Disruption of Bacterial Lipid Membranes by Phenolic Compounds from Plant Extracts:Biophysical Evidence from Microbiological and Langmuir Monolayer Studies

Disruption of Bacterial Lipid Membranes by Phenolic Compounds from Plant Extracts:Biophysical Evidence from Microbiological and Langmuir Monolayer Studies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Disruption of Bacterial Lipid Membranes by Phenolic Compounds from Plant Extracts:Biophysical Evidence from Microbiological and Langmuir Monolayer Studies Agnieszka Gonet-Surówka, Kamil Drożdż, Anita Wnętrzak, Anna Chachaj-Brekiesz, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9356537/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The article presents research on the destruction of the bacterial membrane of celandine (CME) and dandelion (TOE) extracts containing phenolic compounds responsible for their antibacterial activity. The in vitro microbiological tests were done on living cells of different pathogenic bacteria species: Gram-positive ( Staphylococcus aureus and Streptococcus pyogenes ) and Gram-negative ( Escherichia coli and Pseudomonas aeruginosa ). The obtained results clearly demonstrate the antibacterial effect of both extracts. However, in the case of celandine herb extract, the effect was stronger, especially for Gram-positive bacteria. For a deeper understanding of the mechanism of antibacterial action, both studied extracts were subjected to biophysical studies on artificial bacterial lipid membranes, modeled with the Langmuir monolayer technique. The monolayer investigations were performed using two different methodologies. First was based on preparing Langmuir monolayers of model bacterial lipid membranes on subphases containing the tested extracts and analysis of the biophysical parameters of the recorded pressure-area isotherms compared to those without the presence of the herb's extracts. In the other approach, the extracts were introduced into the aqueous subphase and their penetration to model bacterial lipid membranes was monitored. The results of the monolayer experiments, including AFM analysis of the domain structures of the LB transferred films, are in good agreement with the results of biological tests and analytical analyses of the tested extracts, which confirm that the stronger antibacterial effect of celandine extract, associated with a greater amounts of phenolic compounds, has a more destructive effect on the lipid membranes of bacteria compared to dandelion. pathogenic bacteria phenolic compounds celandine extract dandelion extract bacterial lipid model membranes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Introduction The increasing resistance of many bacterial strains to conventional antibiotics poses a significant challenge to medicine in the 21st century. As a result of acquired resistance to drugs, the pathogenicity of many strains of bacteria has increased significantly. In consequence, thousands of patients die in hospitals around the world every year due to infections with antibiotic-resistant bacteria. In recent years, extracts isolated from plants have attracted great interest and are experiencing a real renaissance in scientific research, giving new hope for the treatment of severe problems related to antibiotic resistance. As numerous studies have shown (J.A. Duke 2002 ; I.F.F. Benzie and S.Wachtel-Calor 2011), plant-derived compounds have many medicinal properties, including antibacterial, antiviral, antifungal, anticancer, antioxidant and immunomodulatory effects. Research shows that natural herbs can be a good alternative to synthetic drugs due to their lower toxicity to both the patient and the environment. Great hopes are also placed in the treatment of diseases considered incurable, such as neurodegenerative diseases, endometriosis, or malignant tumors, for which synthetic drugs seem to be ineffective. Plant extracts are characterized by a multi-component composition, and their specific mutual proportions acting synergistically may be the key to their more effective therapeutic action compared to individual medicinal substances either extracted from natural sources or of synthetic origin. A good example is chaga mushroom ( Inonotus obliquus ) extract used in various human cancer cell lines (Chung et al. 2010 ) (Zhao et al. 2015 ). Alcoholic extracts are mostly available on the market but frequently used in folk medicine are aqueous (Lee et al. 2009 ) extracts (containing, among others, polysaccharides (Lu et al. 2021 ), and – interestingly - they were reported to be also effective. The composition of alcohol extracts contains – in addition to bioelements - phenolic acids, sterols, indole compounds and triterpenoids (Sułkowska-Ziaja et al. 2023 ). For anticancer activity of chaga extracts, triterpenoids, especially betulin, are believed to be effective, however, they are nearly insoluble in water. The most likely theory assumes that all the active substances found in the chaga mushroom are naturally present in precisely defined, appropriate proportions. It is possible that the compounds contained in the mushroom somehow activate trace amounts of betulin, which are transferred into the infusion during brewing and delivered to the body, where they begin to affect cancer cells (Camilleri et al. 2024 ). This aspect, however, needs further investigations. Treating infections with herbs has a centuries-old world tradition. Using herbs as medicinal sources dates back to prehistoric times, for example, in tombs in Iraq from about 60,000 years ago, fragments of plants, species such as Ephedra , Centaurea and Sambucus (edelberry) were found (Tyler 2000 ). Many herbs and plants of mainly antimicrobial activities but also showing other health promoting properties were found in various regions of the world as reported in a plethora of papers (for example in India (Srinivasan et al. 2001 ), China (Tan and Vanitha 2004 ), and other regions in the East: Maylasia (Chung et al. 2004 ), Arab Emirates (Tanira et al. 1994 ), Iran (Mahboubi 2388 ), Russia (Shikov et al. 2014 ) and Siberia (Kokoska et al. 2002 ); Africa (A. Ajayi 2008 ), America (Belinda Reynolds 2023), (Cuevas-Cianca et al. 2023 ), (Ruiz-Bustos et al. 2009 ), and Spanish Mediterranean region (Ríos et al. 1987 ). Their components belonging to alkaloids, coumarins, tannins, terpenoids, flavonoids, saponins and polyphenols, have proven antibacterial, antifungal or antiviral effects. What is interesting, their mode of action is twofold. Namely, their bioactive compounds affect cellular metabolic pathways, causing protein and DNA damage (without producing free radicals ROS, which are very dangerous for the body, unlike antibiotics or nanomaterials, including metal nanoparticles or metal oxides, e.g. nanosilver (Parham et al. 2020 ), but also—due to their amphiphilic structure and surface activity—exhibit membrane activity. This latter aspect of their action has not been thoroughly investigated to date. Only a few reports on this topic can be found in the literature regarding individual compounds found in herbal extracts. Regarding antimicrobial activities, the majority of studies are concentrated on terpenoids (Fontanay et al. 2008 ) and (Francis et al. 2002 ). They are proved to adsorb to the membrane and induce cells lysis by membrane fluidization, causing cells lysis (Korchowiec et al. 2015 ) (Orczyk et al. 2020 ) (Broniatowski et al. 2014 ). Although a large body of literature, as mentioned above, describes the antimicrobial activity of the local plants and herbs, the aim of our work was to investigate the antimicrobial properties of two herbal extracts with a wide distribution worldwide: dandelion ( Taraxacum officinale F.H. Wigg) and celandine ( Chelidonium majus L.). A review of the literature in recent decades confirms that the largest number of publications related to medicinal antibacterial plants concerns dandelion (which is in second place in the cited literature, right after the most frequently studied Baikal skullcap ( Scutellaria baicalensis ) (Chen et al. 2021 ). We therefore focused our attention on an in-depth study of celandine, comparing its antibacterial activity with that of dandelion. One reason celandine has been less studied may be its hepatotoxicity (F Pantano 1 2017) when administered orally. However, this does not rule out its potential as an effective antibacterial agent for external (e.g. skin) infections. Celandine, formerly known as the Gift of Heaven ( Coeli donum , hence the generic name Chelidonium ) has been reported to possess antioxidant and antimicrobial activities (Hong et al. 2024 ) due to high contents of phenolic and flavonoid compounds (Zielińska et al. 2018 ). However, it has been less frequently studied compared to dandelion, which has long been known for its antimicrobial effects due its extracts rich in triterpenes and their saponins, sterols and their glycosides, organic acids, saccharides, volatile oils in addition to phenolic acid and flavonoids (Yan et al. 2024 ). In this study, we conducted in vitro experiments, using both living bacterial cells and model lipid membrane systems. The selected pathogenic strains comprised Gram-positive bacteria— Staphylococcus aureus and Streptococcus pyogenes —as well as Gram-negative bacteria— Escherichia coli and Pseudomonas aeruginosa. The model investigations included floating Langmuir monolayers and adsorption experiments. Furthermore, lipid monolayers mimicking Gram-positive and Gram-negative membrane