 2016;16(1):180. doi:10.1186/s12906-016-1142-9
16. Cao DL, Zhang XJ, Xie SQ, Fan SJ, Qu XJ. Application of chloroplast genome in the identification of traditional Chinese medicine Viola philippica. BMC Genomics. 2022;23(1):540. doi:10.1186/s12864-022-08727-x
17. Yang P, Yan F, Yang Y, Meng SY, Hou RJ. Study on chemical constituents of Viola yedoensis (in Chinese). J Xinxiang Med Coll. 2008;(02):185–187.
18. Xie C, Kokubun T, Houghton PJ, Simmonds MS. Antibacterial activity of the Chinese traditional medicine, zi hua di ding. Phytother Res. 2004;18(6):497–500. doi:10.1002/ptr.1497
19. Wang WD, Zhang NN, Chanda W, et al. Antibacterial and anti-biofilm activity of the lipid extract from mantidis ootheca on Pseudomonas aeruginosa. J Zhejiang Univ Sci B. 2018;19(5):364–371. doi:10.1631/jzus.B1700356
20. Pu J, Zhang S, He X, et al. The small RNA AmiL regulates quorum sensing-mediated virulence in Pseudomonas aeruginosa PAO1. Microbiol Spectr. 2022;10(2):e221121. doi:10.1128/spectrum.02211-21
21. Zhao Z, Yang T, Xiang G, et al. A novel small RNA PhaS contributes to polymyxin B-heteroresistance in carbapenem-resistant Klebsiella pneumoniae. Emerg Microbes Infect. 2024;13(1):2366354. doi:10.1080/22221751.2024.2366354
22. Li Y, Ding W, Yin J, et al. 2,3-dimethoxycinnamic acid from a marine actinomycete, a promising quorum sensing inhibitor in Chromobacterium violaceum. Mar Drugs. 2024;22(4):177. doi:10.3390/md22040177
23. Pinzon NM, Ju LK. Analysis of rhamnolipid biosurfactants by methylene blue complexation. Appl Microbiol Biotechnol. 2009;82(5):975–981. doi:10.1007/s00253-009-1896-9
24. Djokic L, Stankovic N, Galic I, et al. Novel quorum quenching ytnp lactonase from bacillus paralicheniformis reduces Pseudomonas aeruginosa virulence and increases antibiotic efficacy in vivo. Front Microbiol. 2022;13:906312. doi:10.3389/fmicb.2022.906312
25. Wu H, Zhan Y, Chen H, et al. Rapid identification of chemical constituents in violae herba by UHPLC-ESI-Q-TOF-MS /MS. Chin J Exp Traditional Med Formulae. 2016;22(24):70–75. doi:10.13422/j.cnki.syfjx.2016240070
26. Zhang P, Wu Q, Chen L, Duan K. Virulence-inhibiting herbal compound falcarindiol significantly reduced mortality in mice infected with Pseudomonas aeruginosa. Antibiotics. 2020;9(3):136. doi:10.3390/antibiotics9030136
27. Papenfort K, Bassler BL. Quorum sensing signal-response systems in gram-negative bacteria. Nat Rev Microbiol. 2016;14(9):576–588. doi:10.1038/nrmicro.2016.89
28. Kalia VC. Quorum sensing inhibitors: an overview. Biotechnol Adv. 2013;31(2):224–245. doi:10.1016/j.biotechadv.2012.10.004
29. Vadakkan K, Sathishkumar K, Mapranathukaran VO, et al. Critical review on plant-derived quorum sensing signaling inhibitors in Pseudomonas aeruginosa. Bioorg Chem. 2024;151:107649. doi:10.1016/j.bioorg.2024.107649
30. Xie C, Veitch NC, Houghton PJ, Simmonds MS. Flavone c-glycosides from Viola yedoensis makino. Chem Pharm Bull. 2003;51(10):1204–1207. doi:10.1248/cpb.51.1204
31. Zhang J, Wang LS, Gao JM, Xu YJ, Li LF, Li CH. Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography-photodiode array detection-electrospray ionisation mass spectrometry. Phytochem Anal. 2012;23(1):16–22. doi:10.1002/pca.1320
32. Wang CK, Colgrave ML, Gustafson KR, Ireland DC, Goransson U, Craik DJ. Anti-hiv cyclotides from the Chinese medicinal herb Viola yedoensis. J Nat Prod. 2008;71(1):47–52. doi:10.1021/np070393g
33. Sharifi-Rad J, Cruz-Martins N, Lopez-Jornet P, et al. Natural coumarins: exploring the pharmacological complexity and underlying molecular mechanisms. Oxid Med Cell Longev. 2021;2021(1):6492346. doi:10.1155/2021/6492346
34. Zeng Z, Qian L, Cao L, et al. Virtual screening for novel quorum sensing inhibitors to eradicate biofilm formation of Pseudomonas aeruginosa. Appl Microbiol Biotechnol. 2008;79(1):119–126. doi:10.1007/s00253-008-1406-5
35. D’Almeida RE, Molina R, Viola CM, et al. Comparison of seven structurally related coumarins on the inhibition of quorum sensing of Pseudomonas aeruginosa and Chromobacterium violaceum. Bioorg Chem. 2017;73:37–42. doi:10.1016/j.bioorg.2017.05.011
