Aqueous extracts of 
ASADH aspartate-semialdehyde dehydrogenase CF Cystic fibrosis CFU colony forming unit CV crystal violet DMSO Dimethyl sulfoxide DPPH 2,2-diphenyl-1-picrylhydrazyl EPS Extracellular polymeric substance FRAP Ferric Reducing Antioxidant Power MRSA methicillin-resistant Staphylococcus aureus QS quorum sensing RMSD root-mean-square deviation SEB Staphylococcus aureus enterotoxin B SCV Small colony variant 

Introduction Bacterial infections are a major contributor to the decline in lung function, respiratory failure, and premature death of cystic fibrosis (CF) patients. CF is a genetic disorder characterized by severe airway complications, leading to the accumulation of thick and viscous mucus, which creates a propitious environment for the development of respiratory chronic infections . Staphylococcus aureus is one of the first pathogens to colonize the airways of CF patients, with a prevalence reaching approximately 75 % in children aged 6–17 years . Meanwhile, Pseudomonas aeruginosa is currently considered the most relevant pathogenic agent responsible for chronic infection development, decreased quality of life, long hospitalizations, and ultimately the high number of deaths [ , , ]. Biofilm formation plays a central role in the long-term persistence and resistance of both S. aureus and P. aeruginosa . Biofilms provide multifactorial mechanisms that contribute to antibiotic resistance, including restricted penetration of antibiotics through the matrix, slow growth of bacteria due to nutritional constraints and restricted oxygen penetration, quorum sensing (QS), expression of biofilm-specific genes, and the presence of persister cells [ , , ]. Additionally, biofilms facilitate interactions between bacterial species in CF lungs, which can enhance resistance to antibiotics through coordinated activities . Depending on the age, 40–95 % of CF patients are coinfected with P. aeruginosa and S. aureus , and these coinfections often result in more severe effects on lung function and poorer clinical prognosis compared to mono-infections, which explain the biofilm relevance in CF disease . A particularly challenging aspect of CF-related infections is the presence of small colony variants (SCV) and the mucoid phenotype. SCV of S. aureus and P. aeruginosa are slow-growing populations that exhibit increased resistance to antibiotics and immune response due to their altered metabolism, ability to survive under the nutrient-depleted and anaerobic conditions of CF lungs, and intracellular survival [ , , , ]. These adaptations allow SCV to evade conventional therapies, contributing to chronic infection and the progressive decline in lung function . Similarly, the mucoid phenotype of P. aeruginosa , which is characterized by excessive alginate production, an exopolysaccharide that creates a protective barrier around the bacteria, is strongly linked to chronic infections and antibiotic resistance. This alginate matrix enhances biofilm formation, complicating treatment and resulting in more severe lung damage [ , , ]. Over the past decades, therapeutic advancements, such as Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulators that target the CFTR protein responsible for regulating chloride and sodium ion transport across cell membranes, and mucoactive drugs have significantly increased the survival of CF patients, with a median age at death rising to 36.9 years in 2023 [ , , , ]. Although these drugs have notably improved disease management , they have no reported effect on bacterial infections. Antibiotics remain the cornerstone of CF treatment, however, they are not specifically designed to target biofilms, underscoring the pressing need for compounds in CF therapeutic regimes that can alter the physiological state of antibiotic-tolerant biofilm cells to enhance antibiotic efficacy . Plants have shown great potential as therapeutic agents, offering anti-inflammatory, anticancer, antioxidant, and antimicrobial properties. They have been a valuable source in providing chemical architectures for the drug discovery pipeline of antibiofilm drugs [ , , ]. For instance, Camellia sinensis (L.) Kuntze (tea plant) was shown to reduce P. aeruginosa biofilm biomass and weaken biofilm structure, while also reducing the expression of several virulence factors . Also, the seeds of Capsicum baccatum var. pendulum (Willd.) Eshbaugh (red pepper) interfered with P. aeruginosa bacterial adhesion to the surface and only allowed the formation of smaller cell clusters without biofilm matrix . Regarding S. aureus , both Melaleuca alternifolia (Maiden & Betche) Cheel (tea tree) and leaves of Duabanga grandiflora (Roxb. Ex DC.) Walp. (Lamphu Tree) reduced the bacterial attachment to the surface, leading to the formation of weak biofilms, with tea tree being able to target methicillin-resistant Staphylococcus aureus (MRSA) . A water extract of Artemisia princeps Pamp. (mugwort) also showed activity against MRSA inhibiting biofilm formation and the expression of some virulence genes, including mecA , sea , agrA , and sarA involved in the biofilm formation . Plant extracts are complex mixtures of a variety of compounds, such as proteins, carbohydrates, lipids, and secondary metabolites. The antibiofilm activity of several plant extracts is often attributed to their secondary metabolites, or phytochemicals, including phenolics. Plant extracts rich in phenolic compounds have shown the ability to inhibit biofilms by different mechanisms; including reducing bacterial virulence , interfering with quorum-sensing  or disrupting mature biofilms . Despite extensive research into antibiofilm drug discovery, no effective treatment specifically targeting biofilms in CF lungs currently exists, and antibiofilm agents are not yet part of standard CF therapeutic regimes. Developing plant-based antibiofilm drugs to prevent biofilm formation remains a significant challenge, representing a novel and promising avenue in drug discovery and development. To the best of our knowledge, this study is the first to investigate the antibiofilm activity of plant extracts specifically against mucoid and SCV strains of P. aeruginosa, and S. aureus . The objective of this study is to identify potent candidate extracts, elucidate their mechanism of action and identify phytochemicals responsible for antibiofilm activity, with the potential to progress these findings into drug discovery and development for CF treatment. The selected plants, eucalyptus ( Eucalyptus globulus Labill.), broom flowers ( Cytisus scoparius (L.) Link), pine ( Pinus pinaster Aiton), moringa ( Moringa oleifera Lam.), cinnamon ( Cinnamomum cassia (L.) J.Presl) and lemongrass ( Cymbopogon citratus (DC.) Stapf), were chosen based on their documented antimicrobial and antioxidant properties, as well as their known antibiofilm activity against bacteria [ , , , ]. 