were transferred onto solid substrates, and their structures were examined by atomic force microscopy (AFM). Notably, effects of the studied extracts on artificial bacterial membranes have not been previously investigated. Results from both microbiological and monolayer studies confirmed the antibacterial activity of the tested herbal extracts, with celandine exhibiting a more pronounced effect. AFM analysis of Langmuir–Blodgett (LB) monolayers revealed distinct differences in the extracts’ impact on Gram-positive versus Gram-negative membrane models, as well as differences between the two extracts in their destructive action on Gram-positive bacteria lipid membranes. Materials and Methods Materials For the preparation of extracts, dried herbs from the company Herbapol (Kraków, Poland) were used. According to the manufacturer, the herbs were grown in a pollution-free region of northern Poland and air-dried at a temperature not exceeding 35°C. The dandelion ( Taraxacum officinale F.H. Wigg ) root, and celandine ( Chelidonium majus L.) aerial part (including the stem, leaves, and flowers, without the root) were used in our experiments. The dried herbs were ground into fine dust in a mortar, then 10 g portions were prepared. The portions were filled with 500 mL of 96% ethanol. They were left for 24 hours, filtered, and the residue was again filled with 300 mL of solvent. Again, it was left for 24 hours, filtered and the residue on the filter was covered with 200 mL of the given solvent. After another 24 hours, filtration was carried out and the filtrate from three days was combined. The solvent was evaporated on a vacuum evaporator to dryness. The dandelion root extract (TOE) or celadine herb extract (CME) were stored at 4°C. The following lipids, as components of bacterial membranes, were employed in the monolayer studies: 1′,3′-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-glycerol (14:0 CL), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were purchased from Sigma–Aldrich in purity > 99% and used without further purification. Chloroform of spectroscopic purity stabilized by ethanol was provided by Sigma–Aldrich. The aqueous solution of NaCl in concentration of 0.1 mol/L was prepared in ultra-pure water of the resistivity of 18.2 MΩ·cm (from HLP system) and was used as a subphase. As a model of bacterial membranes, the two-component lipid systems: CL/DPPG (in mole proportion of 1:1) for Gram-positive membrane model (Hąc-Wydro et al. 2019 ; Barbosa et al. 2019 ) and POPE/DPPG (in mole proportion of 8:2)࿼ membrane model were applied applied (Singleton et al. 1999 ). Methods Antibacterial Activity Testing Four strains of pathogenic bacteria were selected for the study. Two of them were Gram-positive bacteria: Staphylococcus aureus ATCC® 25923 and Streptococcus pyogenes ATCC® 12384. The selected Gram-negative bacteria were: Escherichia coli ATCC® 25922 and Pseudomonas aeruginosa ATCC® 27853. The evaluation of antibacterial activity of isolated plant extracts i n vitro was conducted following the methodology outlined in the EUCAST Broth Microdilution – Reading Guide v 5.0 (1 January 2024). Initially, bacteria were cultured on Columbia Agar (Oxoid) with 5% Sheep Blood for 24 h in 37°C. Microbial inoculum for antibacterial activity testing were prepared by suspending a few colonies in sterile TSB (Tryptic Soya Broth) (Sigma). The densities of the inoculum were adjusted to 0.5 McF with a densitometer (Biosan, Poland). Three bacterial strains: S. aureus , E. coli and P. aeruginosa were subsequently diluted 1:100 to concentration 1,5 x 10 6 CFU/mL in Miller Hinton Broth II (MHB) (Merk, Germany) and S. pyogenes strain were diluted 1:100 in TSB and used for testing immediately after preparation. Plant extracts were dissolved in TSB or MHB to obtain stock solution of 200 mg/mL from which series dilutions were made giving intermediate concentrations ranging from 0.2 mg/mL to 200 mg/mL. The studies were performed in flat-bottom, polystyrene, 96-well microdilution plates (Nest, China). A specific concentration of tested substances diluted in culture medium was placed in the volume of 100 µL in the wells, and 10 µL of bacterial inoculum was added. As a control, pure medium was used to which 10 µL of microbial inoculum was added. Gentamicin (Pol-Aura), a broad-spectrum antibiotic with a concentration range of 0.08 µg/mL to 40 µg/mL was used. All plates were incubated for 24 hours at 36°C. To check the Minimal bactericidal concentrations (MBC) value, each well containing the tested strain was plated on solid medium and incubated for 24 hours at 36°C. After this time, bacteria growth on the medium was checked. The tests were performed in triplicate. The Adsorption and Penetration Experiments The adsorption and penetration experiments were performed in a Teflon cuvette with cylindrical cavity (75 mL capacity) placed above the magnetic stirrer. For adsorption experiments the cuvette was filled with NaCl solution in concentration of 0.1 mol/L in ultrapure water and after several minutes of equilibration the selected volume of ethanolic plant extract was injected into subphase using microsyringe (Hamilton) through a side slot of cuvette. Surface pressure was monitored for 30 minutes with the Wilhelmy method (accuracy of ± 0.01 mN/m). For penetration experiments the chosen lipid mixture mimicking cellular membrane of Gram-positive or Gram-negative bacteria was deposited dropwise on NaCl solution in concentration of 0.1 mol/L in ultrapure water with microsyringe (Hamilton) and stabilized for 5 minutes. The initial surface pressure of spread monolayer was approximately 32 mN/m. Then, the selected volume of ethanolic plant extract was injected into subphase using microsyringe (Hamilton) through a side slot of cuvette and the surface pressure was measured for 40 minutes. Langmuir Monolayer Experiments The experiments were performed employing the Langmuir trough (KSV) with two movable barriers and a total area of 700 cm 2 placed on an anti-vibration table. The surface pressure (π) – molecular area (A) isotherms for lipid films mimicking bacterial membranes were recorded on 0.1 mol/L NaCl solution and 0.1 mol/L NaCl aq. with addition of TOE or CME solutions. The volumes of TOE added to 1 L of subphase were as follows: 25 µL, 50 µL, 100 µL and 400 µL, while CME was added in volumes of: 3.1 µL, 6.3 µL, 12.5 µL and 50 µL. Every time, appropriate volume of chloroform solution of the investigated lipid mixtures was deposited at the air/water interface with Hamilton analytical syringe. 5 min was left for the spreading solvent evaporation and film stabilization after which the monolayers were compressed with constant compression rate of 20 cm 2 ·min − 1 . Surface pressure was measured with the accuracy of 0.1 mN/m with the Wilhelmy electrobalance (KSV). As the surface pressure sensor, a rectangular plate (1.0 cm x 2.5 cm) of ashless filtration paper (Whatman) was applied. All experiments were performed at a constant temperature (20°C), which was controlled (± 0.1°C) by a circulating water system. Each π-A isotherm was repeated at least three times to ensure reproducibility of the curves to 2 Å 2 /molecule and of surface pressure of 0.5 mN/m. Film Deposition and AFM Measurements Samples for atomic force microscopy (AFM) measurements were prepared by transferring Langmuir monolayers of mixed lipid systems mimicking Gram-positive and Gram-negative bacterial membranes from the gas–aqueous interface onto solid support. The transfers were performed using a double-barrier Langmuir–Blodgett trough (NIMA 612D; total trough area: 600 cm²). For deposition, freshly cleaved mica substrates were immersed in the aqueous subphase, and the monolayers were compressed to the target surface pressure of 30 mN/m. After a stabilization period of 10 min at constant pressure, the monolayers were transferred onto mica at a dipping speed of 3 mm/min. For reference systems (bacterial membranes without extracts) the subphase consisted of an aqueous 0.1 mol/L NaCl solution. Modified subphases were prepared by supplementing the 0.1 mol/L NaCl solution with TOE at concentrations of 50 and 400 µL/L, or with CME at concentrations of 3.6, and 50 µL/L (hereinafter referred to as TOE 50, TOE 400, CME 3.6 and CME 50, respectively). Topography of bacterial membranes transferred on mica surface was visualized using Bruker Multimode 8 AFM instrument equipped with Nanoscope V controller. Imaging was performed in Peak-Force Tapping mode using ScanAsyst Air cantilevers (spring constant 0.4 N/m). To minimalize the influence of tip-sample interaction, scanning was performed using relatively low Peak Force values (~ 300 pN). Typical scanning conditions were as follow: Scan rate 0.7–1.0 Hz, Tapping Frequency 4 kHz, Tapping amplitude 30 nm and Lift Height 60 nm. Raw AFM data was using Gwyddion Software. Typically, images were flattened by subtracting the 2nd order XY polynomial, followed by line levelling using median method. For images with relatively large differences in height, additional step consisting on masking procedure and 2nd order polynomial line levelling was applied. If needed, horizontal scars were removed from particular image using Remove Scars function. Images were then exported as 1024 x 1024 pixel .tiff files. Gwyddion software was also used to derive the quantitative topographical information from processed AFM scans. This was done by manually setting the mask on Height-Channel, followed by use of the Statistical