36. More G, Lall N, Hussein A, Tshikalange TE. Antimicrobial constituents of Artemisia afra jacq. ex Willd. against periodontal pathogens. Evid Based Complement Alternat Med. 2012;2012:252758. doi:10.1155/2012/252758
37. Li JY, Cao HY, Sun L, et al. Therapeutic mechanism of Yin-Chen-Hao decoction in hepatic diseases. World J Gastroenterol. 2017;23(7):1125–1138. doi:10.3748/wjg.v23.i7.1125
38. Chen X, Krakauer T, Oppenheim JJ, Howard OM. Yin zi huang, an injectable multicomponent Chinese herbal medicine, is a potent inhibitor of t-cell activation. J Altern Complement Med. 2004;10(3):519–526. doi:10.1089/1075553041323687
39. Liu B, Deng X, Jiang Q, et al. Scoparone improves hepatic inflammation and autophagy in mice with nonalcoholic steatohepatitis by regulating the ros/p38/nrf2 axis and pi3k/akt/mtor pathway in macrophages. Biomed Pharmacother. 2020;125:109895. doi:10.1016/j.biopha.2020.109895
40. Liu C, Li S, Zhang Q, et al. Emerging role of Chinese herbal medicines in the treatment of pancreatic fibrosis. Am J Chin Med. 2019;47(4):709–726. doi:10.1142/S0192415X1950037X
41. Wang Y, Wang M, Chen B, Shi J. Scoparone attenuates high glucose-induced extracellular matrix accumulation in rat mesangial cells. Eur J Pharmacol. 2017;815:376–380. doi:10.1016/j.ejphar.2017.09.039
42. Silva LN, Zimmer KR, Macedo AJ, Trentin DS. Plant natural products targeting bacterial virulence factors. Chem Rev. 2016;116(16):9162–9236. doi:10.1021/acs.chemrev.6b00184
43. Deryabin D, Galadzhieva A, Kosyan D, Duskaev G. Plant-derived inhibitors of ahl-mediated quorum sensing in bacteria: modes of action. Int J Mol Sci. 2019;20(22):5588. doi:10.3390/ijms20225588
44. Harrison AM, Soby SD. Reclassification of Chromobacterium violaceum ATCC 31532 and its quorum biosensor mutant cv026 to chromobacterium subtsugae. AMB Express. 2020;10(1):202. doi:10.1186/s13568-020-01140-1
45. Tajani AS, Amiri TZ, Pourmohammad A, et al. Anti-quorum sensing and antibiofilm activity of coumarin derivatives against Pseudomonas aeruginosa pao1: insights from in vitro and in silico studies. Iran J Basic Med Sci. 2023;26(4):445–452. doi:10.22038/IJBMS.2023.69016.15047
46. Mudaliar SB, Bharath PA. A biomedical perspective of pyocyanin from Pseudomonas aeruginosa: its applications and challenges. World J Microbiol Biotechnol. 2024;40(3):90. doi:10.1007/s11274-024-03889-0
47. Soberon-Chavez G, Gonzalez-Valdez A, Soto-Aceves MP, Cocotl-Yanez M. Rhamnolipids produced by pseudomonas: from molecular genetics to the market. Microb Biotechnol. 2021;14(1):136–146. doi:10.1111/1751-7915.13700
48. Bru JL, Kasallis SJ, Zhuo Q, Hoyland-Kroghsbo NM, Siryaporn A. Swarming of P. aeruginosa: through the lens of biophysics. Biophys Rev. 2023;4(3):31305. doi:10.1063/5.0128140
49. O’May C, Tufenkji N. The swarming motility of Pseudomonas aeruginosa is blocked by cranberry proanthocyanidins and other tannin-containing materials. Appl Environ Microbiol. 2011;77(9):3061–3067. doi:10.1128/AEM.02677-10
50. Clamens T, Rosay T, Crepin A, et al. The aliphatic amidase amie is involved in regulation of Pseudomonas aeruginosa virulence. Sci Rep. 2017;7(1):41178. doi:10.1038/srep41178
51. Lee K, Yoon SS. Pseudomonas aeruginosa biofilm, a programmed bacterial life for fitness. J Microbiol Biotechnol. 2017;27(6):1053–1064. doi:10.4014/jmb.1611.11056
52. Fleming D, Rumbaugh KP. Approaches to dispersing medical biofilms. Microorganisms. 2017;5(2):15. doi:10.3390/microorganisms5020015
53. Saleem M, Nazir M, Ali MS, et al. Antimicrobial natural products: an update on future antibiotic drug candidates. Nat Prod Rep. 2010;27(2):238–254. doi:10.1039/b916096e
54. Sun Y, Sun F, Feng W, et al. Hyperoside inhibits biofilm formation of Pseudomonas aeruginosa. Exp Ther Med. 2017;14(2):1647–1652. doi:10.3892/etm.2017.4641
55. Lee J, Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell. 2015;6(1):26–41. doi:10.1007/s13238-014-0100-x