Materials and methods 2.1 Plant material Eucalyptus leaves ( Eucalyptus globulus Labill.) and broom flowers ( Cytisus scoparius (L.) Link) were collected in Braga, Portugal, in September 2021 and in April 2022, respectively. Pine bark ( Pinus pinaster Aiton) was collected in Ponte de Lima, Portugal, in April 2019. Moringa seeds and leaves ( Moringa oleifera Lam.) were collected in Ceará, Brazil, in December 2017. Cinnamon bark ( Cinnamomum cassia (L.) J.Presl) and lemongrass ( Cymbopogon citratus (DC.) Stapf) were purchased from a local supermarket. The plant parts were washed, dried and milled to a fine powder. 2.2 Extractions Solid-liquid extractions were performed in a water bath at 70 °C and 150 rpm for 1 h, using different solvents aiming at obtaining extracts rich in compounds with different polarities: water, ethanol 70 % (v/v) (EtOH70) or ethanol 96 % (v/v) (EtOH96), in triplicate. The extractions used a solid load of 5 % (2 g of dry plant biomass in 40 mL of solvent). After the extraction process, the product was filtered under vacuum. Liquid extracts were then analysed to determine the extraction yield and the extract concentration, antioxidant activity, and total phenolic content. The remaining extracts were stored at −20 °C until further chemical characterization and microbiology assays. 2.3 Extraction yield The concentration of each extract was determined gravimetrically. Specifically, 1 mL of extract was dried overnight at 105 °C and the weight of the dry extract was measured. The test was made in triplicate. The extraction yield (expressed in %) was calculated as shown in Equation : (1) Extraction yield ( % ) = dried extract ( g ) initial liquid extract ( g ) × 100 2.4 Antioxidant activity 2.4.1 Ferric Reducing Antioxidant Power Antioxidant activity was determined using the Ferric Reducing Antioxidant Power (FRAP) assay . 6-Hidroxy-2,5,7,8-tetra-methylchromone-2-carboxylic acid (Trolox), prepared in methanol, was used as standard. The 2,4,6-Tripyridyl- s -Triazine solution (TPTZ) with 10 mM was prepared in 40 mM HCl and the ferric chloride solution at 20 mM was prepared in water. The FRAP working solution was prepared by mixing 10-vol of acetate buffer at 300 mM (pH 3.6) with 1-vol of TPTZ and 1-vol of ferric chloride. The mixture was composed of 20 μL of liquid extracts resulting from each extraction and 280 μL FRAP working solution. The mixture was incubated at 37 °C for 30 min, protected from light, and the absorbance was measured at 595 nm. Antioxidant activity was expressed as milligrams of Trolox equivalent per milligram of dry extract (mg TEAC/mg extract). Analyses were performed in triplicate. 2.4.2 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity Antioxidant activity was also determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay . Trolox, prepared in methanol, was used as standard. The mixture was composed of 20 μL of liquid extracts resulting from each extraction and 180 μL DPPH solution (150 μM, dissolved in 80 % methanol). The mixture was incubated at room temperature for 30 min, protected from light, and the absorbance was measured at 515 nm. DPPH radical scavenger activity was calculated as shown in Equation : (2) % inhibition = Ac − As Ac × 100 where Ac is absorbance of control and As is absorbance of sample. Results were expressed as milligrams of Trolox equivalent per milligram of dry extract (mg TEAC/mg extract). Analyses were performed in triplicate. 2.5 Total phenolic content Total phenolic content was determined according to the Folin-Ciocalteu method . The reaction mixture was composed of 20 μL of liquid extract resulting from each extraction, 100 μL Folin-Ciocalteu reagent 10 % (1:10 in water) and 80 μL of sodium carbonate 75 g/L. The mixture was incubated at 42 °C for 30 min, protected from light. The absorbance was measured at 750 nm using a microplate reader. Gallic acid was used as standard. Total phenolic content was expressed as milligrams of gallic acid equivalents per milligrams of extract (mg GAE/mg extract), and phenolic extraction yield was expressed as milligrams of gallic acid equivalents per gram of dry matrix (mg GAE/g matrix). Analyses were performed in triplicate. 2.6 HPLC-TOF-MS An exploratory identification of phytochemicals compounds recovered was performed using high-performance liquid chromatography (HPLC) time-of-flight mass spectrometry (HPLC-TOF-MS). The plant extracts were injected into a ZORBAX Eclipse XDB-C18 fast resolution HD (2.1 × 100 mm 1.8 μm de Agilent), and the HPLC separation was performed using an Elute HPLC from Bruker Daltonics. The mobile phases used were 0.1 % formic acid in water (solvent A) and acidified acetonitrile with 0.1 % formic acid (solvent B), with a flow rate of 0.4 mL/min. The linear gradient was as follows: 2 % solvent B over 2 min, 2 %–30 % solvent B over 13 min, 30 %–100 % solvent B over 2 min, 100 % solvent B over 4 min, 100 %–2 % solvent B over 1 min, and 2 % solvent B isocratically for 2 min. An ESI source created ions in negative and positive ion mode. The operating circumstances were as follows: 3000 V capillary voltage, 500 V end plate offset, 8.0 L/min dry gas, 2 bar nebulizer pressure, and a dry heater set to 220 °C. Metabolite identification was based on reliable mass data, isotopic pattern matching (mSigma value), retention duration (where a standard was provided), and substances reported in literature. 2.7 Bacterial strains and culture conditions The antibiofilm activity of the extracts was determined using two strains of S. aureus including ATCC 25923 and a CF clinical isolate with SCV phenotype, named SA-1 and SA-SCV, respectively. Additionally, three P. aeruginosa strains were included: two CF clinical isolates, representing a mucoid phenotype and a SCV phenotype, named PA-Muc and PA-SCV respectively, and a non-CF isolate named PAI. The clinical isolates used in this study were generously provided by various Portuguese Hospitals. These strains were selected based on their phenotypic characteristics to represent the variety of strains commonly isolated from CF patients. Bacteria were routinely cultured on Tryptic Soy Broth (TSB, Liofilchem) or Tryptic Soy Agar (TSA, Liofilchem) at 37 °C. All strains were preserved in cryovials (Nalgene) at −80 ± 2 °C. Prior to each experiment, bacterial cells were grown on TSA plates overnight at 37 °C. 2.8 Biofilm formation inhibition assay Serial concentrations of plant extracts were tested in 96-well microtiter plates with flat bottoms (Orange) using TSB, with 100 μL of extracts in each well. For the ethanolic and hydroethanolic extracts, before the serial dilutions, the solvent was first evaporated using nitrogen and then the extract was dissolved in 5 % of dimethyl sulfoxide (DMSO). Overnight inocula of each bacteria strain were washed twice in sterile water by centrifugation (9000 g , 5 min) and further serial diluted in sterile water. The cell suspensions were added to 96-well microtiter plates (100 μL per well) to obtain a final concentration of 1 × 10 7 CFU/mL. The plate was then incubated for 24 h at 37 °C and 120 rpm to allow biofilm formation. After 24 h of growth, planktonic cells (liquid content of the wells) were removed, and each well was washed twice with distilled water. Biofilm formation was determined using the crystal violet (CV) method . 200 μL of methanol was added to each well to fix the biofilm for 15 min. After the wells dried out, CV (1 %) was added to dye biofilms for 5 min and washed twice with distilled water. 200 μL of glacial acetic acid (33 %) was added and mixed (pipette up and down) with CV retained by biofilm biomass. The absorbance was measured at 570 nm using a microplate reader. Biofilm inhibition was calculated as shown in Equation (3) % inhibition = Au − Ae Au × 100 where Au is the absorbance of untreated biofilm and Ae is the absorbance of biofilm treated with extract. Analyses were performed, at least three times and the results are expressed in percentage of biofilm inhibition. 2.9 Assessment of bacterial adhesion The viability of P. aeruginosa and S. aureus cells adhered after the application of plant extracts was determined through colony-forming unit (CFU). The formation of biofilm was performed in 96-well plates flat bottom (Orange) as described in section . After 24 h of growth, planktonic cells were removed, and each well was washed twice with distilled water. 200 μL of distilled water was added to each well and the plate placed on an ultrasonic water bath for 10 min to release the biofilm cells from the well surface as previously optimized . The content of five wells was transferred to a microcentrifuge tube to make up a volume of 1 mL and vortexed to homogenise the cell suspensions. Afterwards, the cell suspensions were serially diluted and plated on TSA to determine the number of culturable cells adhered to the wells. After overnight growth at 37 °C, CFUs were counted. Analyses were performed in triplicate and the results were expressed as log 10 CFU per volume (mL). 2.10 Assessment of antibiofilm activity by brightfield microscopy Brightfield microscopic assessment of all bacterial biofilms was accomplished to investigate the antibiofilm activity. The methodology described in section was slightly altered to allow the biofilm formation on a coverslip. Briefly, a plastic coverslip (Thermanox) was placed on the bottom of wells in a 24-well plate and 1 mL of the selected concentration of each extract was added to the well on top of the coverslip. Further, 1 mL of bacterial suspension from overnight inocula was added to the wells to make a total volume of 2 mL and a final concentration of 1 × 10 7 CFU/mL. The plate was incubated for 24 h at 37 °C and 120 rpm to allow biofilm formation. After 24 h growth, the well liquid content was removed, leaving the coverslip in place, and each well was washed twice with distilled water. The coverslip was stained using the CV method as described previously with slight alterations. 500 μL of methanol was added to each well to fix the biofilm to the coverslip and after 15 min, methanol was removed. After the coverslips dried out, 500 μL of CV reagent (1 %) was added to dye the biofilm for 5 min. The excess stain was washed off using distilled water, and the coverslip was placed on a microscope glass slide to observe the biofilms under a brightfield microscope (10× magnification) (Olympus BX51TF). 2.11 Targets prediction Putative targets of the most abundant phytochemical, moringin, were predicted using PharmMapper . The parameters set in PharmMapper included a Maximum of 300 Generated Conformations and selecting ‘all targets’ option . The results were ranked by normalized fit-score. 2.12 Molecular docking RCSB-Protein Data Bank  was used to obtain the three-dimensional (3D) structure of proteins from P. aeruginosa and S. aureus indicated by PharmMapper. The 3D structure of the moringin was obtained from PubChem (ID 153557)  in.sdf format. The protein structure was pre-processed using the protein preparation wizard, which involved adding the missing residues in the crystal structure and removing the water molecules surrounding the receptor. Moringin was individually docked with the two proteins using the molecular docking software AutoDock 4.2.6 . The docking procedure followed previously established protocols, with a grid size configured for blind docking. The grid box was set to 126 x 126 x 126 for x, y and z axis, respectively, using Autogrid program in such a way that encompass the entire molecule of moringin. Docking simulation were carried out using the Lamarckian genetic algorithm with default parameters. The docking protocol included 100 different runs (RMSD tolerance of 2.0 Å) with a population size of 150 and a maximum number of evaluations of 25 000 000 (set to ‘long’). After each docking calculation, the generated docked conformations were ranked based on predicted binding energy. The best energy conformation of the moringin (ligand)-protein complex and orientation of the ligand at the binding site were analysed using USCF Chimera 1.8 (RBVI, USA) and Biovia Discovery Studio . 2.13 Statistical analysis All data was analysed using GraphPad Prism software package (GraphPad Software version 8). Statistical analysis was carried out by ANOVA with Tukey's multiple comparison. Differences were considered statistically significant at p < 0.05. 