Quantities built-in function. In this way, average height and roughness of topographical features and surrounding, continuous phase were derived. Exported tiff images were analyzed using ImageJ software. First, the images were binarized by setting they type to 8-bit image ( Image – Type – 8-bit ). Then, threshold was set manually ( Image – Adjust - Threshold ) to ensure that topographical features of interest are marked appropriately. Binarized images were then analyzed using Analyze Particles ( Analyze – Analyze Particles ) function, with Exclude on edges option enabled. This allowed to derive the distribution dimensional (area, perimeter) of and dimensionless (aspect ratio, circularity, solidity) topographical quantities. Fluorescence Microscopy Images Four strains of pathogenic bacteria: S. aureus , S. pyogenes , E. coli and P. aeruginosa were cultured on Columbia Agar (Oxoid) with 5% Sheep Blood for 24 h in 37°C. Microbial inoculum were prepared by suspending a few colonies in sterile TSB (Tryptic Soya Broth) (Sigma). The densities of the inoculum were adjusted to 0.5 McF with a densitometer (Biosan, Poland). Three bacterial strains: S. aureus , E. coli and P. aeruginosa were subsequently diluted 1:100 in Miller Hinton Broth II (MHB) (Merck, Germany) and S. pyogenes strain were diluted in TSB and used for testing immediately after preparation. Plant extracts were dissolved in TSB or MHB to obtain stock solution of 200 mg/mL from which series dilutions were made giving intermediate concentrations ranging from 0.2 mg/mL to 200 mg/mL. A specific concentration of tested substances diluted in culture medium was placed in the test tube, and 100 µL of bacterial inoculum was added. As a control, pure medium was used to which 100 µL of microbial inoculum was added. All test tubes were incubated for 24 hours at 36°C. After 24 hours, the test tubes were centrifuged at 4000 g for 5 minutes. The supernatant was then poured off, and the pellet was resuspended in 1 mL of physiological saline. The sample was centrifuged again, the pellet was resuspended in 100 µL of physiological saline, and then 3 µL of SYTO 9 and 3 ul propidium iodide solution prepared from the LIVE/DEAD BacLight (Thermofisher) kit. The samples were mixed thoroughly and incubated at room temperature in the dark for 15 minutes. 5 µL of the stained bacterial suspensions were trapped between a slide and an 18 mm square coverslip. The samples were examined using a fluorescence microscope (Olympus BX63, Japan) equipped with a 20× objective lens (UPLXAPO 20×/0.8, dry) to evaluate the effects of the plant extracts. The excitation/emission maxima for these dyes was about 480/500 nm for SYTO 9 stain and 490/635 nm for propidium iodide. Chromatographic Analysis with Detection by a High-resolution Mass Spectrometric System (UHPLC-QTOF) All high-resolution mass spectra were analyzed using a UHPLC-QTOF system consisting of Waters Synapt XS mass spectrometer (electrospray ionization mode ESI-QTOF) coupled to a Waters Acquity I-Class Plus chromatographic separation system (Waters Corporation, Milford, MA, USA). The samples were eluted through a 2.1 × 100 mm and 1.7 µm particle size chromatographic column (Phenomenex, Torrance, CA, USA) preceded by a guard column of the same material (2.1 × 5 mm and 1.7 µm particle size). The injection volume was 10 µL, and the flow rate was 0.3 mL/min. The column was thermostated at 40°C. The separation of the analytes was performed with binary gradient elution. The mobile phases were: A − 0.1% formic acid in water and B − 0.1% formic acid in acetonitrile. The gradient profile was: (t (min), % B), (0, 5), (6, 60), (8, 90). The full range of Photodiode Array (PDA) detector spectra were recorded in the 200–700 nm range with a 1.2 nm resolution and a sampling rate of 20 points/s. The conditions for MS detection settings of Waters Synapt XS mass spectrometer were as follows: source temperature 150°C, desolvation temperature 250°C, desolvation gas flow rate 600 L/h, cone gas flow 100 L/h, capillary potential 3.00 kV, and cone potential 30 V. Nitrogen was used for both nebulizing and drying gas. The full scan data ranging from m/z 50 to 1000 were obtained in a resolution MSE scan mode in positive or negative polarity. Leu-enkephalin was used as a mass reference. Data acquisition and analysis software were MassLynx v4.2 and MSe Data Viewer v2.0 (Waters Corporation, Milford, MA, USA). Determination of the Total Phenolic Content (TPC) by the Folin-Ciocalteu Spectrophotometric Method The total phenolic content (TPC) of different plant samples was determined using the Folin-Ciocalteu spectrophotometric method (Singleton et al. 1999 ) with the absorbance of the mixtures measured in a microplate reader Tecan Infinite 200 (Tecan Austria GmbH, Grödig/Salzburg, Austria) at a wavelength of 740 nm. Three repetitions were done for each of the samples. The ethanolic extract of a sample (1 mL) was mixed with 0.5 mL of Folin-Ciocalteu reagent and 2.5 mL of 7.5% Na 2 CO 3 . A blank was prepared in the same way, using ethanol instead of the sample. The prepared mixtures were incubated in a dark place for 120 min, after which the absorbance was measured. The content of total phenols was determined using a calibration curve of standard gallic acid and the results were expressed as milligrams of gallic acid equivalents (mg GAE/g DW) per gram of dried plant sample. Results and Discussion In Vitro Tests on Bacterial Strains The antimicrobial potential of celandine extract is mainly due to the alkaloids (Hong et al., 2024 ) (Zuo et al., 2009 ), while sesquiterpene lactones are mostly responsible for dandelion root antimicrobial efficacy (González-Castejón et al. 2012 ). Futhermore, phenolic compounds (especially flavonoids) present in both extracts, known for their antioxidant properties, may mediate their antimicrobial activity (González-Castejón et al. 2012 ) (Sengul M et al. 2009 ). In literature the authors report the results of the composition of celandine and dandelion extracts using different solvents (Tettey et al. 2014 ), mostly alcohol (ethanol, methanol), hexane, methylene chloride, ethyl acetate, butanol or water. For our investigations, we chose ethanol extracts based on literature reports proving their high content of bioactive components of antimicrobial effects, contrary to water extracts (Kenny et al. 2014b ). The presence of polysaccharides in the water extracts is the most probable cause for the lack of significant (Kenny et al. 2014b ) or even no (Shittu et al. 2025 ) antimicrobial activity of these extracts as sugars may act as bacteria feed source, and in this way promote their proliferation. Minimal bactericidal concentrations (MBC) values were determined for two different extracts (Table 1 ). In the case of greater celandine extract, for Gram-negative bacteria; E. coli MBC was 160 mg/ml, and for P. aeruginosa MBC was lower and amounted to 128 mg/mL. For Gram-positive bacteria incubated with celandine extract, the MBC was 64 mg/mL for S. aureus and 1.75 mg/mL for S. pyogenes. Dandelion extract exhibited generally weaker bactericidal activity, with the highest value for S. aureus of 160 mg/mL and 60 mg/mL for S. pyogenes . For Gram-negative bacteria: E. coli and P. aeruginosa , the MBC value for dandelion extract was 128 mg/mL. Celandine extract has been shown to have greater bactericidal activity, especially against Gram-positive bacteria. Stronger antimicrobial activity of dandelion versus celandine extracts (TOE versus CME, respectively) may be related to a lower amount of bactericidal components present in the latter herb. Table 1 MBC value for celandine and dandelion extracts acting on selected pathogenic bacterial strains. bacteria strain dandelion extract (TOE) celandine extract (CME) Escherichia coli ATCC® 25922 128 mg/mL 160 mg/mL Pseudomonas aeruginosa ATCC® 27853 128 mg/mL 128 mg/mL Staphylococcus aureus ATCC® 25923 160 mg/mL 64 mg/mL Streptococcus pyogenes ATCC® 12384 60 mg/mL 1.75 mg/mL All four strains of bacteria tested were found to be sensitive to alcohol extracts from the plants used. Gram-positive bacteria were found to be more sensitive to the compounds contained in the extracts, particularly the CME herb extract. The differences resulting from the action of the extracts on the two groups of bacteria may result from the composition of the cell membrane structure. Gram-negative bacteria possess different cell envelope components than Gram-positive. Namely, Gram-negative bacteria have two lipid bilayer membranes in their structure: an outer and an inner one, which is separated by a periplasmic space and thin layer of peptidoglycan. In the Gram-negative cell wall, the outer membrane may act as a barrier against the diffusion of many compounds, making the cell more resistant to toxic substances. Gram-positive bacteria have thick layer of peptidoglycan but they do not have an outer membrane. The lack of outer lipid bilayer membranes makes them more sensitive to external factors, especially chemical compounds that can penetrate more easily through peptidoglycan, destroying the cell membrane more quickly and penetrating the interior of the bacterial cell (Silhavy et al. 2010 ). Although the cytoplasmic membrane of both kinds of bacteria is primarily composed of phosphatidylethanolamines (PE) and negative phospholipids (phosphatidylglycerols, PG and cardiolipin, Cl) (López-Lara & Geiger, 2017 ) (Sohlenkamp & Geiger, 2016 )(Gunstone et al. 1994 ), their mutual proportions are different (Epand et al. 2007 ). In Gram-negative bacteria, PE predominates over anionic phospholipids while in Gram-positive inner membrane anionic phospholipids dominate over PE. Interestingly, some Gram-positive species are even completely devoid of PE (Trombe et al. 1979 ) and references therein. This different proportion of anionic-to-zwitterionic phospholipids, resulting in different charge of bacterial cells, contribute to their different interactions with various molecules, including antimicrobial agents (Breijyeh et al. 2020 ). Fluorescence Microscopy Images The LIVE/DEAD BacLight Bacterial Viability Kits utilize mixtures of SYTO® 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO 9 stain generally labels all bacteria in a population — those with intact membranes and those with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO 9 stain fluorescence when both dyes are present. Bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. The staining results of bacterial suspensions incubated in plant extract solutions are presented in Fig. 1 . The control sample consisted of bacterial cells grown in medium. The photos showing the control cells of the four bacterial strains tested show a green coloration of the bacteria, mainly indicating live cells. Bacterial cultures treated with different, appropriate bactericidal doses of extract concentrations after staining show a significant decrease in the number of bacteria and indicate dead or membrane-compromised cells. Adsorption of TOE and CME at the Air/Water Interface and their Penetration to the Model Membranes Based on chromatographic results (Supplementary Figures S1 -S6) it can be seen that some compounds present in the analyzed extracts are insoluble in the aqueous phase, but their amphiphilic structure enables their surface activity. This suggests that the components of TOE and CME can penetrate the membranes. To confirm this, in the first step we performed adsorption experiments. In a typical experiment, the chosen volume of herb extract was injected below the interface of NaCl solution of concentration of 0.1 mol/L, and then the change in surface pressure was monitored overtime. As can be seen based on the course of π – time plots (Fig. 2 ), both TOE and CME form stable insoluble films at the surface of water already a few minutes after injection. The difference between extracts becomes visible when the amount of extract necessary to induce similar increase of surface pressure is considered. Namely, in comparison to the TOE, the CME extract seems to be enriched in surface active compounds; therefore the smaller amount of extract is needed to induce comparable raise of surface pressure. In the next stage of our experiments, we aimed to verify if compounds of TOE and CME are able to penetrate the cell membranes of Gram-positive and Gram-negative bacteria in physiological conditions (Marsh 1996 ). For this purpose on the surface of NaCl solution films of lipid composition mimicking bacterial membranes were equilibrated at the surface pressure of 32 mN/m (biologically relevant conditions (Marsh 1996 ) (Dynarowicz-Latka et al. 2024 ). Then the chosen volume of extract was injected below the film, and the change in surface pressure was monitored overtime. The doses of each extract for those experiments were selected based on the adsorption experiments i.e. the smallest dose causing change in surface pressure in adsorption experiments, and this value multiplied by two, four and sixteen. The measured π – time plots were presented in Fig. 3 . The obtained π - time curves confirm incorporation of extract components to membrane models; however, they show different characteristics depending on the investigated membrane model. In case of Gram-negative membrane model, the incorporation of extract in higher doses occurs immediately after its injection into the subphase and the film remains stable overtime. For smaller doses, after injection, the surface pressure rises rapidly and then a gradual penetration of the extract components into the model membrane is still progressing. The film stabilizes after about 15 minutes after extract injection, which is demonstrated by achieving a constant surface pressure values. In the case of Gram-positive membrane model, the penetration kinetics is diverse, depending on the kind of extract investigated and its dose. However, in all cases it can be noticed that the value of surface pressure of the membrane after the extract injection stabilizes overtime. To compare the scale of the effect of the tested plant extracts on the models of Gram-negative and Gram-positive bacterial membranes, based on the data from Fig. 3 , the dependence of the change in surface pressure (Δπ) on the applied extract dose is presented in Fig. 4 . The Δπ values were calculated based on experimental curves by subtracting of the initial surface pressure value (before the extract injection) from surface pressure value of membrane after 20 minutes of equilibration. For CME, the increase in the surface pressure of bacterial membrane models with the quantity of injected extract is more steep in comparison with the results for TOE. This suggests that both types of membranes are more sensitive to components of CME extract or - more likely - CME is enriched in a larger quantity of surface active compounds in comparison to TOE. Furthermore, the Gram-positive membrane model seems to be more susceptible to incorporation of plant extracts components, whereas analogical TOE or CME cause greater increase of surface pressure in comparison to Gram-negative. This phenomenon can be explained using additional experimental methods such as Langmuir monolayers technique and AFM. Langmuir Monolayer Study To get insight into the effect of TOE and CME on model lipid bacterial membranes, the π-A isotherms were measured on 0.1 mol/L NaCl subphase with different amount of studied extracts. π-A isotherms and compressibility modulus plots are presented in Figs. 5 and 6 . Figures 7 and 8 present the collective graphs showing the changes in the compressibility moduli at 32 mN/m and the shift of the mean molecular area in relation to the value measured on pure 0.1 mol/L subphase (without extracts). The π-A isotherm for Gram-positive membrane registered during the compression of the film on 0.1 mol/L NaCl subphase has a characteristic course (Fig. 5 ). The surface pressure starts to rise at ca. 195 Å 2 /molecule and at ca. 7 mN/m and surface area of ca. 138 Å 2 /molecule a plateau occurs. Further increase in surface pressure is not monotonous, namely the slope of the curve changes twice: at pressures of approx. 16 mN/m and 30 mN/m. The monolayer collapses at a surface pressure of 64 mN/m. The plateau region and the slope changes are reflected as minima on the graphs C s −1 versus π (Fig. 5 ). The addition of both extracts (TOE and CME) causes the shift of isotherms towards larger surfaces area per molecule and a slight increase in surface pressure at which a plateau occurs. In the presence of extracts, no inflection is observed at 16 mN/m. The most visible change in the course of the π-A isotherm is observed for the highest content of extracts in the subphase (namely, 400 µL for TOE and 50 µL for CME). The isotherms are more inclined, and the collapse of the films occurs at a significantly lower pressure (ca. 53 mN/m in both cases). On the other hand, the π-A isotherm for Gram-negative model membrane has a different course. The surface pressure starts to rise at ca. 125 Å 2 /molecule and it increases monotonically up to ca. 45 mN/m, where a characteristic bend in the curve is observed. The collapse occurs at a surface pressure of 48 mN/m. Interestingly, in the C s −1 versus π dependency (Fig. 6 ), an additional minimum at about 37 mN/m is observed, suggesting a change in the slope of the π-A curve (not visible on the isotherm). In the case of the Gram-negative membrane, a shift of the isotherms towards larger surface areas per molecule is also observed with an increase in the amount of extracts in the subphase, as well as a decrease in the value of collapse pressure. As can be seen in Fig. 7 , the addition of the studied herb extracts causes fluidization of the model membrane. In the case of TOE and Gram-positive model, strong fluidization occurs already for the smallest amounts of the extract, i.e. 25 µL and 50 µL. The increase of TOE amount from 50 µL to 100 µL does not affect film packing. For the highest TOE content (400 µL), a decrease in C s −1 value is observed pointing on film physical state change from liquid condensed (LC) to the expanded state (LE). In the case of the Gram-negative membrane, the fluidization effect appears for the volume of 50 µL and occurs linearly with the increase of TOE concentration. For the second of the studied extracts - CME - some differences in the fluidization of the Gram-positive versus Gram-negative model membranes are observed. The decrease in molecular packing is stronger for the Gram-positive membrane. In contrast, in the case of Gram-negative membrane, the strongest fluidization occurs when the CME concentration is changed from 50 µL to 100 µL. Interestingly, further increase in the amount of the extract does not affect the film packing. The observed fluidization of the films in all the studied cases can be related to the increase in the mean molecular area (Fig. 8 ). In summary, the investigated extracts exert a comparable influence on the packing of bacterial films, leading to a reduction in membrane