Plant material Eucalyptus leaves ( Eucalyptus globulus Labill.) and broom flowers ( Cytisus scoparius (L.) Link) were collected in Braga, Portugal, in September 2021 and in April 2022, respectively. Pine bark ( Pinus pinaster Aiton) was collected in Ponte de Lima, Portugal, in April 2019. Moringa seeds and leaves ( Moringa oleifera Lam.) were collected in Ceará, Brazil, in December 2017. Cinnamon bark ( Cinnamomum cassia (L.) J.Presl) and lemongrass ( Cymbopogon citratus (DC.) Stapf) were purchased from a local supermarket. The plant parts were washed, dried and milled to a fine powder. 

Extractions Solid-liquid extractions were performed in a water bath at 70 °C and 150 rpm for 1 h, using different solvents aiming at obtaining extracts rich in compounds with different polarities: water, ethanol 70 % (v/v) (EtOH70) or ethanol 96 % (v/v) (EtOH96), in triplicate. The extractions used a solid load of 5 % (2 g of dry plant biomass in 40 mL of solvent). After the extraction process, the product was filtered under vacuum. Liquid extracts were then analysed to determine the extraction yield and the extract concentration, antioxidant activity, and total phenolic content. The remaining extracts were stored at −20 °C until further chemical characterization and microbiology assays. 

Extraction yield The concentration of each extract was determined gravimetrically. Specifically, 1 mL of extract was dried overnight at 105 °C and the weight of the dry extract was measured. The test was made in triplicate. The extraction yield (expressed in %) was calculated as shown in Equation : (1) Extraction yield ( % ) = dried extract ( g ) initial liquid extract ( g ) × 100 

Antioxidant activity 2.4.1 Ferric Reducing Antioxidant Power Antioxidant activity was determined using the Ferric Reducing Antioxidant Power (FRAP) assay . 6-Hidroxy-2,5,7,8-tetra-methylchromone-2-carboxylic acid (Trolox), prepared in methanol, was used as standard. The 2,4,6-Tripyridyl- s -Triazine solution (TPTZ) with 10 mM was prepared in 40 mM HCl and the ferric chloride solution at 20 mM was prepared in water. The FRAP working solution was prepared by mixing 10-vol of acetate buffer at 300 mM (pH 3.6) with 1-vol of TPTZ and 1-vol of ferric chloride. The mixture was composed of 20 μL of liquid extracts resulting from each extraction and 280 μL FRAP working solution. The mixture was incubated at 37 °C for 30 min, protected from light, and the absorbance was measured at 595 nm. Antioxidant activity was expressed as milligrams of Trolox equivalent per milligram of dry extract (mg TEAC/mg extract). Analyses were performed in triplicate. 2.4.2 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity Antioxidant activity was also determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay . Trolox, prepared in methanol, was used as standard. The mixture was composed of 20 μL of liquid extracts resulting from each extraction and 180 μL DPPH solution (150 μM, dissolved in 80 % methanol). The mixture was incubated at room temperature for 30 min, protected from light, and the absorbance was measured at 515 nm. DPPH radical scavenger activity was calculated as shown in Equation : (2) % inhibition = Ac − As Ac × 100 where Ac is absorbance of control and As is absorbance of sample. Results were expressed as milligrams of Trolox equivalent per milligram of dry extract (mg TEAC/mg extract). Analyses were performed in triplicate. 

Ferric Reducing Antioxidant Power Antioxidant activity was determined using the Ferric Reducing Antioxidant Power (FRAP) assay . 6-Hidroxy-2,5,7,8-tetra-methylchromone-2-carboxylic acid (Trolox), prepared in methanol, was used as standard. The 2,4,6-Tripyridyl- s -Triazine solution (TPTZ) with 10 mM was prepared in 40 mM HCl and the ferric chloride solution at 20 mM was prepared in water. The FRAP working solution was prepared by mixing 10-vol of acetate buffer at 300 mM (pH 3.6) with 1-vol of TPTZ and 1-vol of ferric chloride. The mixture was composed of 20 μL of liquid extracts resulting from each extraction and 280 μL FRAP working solution. The mixture was incubated at 37 °C for 30 min, protected from light, and the absorbance was measured at 595 nm. Antioxidant activity was expressed as milligrams of Trolox equivalent per milligram of dry extract (mg TEAC/mg extract). Analyses were performed in triplicate. 