condensation in both models. However, for CME the effect is achieved at significantly lower doses, which indicates its stronger activity. On the other hand, comparing the activity towards Gram-positive versus Gram-negative strains, the effect of molecular packing loosening is slightly stronger for Gram-positive bacteria. To better investigate the fluidization effect, as well as morphological changes caused by both extracts the selected systems were deposited on a solid surface (mica) and imaged using AFM. The Effect of Extract Addition on the Topography of Model Lipid Membranes To gain visual insight into micro- and nanoscale topographical changes in model bacterial membranes upon the addition of TOE or CME, AFM imaging was performed on Langmuir monolayers transferred onto atomically flat mica substrates. Topographical AFM images of Gram-negative and Gram-positive model membranes treated with different amounts of TOE (50 µL and 400 µL) and CME (3.6 µL and 50 µL) (referred to as TOE 50, TOE 400, CME 3.6 and CME 50, respectively) are presented in Figs. 9 and 10 . For the Gram-negative model membrane control sample (Fig. 9 A–C), two distinct types of domains protruding above the general membrane plane are clearly observed. AFM height analysis shows that, independent of their lateral size, the domains protrude on average by approximately 0.9 nm. The bimodal nature of the domains is reflected in the area distribution shown in Fig. 11 . This distribution is relatively broad, extending up to ~ 0.2 µm², which is primarily associated with the presence of large, nearly circular domains. A similar trend is observed in the distribution of domain perimeters. In contrast, smaller domains exhibit significantly less regular shapes, as indicated by the dimensionless shape descriptors presented in Fig. 12 . The aspect ratio (AR) within the interquartile range (Q1–Q3) is approximately 1.5, although more elongated structures with AR values up to 4 are also present. This behavior is consistent with the circularity distribution, which is centered around 0.8 but extends to values as low as 0.4 or lower. In addition, the domains are not fully compact, as the presence of internal voids (“holes”) is clearly visible in Fig. 9 C. Upon the addition of TOE and CME, the AFM topography is preserved and no qualitative changes in the overall morphology are observed. However, AFM images (Fig. 9 D-F and G-I for TOE 50 and 400, respectively) and the data presented in Fig. 11 reveal a significant increase in domain size. This effect is observed for both large (Fig. 11 , first row) and small domains (Fig. 11 , second row), as evidenced by shifts in the area and perimeter distributions. Simultaneously, the number of domains per unit area decreases by factors of 13.5 and 7.6 for TOE 50 and TOE 400, respectively (see Table 2 for exact values). In addition to size changes, domain shape is modified upon extract addition. Domain boundaries become more irregular and jagged. While the AR remains largely unchanged at the lower TOE concentration, it increases at the higher concentration. In contrast, circularity decreases significantly in both cases. This apparent discrepancy arises from the definition of AR, which is based on a best-fitting ellipse and is therefore less sensitive to boundary roughness, whereas circularity depends explicitly on the perimeter and is strongly affected by edge irregularities. Together, these observations indicate that the addition of TOE extract promotes domain fusion. Analysis of domain area and perimeter suggests that the fusion mechanism is at least twofold: small domains are able to merge with one another, as directly observed in AFM images (Fig. 9 , panels C, F, and I), while larger domains can undergo self-fusion and/or incorporate smaller domains. Fusion is accompanied by a decrease in solidity, as shown in Fig. 12 . Despite substantial changes in lateral dimensions and shape descriptors, the height difference between domains and the surrounding continuous phase remains relatively constant at approximately 1.1 nm, further supporting a fusion-driven mechanism. It should be noted that AFM is chemically blind; therefore, the chemical origin of the fusion process cannot be resolved solely from topographical imaging. Similar overall behavior is observed upon the addition of CME extract; however, the fusion process is less pronounced. This is reflected in the domain area and perimeter distributions shown in Fig. 11 . As summarized in Table 2 , for the lower CME amount (6.3 µl/1L), the number of domains per unit area is comparable to that observed for TOE 50, while the area of small domains is significantly reduced. Notably, the AR remains at a similar level for both investigated CME concentrations. Domain fusion also leads to changes in circularity (Fig. 12 ): for CME 6.3, the interquartile range (Q1-Q3) shifts toward lower values and is centered around ~ 0.3, whereas for CME 50 the distribution, although broad, is centered near 0.7. A similar trend is observed for solidity. As with TOE, the addition of CME extract does not significantly affect the height difference between domains and the continuous phase (Table 2 ). Taken together, these results demonstrate that the concentration of active compounds in both extracts plays a crucial role in domain fusion in Gram-negative model membranes. At higher concentrations, smaller domains are observed, which can be rationalized in terms of nucleation-controlled kinetics. Assuming that both extracts contain active components capable of modifying domain surface charge, increased concentration may enhance nucleation, thereby suppressing domain coalescence and stabilizing smaller clusters. Alternatively, higher concentrations may reduce lateral domain mobility. Although the precise mechanism responsible for the suppression of domain fusion cannot be unambiguously determined, the results clearly indicate that the system is kinetically limited, despite sufficient equilibration prior to transfer onto the mica substrate. For Gram-positive model membranes, AFM imaging revealed the presence of circular and elliptical domains depressed, on average, 1.5 nm below the general plane. Clearly, these are not discontinuities in layer, as this difference is significantly smaller than thickness of a lipid monolayer. Similar to Gram-negative membranes, these domains exhibit bimodal characteristics, with a relatively broad area distribution, as determined from 5 µm × 5 µm AFM scans. Smaller domains are semi-elliptical, as visible in high-resolution AFM scans (Fig. 10 C) and indicated by the distributions of their AR and circularity (Fig. 12 ). The continuous phase surrounding these topographical features is smooth, with a RMS roughness of 90 pm. Despite the concentrations used, the addition of TOE extract preserves the overall sample topography (Fig. 10 , A–I). However, as deducted from AFM images and changes in N s (Table 2 ), upon addition of TOE extract at a concentration of 50 µl/1L, the number of domains decreases, whereas almost no changes are observed for samples treated with TOE extract at a concentration of 400 µl/1L. Surprisingly, at the lower concentration, the decrease in the number of domains per unit area is not associated with their fusion, as the area of smaller domains also decreases (Fig. 11 ). An adverse effect is observed for the TOE 400 sample, where both area and perimeter distributions are not only significantly broader but also shifted toward higher values. Simultaneously, no changes in dimensionless shape descriptors are observed for TOE 50. For TOE 400, the AR remains unchanged, but circularity decreases significantly (Fig. 12 ). Compared to the control sample (Fig. 10 A-C), the domains exhibit increased boundary complexity and attained a more lobed shape. Considering the lack of changes in N s alongside a simultaneous increase in domain area, it can be postulated that the active ingredients of TOE interact with components of the continuous phase. This interaction results in the formation of clusters that are deposited within the membrane, for example near the edges of domains formed in this way, as seen in Fig. 10 H and I. For samples treated with CME extract, the influence is highly concentration-dependent. Comparison of AFM images obtained for the control sample (Fig. 10 A–C) and CME 6.3 reveals no detectable effect. However, quantitative data presented in Table 2 and Figs. 11 and 12 clearly show that upon addition of CME extract, both the number of domains and their size increase, without significant changes in their dimensionless shape descriptors. The most probable explanation for this observation is that the active ingredients of CME extracts interact with membrane components, altering the delicate interplay between domain composition and line tension within the membrane, which leads to the formation of a higher number of larger domains. This effect may be responsible for the topography observed for Gram-positive membranes treated with CME 50 extract (Fig. 10 M-O), where the nanoscale domain character is no longer preserved and characteristic interconnected patches are visible. Due to the continuous nature of these patches, determination of their shape descriptors was not feasible. The height difference between these patches and the continuous phase is approximately 3 nm, which corresponds to the thickness of a single lipid monolayer. Table 2 Topographical characteristics of lipid bacterial membranes treated with different amounts of TOE and CME derived from AFM imaging: number of domains per unit area ( N s ), average difference between height of domains and surrounding, continuous phase (Δ z ) and roughness, expressed as Root Mean Square (RMS). All data is presented as mean ± standard deviation (if applicable). membrane model N s [um − 2 ] Δ z [nm] RMS [pm], continuous layer RMS [pm], features* Gram(-) 166 ± 20 0.9 ± 0.1 194 264 Gram(-) with 50 µL TOE 12.3 ± 2.9 1.3 ± 0.2 136 124 Gram(-) with 400 µL TOE 21.8 ± 9.9 1.0 ± 0.1 155 181 Gram(-) with 6.3 µL CME 13.0 ± 3.6 1.2 ± 0.2 217 150 Gram(-) with 50 µL CME 110 ± 23 1.1 ± 0.2 211 216 Gram(+) 26.8 ± 6.2 -1.5 ± 0.2 90 204 Gram(+) with 50 µL TOE 12.1 ± 3.1 -1.5 ± 0.2 119 251 Gram(+) with 400 µL TOE 32.8 ± 6.7 -2.1 ± 0.3 94 1500 Gram(+) with 6.3 µL CME 57.8 ± 9.0 -1.1 ± 0.1 94 250 Gram(+) with 50 µL CME n. a. 2.7 ± 0.3 87 241 *Protruding domains (Gram-negative: or depressed domains (Gram-positive: Chromatographic Profiles of the Chelidonium majus and Extractives Detected by a High-resolution Mass Spectrometric System (UHPLC-QTOF) The identified compounds in extracts of Chelidonium majus and Taraxacum officinale by the UHPLC-QTOF system with high-resolution mass spectrometric detection are reported in Supplementary Tables S1-S6, based on previous reports and representative chromatograms, are shown in Fig. 13 . The most prominent were isoquinoline alkaloids identified in Chelidonium majus extract in positive electrospray ionization mode. Mass spectrometric data were used to determine elemental composition of the alkaloids (Supplementary Table S1 ), which can be characterized by their the protonated adducts ([M + H] + ) ( A1 , A2 , A3 , and A10 ) as well as molecular ions [M] + ( A5 , A6 , A9 , A11 , and A12 ) which are already charged in solution. Unknown compounds A4 , A7 and A8 where tentatively assigned as Na + adducts. The highest MS signal in Chelidonium majus extract was observed for coptisine A5 and the other more abundant peaks were detected for A1 , A2 , A4 , and A6 (Supplementary Table S4). Seven isoquinoline alkaloids (Qiao et al. 2009 ; Zielińska et al. 2020 ), two protopine derivatives (protopine A1 , allocryptopine A3 ), two protoberberine derivatives (coptisine A5 and berberine A11 ), and three phenanthridine derivatives (chelidonine A2 , sanguinarine A9 , chelerythrine A12 ) were readily detected (Supplementary Table S1 ). The other rare compounds were tentatively identified as demethyleneberberine A6 (Bahadur and Shukla 1983 ), and Pessoine A10 (Farrow et al. 2012 ). In Taraxacum officinale root extract, the alkaloid MS signals were much lower but still with the highest peak detected for coptisine A5 . In the case of allocryptopine A3 , unknown A7 , sanguinarine A9 , and chelerythrine A12 , the signals were completely not observed (Tables S1 and S4, Supplementary material). Several flavonoids were found in extract of Chelidonium majus at low levels. Two monoglycosides of quercetin and kaempferol ( B10 and B13 ) and three diglycosides (rutinosides; B8 , B11 and B12 ) were assigned according to (Grosso et al. 2014 ) and one triglycoside of kaempferol B4 (Krizhanovska et al. 2021 ) were identified by HRMS measurements and comparison with previous reports. The highest signals were observed for quercetin 3- O -rutinoside B8 and isorhamnetin 3- O -rutinoside B12 (Supplementary Table S5). Another group of three isomeric kaempferol triglycosides was tentatively identified as robinin and its possible isomers ( B5 , B6 and B7 ) according to (Chen et al. 2023 ). A presence of two structures of rare iridoids ( B2 and B3 ) was indicated, presumably isomeric to barlerine (8- O -acetyl shanzhiside methyl ester), based on characteristic molecular formula (C 19 H 27 O 12 ) inferred from m/z value of the [M-H] − ion close to 447.1503 determined by HRMS (Alipieva et al. 2007 ). The presence of quinic B1 and p -coumaric B9 acids was also acknowledged. Compounds C1 ([M-H] − at m/z 191), C2 and C3 ([M-H] − at m/z 353) detected in Taraxacum officinale root extract corresponded to quinic acid, 3- O -caffeoylquinic acid and 5- O -caffeoylquinic acid, respectively, based on the elution order described in previous studies (Garcia-Perez et al. 2025 ). Similarly, compounds C9 , C10 and C12 ([M-H] − at m/z 515) were attributed to 3,4- O -dicaffeoylquinic acid, 3,5- O -dicaffeoylquinic acid and 4,5- O -dicaffeoylquinic acid, respectively. The following peaks of quinic derivatives, C2 , C3 and C12 , were highly abundant in the chromatograms after the MS and PDA detection. Further analysis of the MS results at negative ionization revealed several chromatographic peaks ( C4 - C8, C11 and C14 ) detected at nominal m/z 447 Da (PDA absorption: λ max ~ 222 and 275 nm) with C4 and C5 showing the highest signals (Supplementary Tables S3 and S6). The HRMS analysis gave an elemental composition of C 22 H 23 O 10 observed at m/z 447.1265 (calculated m/z 447.1286) for all the peaks. This indicated a presence of di-4-hydroxyphenylacetic acid inositols identified previously in Taraxacum officinale (Kenny et al. 2014a ). Likewise, peaks C16-C18 were detected at nominal m/z 581 Da (λ max ~ 222 and 275 nm) giving rise to tentative identification of isomeric tri-4-hydroxyphenylacetic acid inositols (Kenny et al. 2014a ) of ionic molecular formula C 30 H 29 O 12 observed at m/z 581.1672 (calculated m/z 581.1654). A group of known sesquiterpenoids was attributed to compounds C13 , C15 , C20 , and C21 ( Michalska et al. 2021 ). Peak C13 was assigned to highly abundant 14- O - β -D-glucosyl-taraxinic acid (Kashiwada et al. 2001 ), based on accurate mass measurement which gave an elemental composition of detected negative ion observed at m/z 423.1642 (calculated m/z 423.1650) as C 21 H 27 O 9 . Similar derivative was attributed to C15 as 14- O - β -D-glucosyl-11,13-dihydro-taraxinic acid with ionic molecular formula C 21 H 29 O 9 detected at m/z 425.1789 (calculated m/z 425.1806). Similarly, the presence of free taraxinic acid C20 (ionic molecular formula C 15 H 17 O 4 ) and 11,13-dihydro-taraxinic acid C21 (ionic molecular formula C 15 H 19 O 4 ) was acknowledged with C21 demonstrating much higher signal (Tables S3 and S6, Supplementary material). Peak C19 was assigned to ferulic acid (obtained ionic molecular formula C 10 H 9 O 4 ) (Tanasa (Acretei) et al. 2025). Determination of the Total Phenolic Content (TPC) by the Folin-Ciocalteu Spectrophotometric Method To support data from UHPLC-QTOF technique, the Total Phenolic Content (Gallic Acid Equivalents (GAE)) was determined for the investigated extracts. The Total Phenolic Content was equal to 0.359 +/- 0.016 (mg GAE/g DW) for Chelidonium majus extract, whereas for Taraxacum officinale it was equal to 0.0852 +/- 0.0040 (mg GAE/g DW). This four times greater phenolic content corroborates with increased antimicrobic activity of celadine versus dandelion. Conclusions Both tested extracts, celandine (CME) and dandelion (TOE), showed antibacterial activity in microbiological tests, with the celandine extract proving more effective. Among the bacterial strains tested, Streptococcus pyogenes ATCC® 12384 was found to be the most sensitive, while Escherichia coli ATCC® 25922 showed the least sensitivity to the extracts used. Complementary experiments performed on bacterial artificial membranes, modeled with the Langmuir monolayer technique, confirmed the stronger fluidization effect exerted by celandine compared to dandelion extract. This can be explained comparing the content of active antimicrobial substances in both extracts. The higher content of phenolic compounds in celadine compared to dandelion extracts confirms the results obtained for monolayers. AFM imaging combined with quantitative image analysis revealed that the interactions of active compounds from dandelion and celandine extracts differ between Gram-negative and Gram-positive membranes. Although concentration-dependent effects are observed for both extracts, the overall topography of Gram-negative membranes remains conserved, with changes occurring primarily at the domain level, most likely due to domain fusion. In contrast, for Gram-positive membranes, the actions of TOE and CME extracts are markedly different. TOE affects membrane topography under both investigated conditions, leading to the formation of deposits within the monolayer. The action of CME is more complex: at lower concentrations, changes occur mainly in the number and area of domains, whereas at higher concentrations, interactions between active compounds and lipid membrane components result in drastic alterations of membrane topography, such that the overall membrane structure is no longer preserved. Declarations Supplementary material Additional tables presenting chromatographic, spectrophotometric and high-resolution mass spectrometric data. Author Contribution A.G-S. and P.D-L. wrote the main manuscript text. A.G-S. , A.W. and A.Ch-B. , D.L. , S.W., Ł.K. , K. D., E.Ł. wrote the experimental parts, did experiments, analysed the results and prepared figures. P.D-L and M.B-W. supervised the mauscript. All authors reviewed the manuscript. 