2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity Antioxidant activity was also determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay . Trolox, prepared in methanol, was used as standard. The mixture was composed of 20 μL of liquid extracts resulting from each extraction and 180 μL DPPH solution (150 μM, dissolved in 80 % methanol). The mixture was incubated at room temperature for 30 min, protected from light, and the absorbance was measured at 515 nm. DPPH radical scavenger activity was calculated as shown in Equation : (2) % inhibition = Ac − As Ac × 100 where Ac is absorbance of control and As is absorbance of sample. Results were expressed as milligrams of Trolox equivalent per milligram of dry extract (mg TEAC/mg extract). Analyses were performed in triplicate. 

Total phenolic content Total phenolic content was determined according to the Folin-Ciocalteu method . The reaction mixture was composed of 20 μL of liquid extract resulting from each extraction, 100 μL Folin-Ciocalteu reagent 10 % (1:10 in water) and 80 μL of sodium carbonate 75 g/L. The mixture was incubated at 42 °C for 30 min, protected from light. The absorbance was measured at 750 nm using a microplate reader. Gallic acid was used as standard. Total phenolic content was expressed as milligrams of gallic acid equivalents per milligrams of extract (mg GAE/mg extract), and phenolic extraction yield was expressed as milligrams of gallic acid equivalents per gram of dry matrix (mg GAE/g matrix). Analyses were performed in triplicate. 

HPLC-TOF-MS An exploratory identification of phytochemicals compounds recovered was performed using high-performance liquid chromatography (HPLC) time-of-flight mass spectrometry (HPLC-TOF-MS). The plant extracts were injected into a ZORBAX Eclipse XDB-C18 fast resolution HD (2.1 × 100 mm 1.8 μm de Agilent), and the HPLC separation was performed using an Elute HPLC from Bruker Daltonics. The mobile phases used were 0.1 % formic acid in water (solvent A) and acidified acetonitrile with 0.1 % formic acid (solvent B), with a flow rate of 0.4 mL/min. The linear gradient was as follows: 2 % solvent B over 2 min, 2 %–30 % solvent B over 13 min, 30 %–100 % solvent B over 2 min, 100 % solvent B over 4 min, 100 %–2 % solvent B over 1 min, and 2 % solvent B isocratically for 2 min. An ESI source created ions in negative and positive ion mode. The operating circumstances were as follows: 3000 V capillary voltage, 500 V end plate offset, 8.0 L/min dry gas, 2 bar nebulizer pressure, and a dry heater set to 220 °C. Metabolite identification was based on reliable mass data, isotopic pattern matching (mSigma value), retention duration (where a standard was provided), and substances reported in literature. 

Bacterial strains and culture conditions The antibiofilm activity of the extracts was determined using two strains of S. aureus including ATCC 25923 and a CF clinical isolate with SCV phenotype, named SA-1 and SA-SCV, respectively. Additionally, three P. aeruginosa strains were included: two CF clinical isolates, representing a mucoid phenotype and a SCV phenotype, named PA-Muc and PA-SCV respectively, and a non-CF isolate named PAI. The clinical isolates used in this study were generously provided by various Portuguese Hospitals. These strains were selected based on their phenotypic characteristics to represent the variety of strains commonly isolated from CF patients. Bacteria were routinely cultured on Tryptic Soy Broth (TSB, Liofilchem) or Tryptic Soy Agar (TSA, Liofilchem) at 37 °C. All strains were preserved in cryovials (Nalgene) at −80 ± 2 °C. Prior to each experiment, bacterial cells were grown on TSA plates overnight at 37 °C. 

Biofilm formation inhibition assay Serial concentrations of plant extracts were tested in 96-well microtiter plates with flat bottoms (Orange) using TSB, with 100 μL of extracts in each well. For the ethanolic and hydroethanolic extracts, before the serial dilutions, the solvent was first evaporated using nitrogen and then the extract was dissolved in 5 % of dimethyl sulfoxide (DMSO). Overnight inocula of each bacteria strain were washed twice in sterile water by centrifugation (9000 g , 5 min) and further serial diluted in sterile water. The cell suspensions were added to 96-well microtiter plates (100 μL per well) to obtain a final concentration of 1 × 10 7 CFU/mL. The plate was then incubated for 24 h at 37 °C and 120 rpm to allow biofilm formation. After 24 h of growth, planktonic cells (liquid content of the wells) were removed, and each well was washed twice with distilled water. Biofilm formation was determined using the crystal violet (CV) method . 200 μL of methanol was added to each well to fix the biofilm for 15 min. After the wells dried out, CV (1 %) was added to dye biofilms for 5 min and washed twice with distilled water. 200 μL of glacial acetic acid (33 %) was added and mixed (pipette up and down) with CV retained by biofilm biomass. The absorbance was measured at 570 nm using a microplate reader. Biofilm inhibition was calculated as shown in Equation (3) % inhibition = Au − Ae Au × 100 where Au is the absorbance of untreated biofilm and Ae is the absorbance of biofilm treated with extract. Analyses were performed, at least three times and the results are expressed in percentage of biofilm inhibition. 

Assessment of bacterial adhesion The viability of P. aeruginosa and S. aureus cells adhered after the application of plant extracts was determined through colony-forming unit (CFU). The formation of biofilm was performed in 96-well plates flat bottom (Orange) as described in section . After 24 h of growth, planktonic cells were removed, and each well was washed twice with distilled water. 200 μL of distilled water was added to each well and the plate placed on an ultrasonic water bath for 10 min to release the biofilm cells from the well surface as previously optimized . The content of five wells was transferred to a microcentrifuge tube to make up a volume of 1 mL and vortexed to homogenise the cell suspensions. Afterwards, the cell suspensions were serially diluted and plated on TSA to determine the number of culturable cells adhered to the wells. After overnight growth at 37 °C, CFUs were counted. Analyses were performed in triplicate and the results were expressed as log 10 CFU per volume (mL). 

Assessment of antibiofilm activity by brightfield microscopy Brightfield microscopic assessment of all bacterial biofilms was accomplished to investigate the antibiofilm activity. The methodology described in section was slightly altered to allow the biofilm formation on a coverslip. Briefly, a plastic coverslip (Thermanox) was placed on the bottom of wells in a 24-well plate and 1 mL of the selected concentration of each extract was added to the well on top of the coverslip. Further, 1 mL of bacterial suspension from overnight inocula was added to the wells to make a total volume of 2 mL and a final concentration of 1 × 10 7 CFU/mL. The plate was incubated for 24 h at 37 °C and 120 rpm to allow biofilm formation. After 24 h growth, the well liquid content was removed, leaving the coverslip in place, and each well was washed twice with distilled water. The coverslip was stained using the CV method as described previously with slight alterations. 500 μL of methanol was added to each well to fix the biofilm to the coverslip and after 15 min, methanol was removed. After the coverslips dried out, 500 μL of CV reagent (1 %) was added to dye the biofilm for 5 min. The excess stain was washed off using distilled water, and the coverslip was placed on a microscope glass slide to observe the biofilms under a brightfield microscope (10× magnification) (Olympus BX51TF). 