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J Appl Pharm Sci. https://doi.org/10.7324/JAPS.2014.40309 Trombe M-C, Lanéelle M-A, Lanéelle G (1979) Lipid composition of aminopterin-resistant and sensitive strains of Streptococcus pneumoniae. Effect of aminopterin inhibition. Biochimica et Biophysica Acta (BBA) -. Lipids Lipid Metabolism 574:290–300. https://doi.org/10.1016/0005-2760(79)90010-9 Tyler VE (2000) Herbal medicine: from the past to the future. Public Health Nutr 3:447–452. https://doi.org/10.1017/S1368980000000525 Yan Q, Xing Q, Liu Z et al (2024) The phytochemical and pharmacological profile of dandelion. Biomed Pharmacother 179:117334. https://doi.org/10.1016/j.biopha.2024.117334 Zhao F, Mai Q, Ma J et al (2015) Triterpenoids from Inonotus obliquus and their antitumor activities. Fitoterapia 101:34–40. https://doi.org/10.1016/j.fitote.2014.12.005 Zielińska S, Czerwińska ME, Dziągwa-Becker M et al (2020) Modulatory Effect of Chelidonium majus Extract and Its Alkaloids on LPS-Stimulated Cytokine Secretion in Human Neutrophils. Molecules 25:842. https://doi.org/10.3390/molecules25040842 Zielińska S, Jezierska-Domaradzka A, Wójciak-Kosior M et al (2018) Greater Celandine’s Ups and Downs – 21 Centuries of Medicinal Uses of Chelidonium majus From the Viewpoint of Today’s Pharmacology. Front Pharmacol 9. https://doi.org/10.3389/fphar.2018.00299 Zuo GY, Meng FY, Hao XY et al (2009) Antibacterial Alkaloids from Chelidonium Majus Linn (Papaveraceae) Against Clinical Isolates of Methicillin-Resistant Staphylococcus Aureus. J Pharm Pharm Sci 11:90. https://doi.org/10.18433/J3D30Q Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9356537","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633543559,"identity":"c77ff3ec-ac81-4630-8dae-e2ccd50623c3","order_by":0,"name":"Agnieszka Gonet-Surówka","email":"data:image/png;base64,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","orcid":"","institution":"Jagiellonian University","correspondingAuthor":true,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Gonet-Surówka","suffix":""},{"id":633543560,"identity":"999c9b6a-cb0d-47bb-81b0-2bb90ec53a81","order_by":1,"name":"Kamil Drożdż","email":"","orcid":"","institution":"Faculty of Medicine Collegium Medicum Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Kamil","middleName":"","lastName":"Drożdż","suffix":""},{"id":633543561,"identity":"46498468-7abb-4677-9caf-1877cd90d6ad","order_by":2,"name":"Anita Wnętrzak","email":"","orcid":"","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Anita","middleName":"","lastName":"Wnętrzak","suffix":""},{"id":633543562,"identity":"eabe75c1-744d-40ce-bc94-3b6948897cbe","order_by":3,"name":"Anna Chachaj-Brekiesz","email":"","orcid":"","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Chachaj-Brekiesz","suffix":""},{"id":633543563,"identity":"56f46ffe-8dfd-4caa-a218-16f0c22cf0aa","order_by":4,"name":"Dawid Lupa","email":"","orcid":"","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Dawid","middleName":"","lastName":"Lupa","suffix":""},{"id":633543564,"identity":"70b740c0-c4cc-4e3a-a79b-42f3a8fc9674","order_by":5,"name":"Sławomir Wybraniec","email":"","orcid":"","institution":"Univeristy of Technology in Krakow","correspondingAuthor":false,"prefix":"","firstName":"Sławomir","middleName":"","lastName":"Wybraniec","suffix":""},{"id":633543565,"identity":"4ef0cc29-3cda-4e34-a8d6-1f6adb0b73cd","order_by":6,"name":"Łukasz Kozioł","email":"","orcid":"","institution":"Univeristy of Technology in Krakow","correspondingAuthor":false,"prefix":"","firstName":"Łukasz","middleName":"","lastName":"Kozioł","suffix":""},{"id":633543566,"identity":"ab18e551-bcda-40c8-a4d1-2ed2b27aac5a","order_by":7,"name":"Ewa Łopuszyńska","email":"","orcid":"","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Łopuszyńska","suffix":""},{"id":633543567,"identity":"8d893fa2-df89-4659-b858-12bc926c9b12","order_by":8,"name":"Monika Brzychczy-Włoch","email":"","orcid":"","institution":"Faculty of Medicine Collegium Medicum Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Monika","middleName":"","lastName":"Brzychczy-Włoch","suffix":""},{"id":633543568,"identity":"c794c313-92e2-4fda-a0e5-92aa80a802fe","order_by":9,"name":"Patrycja Dynarowicz-Latka","email":"","orcid":"","institution":"Jagiellonian University","correspondingAuthor":false,"prefix":"","firstName":"Patrycja","middleName":"","lastName":"Dynarowicz-Latka","suffix":""}],"badges":[],"createdAt":"2026-04-08 11:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9356537/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9356537/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108960096,"identity":"80276dc3-f590-4c27-8510-4fd473954ff1","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":495605,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy of bacteria stained with LIVE/DEAD BacLight (SYTO® 9 and propidium iodide). Bacterial cultures: \u003cem\u003eStaphylococcus aureus ATCC® 25923\u003c/em\u003e, \u003cem\u003eStreptococcus pyogenes ATCC® 12384\u003c/em\u003e, \u003cem\u003eEscherichia coli ATCC® 25922\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa ATCC® 27853 \u003c/em\u003ewere grown in control medium with and without the addition of celandine or dandelion extracts. Green fluorescence indicates live cells; orange indicates dead or membrane-compromised cells. Scale bar: 50 µm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/c44782126aef6724c4c9f109.png"},{"id":108978315,"identity":"c17c7a7c-aea3-4888-97ba-4697faac1ff3","added_by":"auto","created_at":"2026-05-11 11:36:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1012815,"visible":true,"origin":"","legend":"\u003cp\u003eThe adsorption of dandelion root extract (TOE) (a) or celadine herb extract (CME) (b) into the air/water interface.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/da0b31722d6be8d2daf2fa76.png"},{"id":108960098,"identity":"e52dba97-fd38-460f-89de-7ff6ff8f1620","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101486,"visible":true,"origin":"","legend":"\u003cp\u003eThe surface pressure – time curves for Gram-negative (Gram(-)) (a, b) and Gram-positive (Gram(+)) (c, d) bacterial membrane models measured before and after injection of \u0026nbsp;dandelion root extract (TOE) (a, c) or celadine herb extract (CME) (b, d) into the subphase.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/308c48ae4d9e6f4fc446f1b0.png"},{"id":108978047,"identity":"be337ef8-518d-4648-be20-408ff5fc726a","added_by":"auto","created_at":"2026-05-11 11:33:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":838540,"visible":true,"origin":"","legend":"\u003cp\u003eThe dependence of surface pressure change of bacterial lipid membrane models Gram-negative (Gram(-)) and Gram-positive (Gram(+)) from the applied dose of dandelion root extract (TOE) or celadine herb extract (CME).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/c5a468f55c4523764a108da4.png"},{"id":108960100,"identity":"4cb3aad9-cc96-42e1-bd99-7c0a7c071797","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1757219,"visible":true,"origin":"","legend":"\u003cp\u003eThe π-A isotherms and the compressional modulus \u003cem\u003eversus\u003c/em\u003ethe surface pressure plots for the monolayers mimicking Gram-positive (Gram(+)) bacteria lipid membranes spread on 0.1 mol/L and on dandelion root extract (TOE) (a, c) or celadine herb extract (CME) solutions (b, d).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/f47e7260c3d2ea895e29636b.png"},{"id":108960106,"identity":"567def67-7926-4949-bd71-38d094c2fcd9","added_by":"auto","created_at":"2026-05-11 08:37:40","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1635273,"visible":true,"origin":"","legend":"\u003cp\u003eThe π-A isotherms and the compressional modulus \u003cem\u003eversus\u003c/em\u003ethe surface pressure plots for the monolayers mimicking Gram-negative (Gram(-)) bacteria lipid membranes spread on 0.1 mol/L and on dandelion root extract (TOE) (a, c) or celadine herb extract (CME) solutions (b, d).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/f8850df9eb1694600f66927f.png"},{"id":108960101,"identity":"c14bb3bb-4641-4f0a-ac84-0936716cb2cb","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":185498,"visible":true,"origin":"","legend":"\u003cp\u003eThe compressional modulus changes for the films imitating Gram-positive (Gram(+)) and Gram-negative (Gram(-)) model lipid membranes in the presence of herb extracts (a) (a) dandelion root extract (TOE) or celadine herb extract (CME) (b) ME calculated at 32 mN/m.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/5516ff11bffebe82c0f123bd.png"},{"id":108960107,"identity":"53feeaba-0a68-476b-a9b7-6010154771b4","added_by":"auto","created_at":"2026-05-11 08:37:40","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1381029,"visible":true,"origin":"","legend":"\u003cp\u003eThe shift of mean molecular area for the films imitating Gram-positive (Gram(+)) and Gram-negative (Gram(-)) model lipid membranes in the presence of herb extracts (a) dandelion root extract (TOE) (a) or celadine herb extract (CME) (b) calculated at 32 mN/m.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/68126b0a3159cd7963793590.png"},{"id":108960103,"identity":"ce741235-0b84-49b2-9f91-b996396e0953","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":170479,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative AFM images presenting changes in topography in the monolayers mimicking Gram-negative (Gram(-)) bacteria lipid membranes resulting from interaction of lipid molecules with active compounds of dandelion and celandine extracts: control sample (A-C), dandelion extract TOE 50 (D-F), dandelion extract TOE 400 (G-I), celandine extract CME 6.3 (J-L) and celandine extract CME 50 (M-O). Each image is 512 x 512 pixels. The scale bar corresponds to all images in a column. The false-color scale is applicable to all presented images.