Targets prediction Putative targets of the most abundant phytochemical, moringin, were predicted using PharmMapper . The parameters set in PharmMapper included a Maximum of 300 Generated Conformations and selecting ‘all targets’ option . The results were ranked by normalized fit-score. 

Molecular docking RCSB-Protein Data Bank  was used to obtain the three-dimensional (3D) structure of proteins from P. aeruginosa and S. aureus indicated by PharmMapper. The 3D structure of the moringin was obtained from PubChem (ID 153557)  in.sdf format. The protein structure was pre-processed using the protein preparation wizard, which involved adding the missing residues in the crystal structure and removing the water molecules surrounding the receptor. Moringin was individually docked with the two proteins using the molecular docking software AutoDock 4.2.6 . The docking procedure followed previously established protocols, with a grid size configured for blind docking. The grid box was set to 126 x 126 x 126 for x, y and z axis, respectively, using Autogrid program in such a way that encompass the entire molecule of moringin. Docking simulation were carried out using the Lamarckian genetic algorithm with default parameters. The docking protocol included 100 different runs (RMSD tolerance of 2.0 Å) with a population size of 150 and a maximum number of evaluations of 25 000 000 (set to ‘long’). After each docking calculation, the generated docked conformations were ranked based on predicted binding energy. The best energy conformation of the moringin (ligand)-protein complex and orientation of the ligand at the binding site were analysed using USCF Chimera 1.8 (RBVI, USA) and Biovia Discovery Studio . 

Statistical analysis All data was analysed using GraphPad Prism software package (GraphPad Software version 8). Statistical analysis was carried out by ANOVA with Tukey's multiple comparison. Differences were considered statistically significant at p < 0.05. 

Results 3.1 Extraction yield, antioxidant activity and phenolic content This study was designed to obtain a diverse range of extracts from six plants, using solvents with varying polarities (water > EtOH70 > EtOH96) to maximize the potential for identifying a potent antibiofilm plant extract. Extracts with higher phenolic content often exhibit greater antioxidant activity, which can correlate with enhanced antibiofilm effects. Before evaluating antibiofilm activity, the effects of the extraction procedures on extraction yield, antioxidant activity, and phenolic content were analysed ( ). Eucalyptus leaf extracts showed the highest phenolic yields when extracted with water and EtOH70, but lower yields with EtOH96. Despite the lower total phenolic content of the EtOH96 extract, its antioxidant activity was highest along with water and EtOH70 extracts. Moringa seed extracts showed extremely low phenolic extraction yield and content, along with no notable antioxidant activity. Likewise, moringa leaf extracts showed low phenolic content and antioxidant activity, despite having moderate phenolic yield when extracted with water. Cinnamon bark extracts showed the lowest values across all parameters when using EtOH96 and showed moderate values of antioxidant activity with the other two solvents. Pine bark extracts displayed higher values across all parameters tested when extracted with EtOH70. Both broom flower and lemongrass extracts showed low phenolic yield and content and antioxidant activity across all solvents, with EtOH70 performing slightly better in extracting phenolic compounds and antioxidant activity, and overall extraction yield. For lemongrass, though the extraction yield was higher when using water or EtOH70, the highest phenolic content was obtained when using EtOH96. Overall, our results indicated that a high phenolic content generally corresponds to a high antioxidant activity across all biomasses. However, some exceptions were observed, suggesting that other components in the extracts may be responsible for the antioxidant activity. Additionally, there was no specific correlation between the antioxidant activity and the solvent polarity, as it depended on the biomass being extracted. 3.2 Assessment of antibiofilm activity and chemical composition of the extracts The antibiofilm effect of the plant extracts was determined, revealing significant variations among the extracts and also between the solvents used for extraction. Antibiofilm activity was considered promising if an extract inhibited, at least, 75 % of biofilm formation. Aqueous extracts ( ) showed superior inhibition rates of biofilm formation for all biomasses tested compared to the ethanolic extracts (Supplemental material, ). With a few exceptions, aqueous extracts showed a dose-dependent inhibitory effect on the biofilm formation, achieving higher inhibition rates at the highest concentrations. The antibiofilm effect also depended on the strain tested for both species. The extracts from eucalyptus showed the strongest reduction of biofilm formation of S. aureus with approximately 100 % inhibition rate at a concentration of 3.9 mg/L ( A). The extracts from moringa seeds and cinnamon showed the strongest reduction of biofilm formation in both P. aeruginosa and S. aureus at 3.3 and 1.6 mg/mL, respectively ( B and D). Weak biofilm inhibition was observed for the extracts of moringa leaves, pine bark, broom flower and lemongrass ( C–E, F and G). In an attempt to identify the compounds responsible for the antibiofilm activity in aqueous extracts, a HPLC-TOF-MS analysis focused on phenolic compounds was conducted. Detailed mass spectra data, absorbance spectra, and retention times were compared with the available literature. The names, molecular weight, molecular formula, mass-to-charge ratio and mSigma of each component in the extracts are presented in . The moringa seed and leaf extracts were rich in moringin and benzaldehyde, with adenosine, hydrocinnamic acid and citric acid also in abundance in the extracts. In the extracts from cinnamon bark, coumarin, quinic acid, and moringin were the most abundant compounds. A strong presence of moringin was also found in lemongrass extract, but kaempherol was the most abundant. The broom flower extract was constituted mostly by chrysin, anhydroglycinol and kaempherol, while the eucalyptus extract contained several abundant compounds, including quinic acid, citric acid, 3,4-dihydroxybenzoic acid, azelaic acid, kaempherol and pyrogallol. Notably, 6-gingerol was found in all extracts analysed. 3.3 Effect on bacterial adhesion and biofilm formation Based on significant antibiofilm activity against both species, aqueous extracts of moringa seeds and cinnamon were selected for further investigation to elucidate their potential mechanism of action. Their activity against both species is particularly pertinent for CF management disease and the reason for their drug discovery studies. Quantitative assessment of bacterial adhesion to the surface was initially performed by CFU counting. Obtained results revealed that the surfaces were populated by an increased number of bacterial cells, with no differences observed in P. aeruginosa with respect to the control group (untreated biofilms) ( ). However, extracts from moringa seeds and cinnamon bark reduced the adhered bacterial load of S. aureus by 3 log. In situ visualization of surfaces by brightfield microscopy confirmed an increased number of bacteria adhered to surfaces and the absence of biofilm formation or aggregates. Bacterial cells treated with extracts of moringa seeds ( F, G, H, I, J) and cinnamon bark (K, L, M, N, O) were scattered across the surface, in opposite to the densely packed biofilm structures observed in the control group ( A–E). It should be noted that both extracts, especially moringa, left residues after 24 h that deposited on surfaces and were stained by CV (indicated by red arrows in ), which may be mistaken for biofilms or cell aggregates in brightfield microscopy images (Supplemental material, ). 3.4 Target prediction and molecular docking The compound moringin, abundant in both moringa seed and cinnamon extracts, is likely a key contributor to the antibiofilm properties of these extracts. Given the limited research on the antibiofilm properties of moringin and to lay the groundwork for future research in this compound, we sought to identify its potential targets in P. aeruginosa and S. aureus and perform molecular docking to assess the plausibility of the proposed targets and their interaction with moringin. To explore its potential molecular targets, the PharmMapper web server was used for reverse pharmacophore mapping. PharmMapper compared the pharmacophores of the moringin against an in-built database of pharmacophore models, providing target information for 300 proteins along with their fit-score and number of pharmacophoric features. Target selection by PharmMapper was further refined using protein data retrieved from the RCSB-Protein Data Bank  with a focus on the proteins relevant to P. aeruginosa and S. aureus . shows the potential targets for moringin after filtering for proteins associated with these bacterial species. Aspartate-semialdehyde dehydrogenase (ASADH, PDB ID: 2HJS ) was identified as a potential target for P. aeruginosa, while the HLA-DR4 complexed with peptide and SEB ( Staphylococcus aureus enterotoxin B) (PDB ID: 1D5M ) was associated to S. aureus . HLA-DR4 is a human MHC class II receptor that binds bacterial enterotoxins to mediate immune responses . However, as our experiments were conducted in vitro using bacterial biofilms without a host immune component, the relevance of this complex in our study was not supported. Consequently, further computational investigations involving HLA-DR4 are presented in the supplemental material. Molecular docking simulations were conducted to predict the binding modes and affinities of moringin with ASADH and HLA-DR4 complexed with peptide and SEB, separately. Moringin demonstrated varying degrees of interaction with the target proteins (Supplemental material, ). Cluster analysis indicated that optimal binding conformations were obtained in the 33rd and 55th runs, which correspond to the lowest binding energy ( ), with 2 and 4 conformations identified for moringin-ASADH and moringin-HLA-DR4 complexes, respectively. The estimated inhibition constant (Ki) provided further insights into the compounds' inhibitory potency, with lower Ki values indicating stronger inhibitory potential. Moringin exhibited significantly low Ki values of 8.97 and 99.70 μM, for ASADH and HL4-DR4 complex, respectively, suggesting potent inhibitory activity. The docking results are visually depicted in and , illustrating the interactions between the moringin and ASADH and HLA-DR4 complex, respectively. Moringin formed several hydrogen bonds with the active sites, including Ala12, Ala50, Asp25 in ASADH and Thr130, Thr129 in the HLA-DR4 complex. 

Extraction yield, antioxidant activity and phenolic content This study was designed to obtain a diverse range of extracts from six plants, using solvents with varying polarities (water > EtOH70 > EtOH96) to maximize the potential for identifying a potent antibiofilm plant extract. Extracts with higher phenolic content often exhibit greater antioxidant activity, which can correlate with enhanced antibiofilm effects. Before evaluating antibiofilm activity, the effects of the extraction procedures on extraction yield, antioxidant activity, and phenolic content were analysed ( ). Eucalyptus leaf extracts showed the highest phenolic yields when extracted with water and EtOH70, but lower yields with EtOH96. Despite the lower total phenolic content of the EtOH96 extract, its antioxidant activity was highest along with water and EtOH70 extracts. Moringa seed extracts showed extremely low phenolic extraction yield and content, along with no notable antioxidant activity. Likewise, moringa leaf extracts showed low phenolic content and antioxidant activity, despite having moderate phenolic yield when extracted with water. Cinnamon bark extracts showed the lowest values across all parameters when using EtOH96 and showed moderate values of antioxidant activity with the other two solvents. Pine bark extracts displayed higher values across all parameters tested when extracted with EtOH70. Both broom flower and lemongrass extracts showed low phenolic yield and content and antioxidant activity across all solvents, with EtOH70 performing slightly better in extracting phenolic compounds and antioxidant activity, and overall extraction yield. For lemongrass, though the extraction yield was higher when using water or EtOH70, the highest phenolic content was obtained when using EtOH96. Overall, our results indicated that a high phenolic content generally corresponds to a high antioxidant activity across all biomasses. However, some exceptions were observed, suggesting that other components in the extracts may be responsible for the antioxidant activity. Additionally, there was no specific correlation between the antioxidant activity and the solvent polarity, as it depended on the biomass being extracted. 

Assessment of antibiofilm activity and chemical composition of the extracts The antibiofilm effect of the plant extracts was determined, revealing significant variations among the extracts and also between the solvents used for extraction. Antibiofilm activity was considered promising if an extract inhibited, at least, 75 % of biofilm formation. Aqueous extracts ( ) showed superior inhibition rates of biofilm formation for all biomasses tested compared to the ethanolic extracts (Supplemental material, ). With a few exceptions, aqueous extracts showed a dose-dependent inhibitory effect on the biofilm formation, achieving higher inhibition rates at the highest concentrations. The antibiofilm effect also depended on the strain tested for both species. The extracts from eucalyptus showed the strongest reduction of biofilm formation of S. aureus with approximately 100 % inhibition rate at a concentration of 3.9 mg/L ( A). The extracts from moringa seeds and cinnamon showed the strongest reduction of biofilm formation in both P. aeruginosa and S. aureus at 3.3 and 1.6 mg/mL, respectively ( B and D). Weak biofilm inhibition was observed for the extracts of moringa leaves, pine bark, broom flower and lemongrass ( C–E, F and G). In an attempt to identify the compounds responsible for the antibiofilm activity in aqueous extracts, a HPLC-TOF-MS analysis focused on phenolic compounds was conducted. Detailed mass spectra data, absorbance spectra, and retention times were compared with the available literature. The names, molecular weight, molecular formula, mass-to-charge ratio and mSigma of each component in the extracts are presented in . The moringa seed and leaf extracts were rich in moringin and benzaldehyde, with adenosine, hydrocinnamic acid and citric acid also in abundance in the extracts. In the extracts from cinnamon bark, coumarin, quinic acid, and moringin were the most abundant compounds. A strong presence of moringin was also found in lemongrass extract, but kaempherol was the most abundant. The broom flower extract was constituted mostly by chrysin, anhydroglycinol and kaempherol, while the eucalyptus extract contained several abundant compounds, including quinic acid, citric acid, 3,4-dihydroxybenzoic acid, azelaic acid, kaempherol and pyrogallol. Notably, 6-gingerol was found in all extracts analysed. 

Effect on bacterial adhesion and biofilm formation Based on significant antibiofilm activity against both species, aqueous extracts of moringa seeds and cinnamon were selected for further investigation to elucidate their potential mechanism of action. Their activity against both species is particularly pertinent for CF management disease and the reason for their drug discovery studies. Quantitative assessment of bacterial adhesion to the surface was initially performed by CFU counting. Obtained results revealed that the surfaces were populated by an increased number of bacterial cells, with no differences observed in P. aeruginosa with respect to the control group (untreated biofilms) ( ). However, extracts from moringa seeds and cinnamon bark reduced the adhered bacterial load of S. aureus by 3 log. In situ visualization of surfaces by brightfield microscopy confirmed an increased number of bacteria adhered to surfaces and the absence of biofilm formation or aggregates. Bacterial cells treated with extracts of moringa seeds ( F, G, H, I, J) and cinnamon bark (K, L, M, N, O) were scattered across the surface, in opposite to the densely packed biofilm structures observed in the control group ( A–E). It should be noted that both extracts, especially moringa, left residues after 24 h that deposited on surfaces and were stained by CV (indicated by red arrows in ), which may be mistaken for biofilms or cell aggregates in brightfield microscopy images (Supplemental material, ). 

Target prediction and molecular docking The compound moringin, abundant in both moringa seed and cinnamon extracts, is likely a key contributor to the antibiofilm properties of these extracts. Given the limited research on the antibiofilm properties of moringin and to lay the groundwork for future research in this compound, we sought to identify its potential targets in P. aeruginosa and S. aureus and perform molecular docking to assess the plausibility of the proposed targets and their interaction with moringin. To explore its potential molecular targets, the PharmMapper web server was used for reverse pharmacophore mapping. PharmMapper compared the pharmacophores of the moringin against an in-built database of pharmacophore models, providing target information for 300 proteins along with their fit-score and number of pharmacophoric features. Target selection by PharmMapper was further refined using protein data retrieved from the RCSB-Protein Data Bank  with a focus on the proteins relevant to P. aeruginosa and S. aureus . shows the potential targets for moringin after filtering for proteins associated with these bacterial species. Aspartate-semialdehyde dehydrogenase (ASADH, PDB ID: 2HJS ) was identified as a potential target for P. aeruginosa, while the HLA-DR4 complexed with peptide and SEB ( Staphylococcus aureus enterotoxin B) (PDB ID: 1D5M ) was associated to S. aureus . HLA-DR4 is a human MHC class II receptor that binds bacterial enterotoxins to mediate immune responses . However, as our experiments were conducted in vitro using bacterial biofilms without a host immune component, the relevance of this complex in our study was not supported. Consequently, further computational investigations involving HLA-DR4 are presented in the supplemental material. Molecular docking simulations were conducted to predict the binding modes and affinities of moringin with ASADH and HLA-DR4 complexed with peptide and SEB, separately. Moringin demonstrated varying degrees of interaction with the target proteins (Supplemental material, ). Cluster analysis indicated that optimal binding conformations were obtained in the 33rd and 55th runs, which correspond to the lowest binding energy ( ), with 2 and 4 conformations identified for moringin-ASADH and moringin-HLA-DR4 complexes, respectively. The estimated inhibition constant (Ki) provided further insights into the compounds' inhibitory potency, with lower Ki values indicating stronger inhibitory potential. Moringin exhibited significantly low Ki values of 8.97 and 99.70 μM, for ASADH and HL4-DR4 complex, respectively, suggesting potent inhibitory activity. The docking results are visually depicted in and , illustrating the interactions between the moringin and ASADH and HLA-DR4 complex, respectively. Moringin formed several hydrogen bonds with the active sites, including Ala12, Ala50, Asp25 in ASADH and Thr130, Thr129 in the HLA-DR4 complex. 

Discussion The efficacy of plant-based antibiofilm extracts, fractions or compounds have been extensively studied, however, research focused on their effectiveness against mucoid and SCV biofilms formed by P. aeruginosa and S. aureus remains limited. Therefore, this study aimed to address this gap by evaluating the potential of 21 plant extracts of seven different biomasses to inhibit bacterial biofilm formation, chemically characterizing their composition, identifying promising extracts, and gaining insights into their mechanism of action. This work paves the way for further studies on developing drugs to combat biofilms present in CF lungs. The results indicated substantial variability in antioxidant activity across different extracts varying with the solvent and the biomass used. Extractions with ethanol 96 % generally yielded lower antioxidant activities because phenolic compounds, which are well-known for their potential for scavenging free radicals, are typically better extracted with hydroethanolic solvents with intermediate ethanol concentration (50–80 %) . The antioxidant activity of the phenolic compounds is due to the availability of their phenolic hydroxyl groups that can donate their electron or hydrogen, thereby forming stable end products . In this study, a higher extraction yield of phenolic compounds did not constantly correspond to a higher content of phenolic compounds in the extract, suggesting that other compounds were also co-extracted. Given different solvents produced extracts with distinct chemical compositions, it is not surprising that varying levels of biofilm inhibition were observed. Our screening first systematically compared the antibiofilm activity of ethanolic versus aqueous extracts. In general, ethanolic extracts are described as excellent antibiofilm agents, in opposite to the weak activity of aqueous extracts . For instance, Alam et al. (2020) reported superior antibiofilm activity of ethanolic and methanolic extracts of Bergenia ciliata compared to the aqueous extract that showed the lowest activity . The increased activity of ethanolic extracts is often associated with the higher solubility of phenolic compounds in hydroethanolic mixtures, as previously mentioned [ , , ]. However, our findings did not support this general reported trend. For the assayed plants, aqueous extracts outperformed ethanolic extracts in antibiofilm activity. Water-based extracts are particularly attractive for clinical applications due to the lower toxicity of water compared to organic solvents. Similar to our findings, several studies have been previously reported higher antibiofilm activity for aqueous extracts . For instance, the aqueous extract of the Cochlospermum regium flowers showed higher antibiofilm activity against S. aureus than the ethanolic extract . The high extraction yield of water can be attributed to the solvent capability to dissolve polar molecules, such as anthocyanins, tannins, saponins, lectins and polypeptides, which may contribute to the extract's superior antibiofilm activity . Among the aqueous extracts tested, moringa seed and cinnamon extracts exhibited notable antibiofilm activity against P. aeruginosa and S. aureus . This is particularly significant in CF, as these two species are frequently co-isolated and their interactions can exacerbate infections by increasing biofilm pathogenesis and persistence . The ability of these extracts to inhibit biofilm formation in both species highlights their potential clinical relevance. Both moringa seeds and cinnamon extracts allowed bacterial adhesion but seemed to discourage the formation of biofilms as no aggregates were observed. This finding led to hypothesize that extracts might act on inhibition of EPS production and/or QS. Similar mechanisms have been reported in other studies, such as downregulation of QS genes and reduction of EPS production in P. aeruginosa by Agrimonia pilosa Ledeb. , and reduction of bacterial attachment to the surface and decreased expression of virulence factors that are regulated by QS, such as pyocyanin, pyoverdine and swimming mobility by Camelia sinensis (L.) Kuntze . Moringa is a member of the Moringaceae family and most of its parts including roots, leaves, seeds have been historically used in folk medicine to help cure several diseases. Various studies have confirmed the therapeutic proprieties of moringa, including its antibiofilm activity [ , , , , ]. For instance, an aqueous extract of moringa seeds showed antibiofilm activity against S. aureus by destroying the structure of mature biofilms formed in polyvinyl chloride surfaces . Although the moringa extract and the culture medium (milk) used in that study differ from our study, similar activity with the aqueous extract of moringa seeds against mucoid and SCV P. aeruginosa and S. aureus was found. Given the promising antibiofilm potential of moringa extract, some studies have isolated and tested compounds from moringa seeds. For instance, Onsare et al. (2015) reported the antibiofilm activity of flavonoids isolated from the seed coat of moringa against S. aureus , P. aeruginosa and Candida albicans . Likewise, a lectin isolated from these seeds inhibited biofilm formation in other Gram-negative bacteria such as Serratia marcescens and Bacillus spp . In our study, HPLC-TOF-MS identified benzaldehyde and moringin as the most abundant compounds in the moringa seed extract. The presence of these compounds aligns with previous reports . Benzaldehyde, an aromatic aldehyde with a benzene ring, has shown both antimicrobial and antibiofilm activity against S. aureus , E. coli and P. aeruginosa . Leitão et al. (2024) demonstrated that benzaldehyde blocked QS in P. aeruginosa and increased its susceptibility to ciprofloxacin . Moringin (or [4-(alpha-L-rhamnosyloxy) benzyl isothiocyanate]), an isothiocyanate compound derived from glucosinolates, has also shown antibacterial activity against S. aureus  and Listeria monocytogenes . Despite moringin's higher solubility in ethanol, our aqueous extract showed a strong presence of this compound, likely due to co-extraction with other compounds. Further, isothiocyanates (including moringin) are highly reactive compounds that result from the hydrolysis of glucosinolates (e.g., glucomoringin) and can easily bound to other molecules that have nucleophiles such as hydroxyl, amino or thyol groups. Although moringin is more soluble in ethanol in its free form, its extraction from moringa by-products may be possible using water under certain conditions. This may be due to various complexes formed between various compounds present in this biomass, such as polysaccharides and proteins. For instance, Leone et al. reported 18.4 g/100 g dry weight of carbohydrates and 31 g/100 g dry weight of protein . Moringin can easily bound both to polysaccharides and/or to proteins and be extracted as a complex. Additionally, moringin seeds have a high amount of saponins, that usually mat act as natural surfactant and facilitate the extraction of moringin . This may explain the strong antibiofilm activity of the aqueous extract of moringa seeds. Cinnamon, also known as Chinese cinnamon, is an evergreen tree commonly found in China and one of the oldest spices in the world. In addition to its role as a culinary spice, it has been traditionally used in various cultures as a remedy for colds and gastrointestinal problems . Cinnamon bark has shown antibiofilm activity against Gram-negative bacteria, such as P. aeruginosa , E. coli, Acinetobacter baumannii and periodontal pathogens [ , , , ]. For instance, cinnamon oil inhibited biofilm formation in P. aeruginosa , making the bacterial cells appear scattered on the surface . Similarly, an ethanolic extract reduced EPS and disrupted E. coli biofilm structure, only allowing to form scattered microcolonies . However, in our study, antibiofilm activity was associated with the aqueous extract of cinnamon rather than the ethanolic extract, which may be a result of the extraction process. Several bioactive compounds from cinnamon extracts with described antibiofilm activity were found in our HPLC-TOF-MS analysis. Quinic acid and coumarin were the most abundant compounds, confirming the composition found in the literature . Quinic acid has been shown to reduce biofilm formation in P. aeruginosa by downregulating the QS system , while coumarin has been reported as an inhibitor of several virulence factors involved in biofilm formation in P. aeruginosa . Moringin was also detected in the aqueous extract of cinnamon. Its increased abundance in the most effective antibiofilm extracts, moringa seeds and cinnamon, against both P. aeruginosa and S. aureus suggest moringin may play a central role in their antibiofilm activity. However, the aqueous extract of moringa leaves, despite having a high concentration of moringin, did not show antibiofilm activity. These distinct activities among extracts with high moringin content might be attributed to moringin interaction with diverse compounds present in each of the extracts. For instance, compounds in lower proportions in the moringa leaf extract might exert an antagonistic effect inhibiting the antibiofilm activity of moringin. Likewise, compounds unique to the extracts of moringa seeds and cinnamon, and absent in moringa leaf extract, might be enhancing moringin activity through synergistic or additive interactions. Additionally, other components not identified in this study, such as proteins or carbohydrates, might also influence the moringin bioavailability . To explore if moringin could be responsible for the antibiofilm activity, we used PharmMapper to identify potential bacterial targets of this compound. PharmMapper is an open web server that employs reverse pharmacophore mapping to identify potential drug targets . It automatically identifies the optimal mapping poses of the query molecule against all pharmacophore models in PharmTargetDB, providing a list of the top N best-fitted hits along with target annotations and the aligned poses of the respective molecules. In this study, PharmMapper identified ASADH and HLA-DR4 complexed with peptide and SEB as the main targets in P. aeruginosa and S. aureus , respectively. ASADH is a crucial enzyme involved in the biosynthesis of essential amino acids and metabolites in microorganisms . Inhibition of ASADH has been proposed as a potential strategy for developing drugs to combat multidrug-resistant organisms [ , , ]. Since humans and other mammals lack a homolog of ASADH and do not rely on the aspartate biosynthetic pathway, ASADH represents a particularly promising target in microorganisms. However, when microorganisms are deprived of essential amino acids, they often activate alternative transport mechanisms to obtain the amino acid from the environment , which may explain the P. aeruginosa viability after exposure to moringa and cinnamon extracts. The potential of distinct phytochemicals, such as rosmarinic acid and curcumin, to inhibit ASADH has been previously reported . Molecular docking analysis supported moringin binding to ASADH. Notably, proper intermolecular hydrogen bonding interactions were observed, which is crucial for stable binding and effective inhibition. Moreover, the docking results showed a lower inhibition constant, suggesting that moringin exhibits potent inhibitory effects on this target protein. The identification of HLA-DR4 complexed with peptide and SEB as a potential target of moringin by PharmMapper suggests a possible immunomodulatory activity, which could be particularly relevant in complex diseases such CF. However, as our experiments were conducted in vitro without host immune components, it is likely that moringin also interacts with other bacterial targets that remain unidentified. This finding highlights the importance of further investigating moringin's activity against biofilm-associated bacteria to better understand its mechanisms of action and therapeutic potential. In conclusion, our study demonstrated that aqueous extracts from moringa seeds and cinnamon bark have significant potential as inhibitors of biofilm formation in S. aureus and P. aeruginosa clinical isolates with diversified phenotypes, including SCV and mucoid phenotype. The strong antibiofilm activity observed may be attributed to compounds in higher proportions such as coumarin, quinic acid, and moringin in cinnamon, and benzaldehyde and moringin in moringa seeds, based on literature reports. Notably, moringin, a compound highly prevalent in both extracts, might play a central role in the observed antibiofilm activity. Computational analyses suggested that moringin could be a potential inhibitor of aspartate-semialdehyde dehydrogenase in P. aeruginosa and potentially interact with an unknown target in S. aureus . Although our findings highlighted that moringin is a promising antibiofilm agent against mucoid and SCV P. aeruginosa and S. aureus , the current study is limited to computational predictions and in vitro screening. Further experimental studies are necessary to validate the identified target in P. aeruginosa and identify the specific target in S. aureus , elucidate the mechanisms of action, and assess the therapeutic potential of moringin in treating biofilm-associated infections. Additionally, future work should focus on investigating potential synergistic or antagonistic interactions among extract components to better understand the key contributors of plant-based antibiofilm activity. 

Eduarda Silva: Writing – review & editing, Writing – original draft, Investigation, Formal analysis, Data curation. Pedro Ferreira-Santos: Writing – review & editing, Data curation. José A. Teixeira: Writing – review & editing. Maria Olivia Pereira: Writing – review & editing. Cristina M.R. Rocha: Writing – review & editing, Validation, Conceptualization. Ana Margarida Sousa: Writing – review & editing, Validation, Supervision. 

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