\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/543df36b0300987125bea3ce.jpeg"},{"id":108960105,"identity":"f0c6c207-2f3d-41a0-8fc9-e923b02cbc6c","added_by":"auto","created_at":"2026-05-11 08:37:40","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":125539,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative AFM images presenting changes in topography in the monolayers mimicking Gram-positive (Gram(+)) bacteria lipid membranes resulting from interaction of lipid molecules with active compounds of dandelion and celandine extracts: control sample (A-C), dandelion extract TOE 50 (D-F), dandelion extract TOE 400 (G-I), celandine extract CME 6.3 (J-L) and celandine extract CME 50 (M-O). Each image is 512 x 512 pixels. The scale bar corresponds to all images in a column. The false-color scale is applicable to all presented images.\u003c/p\u003e","description":"","filename":"image10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/882e82efaa089073a14cce26.jpeg"},{"id":108960102,"identity":"18ead203-58a4-4479-87f0-1a45db9334b1","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1049136,"visible":true,"origin":"","legend":"\u003cp\u003eDimensional topographical quantities of Gram-negative (Gram(-)) and Gram-positive (Gram(+)) bacterial lipid membranes treated with different amount of dandelion (TOE 50 and TOE 400) and celandine extracts (CME 6.3 and CME 50). Size distribution of bigger and smaller domains was determined based on 25 µm\u003csup\u003e2\u003c/sup\u003e and 4 µm\u003csup\u003e2\u003c/sup\u003e AFM scans, respectively. Black point represent the scatter of experimental data. Rectangular boxes represent the interquartile range (25\u003csup\u003eth\u003c/sup\u003e–75\u003csup\u003eth\u003c/sup\u003e percentiles) of the data. Whiskers extend to the most extreme data points within 1.5 times the interquartile range from the quartiles Q1 and Q3.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/89dc8351b6a0b021ff5cf6aa.png"},{"id":108978224,"identity":"f780b8a4-ddef-462a-822a-4a2452a3db97","added_by":"auto","created_at":"2026-05-11 11:35:11","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":988787,"visible":true,"origin":"","legend":"\u003cp\u003eDimensionless topographical quantities of Gram-negative (Gram(-)) and Gram-positive (Gram(+)) bacterial lipid membranes treated with different amount of dandelion (TOE 50 and TOE 400) and celandine extracts (CME 6.3 and CME 50). Black point represent the scatter of experimental data. Rectangular boxes represent the interquartile range (25\u003csup\u003eth\u003c/sup\u003e–75\u003csup\u003eth\u003c/sup\u003e percentiles) of the data. Whiskers extend to the most extreme data points within 1.5 times the interquartile range from the quartiles Q1 and Q3.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/cd3bcdfbd9c28f97155f6214.png"},{"id":108978316,"identity":"1b2fe8e6-6246-4617-a447-437ef24a0524","added_by":"auto","created_at":"2026-05-11 11:36:12","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":319170,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChromatograms of Taraxacum officinale\u003c/em\u003e F. H. Wigg. Root (a) and \u003cem\u003eChelidonium majus\u003c/em\u003e L. aerliar part (b\u003cem\u003e) extracts monitored by PDA detection.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/773af6c6d44a7d9261a373f8.png"},{"id":108980015,"identity":"9bc2a484-f1f3-4f44-a92b-ba123a41769f","added_by":"auto","created_at":"2026-05-11 12:03:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9969855,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/5b185aec-132d-47b5-aee5-72b02bb15274.pdf"},{"id":108960095,"identity":"0168c099-b6b5-476b-8cd3-6229864cb0d3","added_by":"auto","created_at":"2026-05-11 08:37:39","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":45681,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9356537/v1/8eb346b29afd53de2992ea4b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Disruption of Bacterial Lipid Membranes by Phenolic Compounds from Plant Extracts:Biophysical Evidence from Microbiological and Langmuir Monolayer Studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe increasing resistance of many bacterial strains to conventional antibiotics poses a significant challenge to medicine in the 21st century. As a result of acquired resistance to drugs, the pathogenicity of many strains of bacteria has increased significantly. In consequence, thousands of patients die in hospitals around the world every year due to infections with antibiotic-resistant bacteria. In recent years, extracts isolated from plants have attracted great interest and are experiencing a real renaissance in scientific research, giving new hope for the treatment of severe problems related to antibiotic resistance.\u003c/p\u003e \u003cp\u003eAs numerous studies have shown (J.A. Duke \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; I.F.F. Benzie and S.Wachtel-Calor 2011), plant-derived compounds have many medicinal properties, including antibacterial, antiviral, antifungal, anticancer, antioxidant and immunomodulatory effects. Research shows that natural herbs can be a good alternative to synthetic drugs due to their lower toxicity to both the patient and the environment. Great hopes are also placed in the treatment of diseases considered incurable, such as neurodegenerative diseases, endometriosis, or malignant tumors, for which synthetic drugs seem to be ineffective. Plant extracts are characterized by a multi-component composition, and their specific mutual proportions acting synergistically may be the key to their more effective therapeutic action compared to individual medicinal substances either extracted from natural sources or of synthetic origin. A good example is chaga mushroom (\u003cem\u003eInonotus obliquus\u003c/em\u003e) extract used in various human cancer cell lines (Chung et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) (Zhao et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Alcoholic extracts are mostly available on the market but frequently used in folk medicine are aqueous (Lee et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) extracts (containing, among others, polysaccharides (Lu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u0026ndash; interestingly - they were reported to be also effective. The composition of alcohol extracts contains \u0026ndash; in addition to bioelements - phenolic acids, sterols, indole compounds and triterpenoids (Sułkowska-Ziaja et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For anticancer activity of chaga extracts, triterpenoids, especially betulin, are believed to be effective, however, they are nearly insoluble in water. The most likely theory assumes that all the active substances found in the chaga mushroom are naturally present in precisely defined, appropriate proportions. It is possible that the compounds contained in the mushroom somehow activate trace amounts of betulin, which are transferred into the infusion during brewing and delivered to the body, where they begin to affect cancer cells (Camilleri et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This aspect, however, needs further investigations.\u003c/p\u003e \u003cp\u003eTreating infections with herbs has a centuries-old world tradition. Using herbs as medicinal sources dates back to prehistoric times, for example, in tombs in Iraq from about 60,000 years ago, fragments of plants, species such as \u003cem\u003eEphedra\u003c/em\u003e, \u003cem\u003eCentaurea\u003c/em\u003e and \u003cem\u003eSambucus\u003c/em\u003e (edelberry) were found (Tyler \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Many herbs and plants of mainly antimicrobial activities but also showing other health promoting properties were found in various regions of the world as reported in a plethora of papers (for example in India (Srinivasan et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), China (Tan and Vanitha \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), and other regions in the East: Maylasia (Chung et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), Arab Emirates (Tanira et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), Iran (Mahboubi \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2388\u003c/span\u003e), Russia (Shikov et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Siberia (Kokoska et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e); Africa (A. Ajayi \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), America (Belinda Reynolds 2023), (Cuevas-Cianca et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), (Ruiz-Bustos et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and Spanish Mediterranean region (R\u0026iacute;os et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Their components belonging to alkaloids, coumarins, tannins, terpenoids, flavonoids, saponins and polyphenols, have proven antibacterial, antifungal or antiviral effects. What is interesting, their mode of action is twofold. Namely, their bioactive compounds affect cellular metabolic pathways, causing protein and DNA damage (without producing free radicals ROS, which are very dangerous for the body, unlike antibiotics or nanomaterials, including metal nanoparticles or metal oxides, e.g. nanosilver (Parham et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but also\u0026mdash;due to their amphiphilic structure and surface activity\u0026mdash;exhibit membrane activity. This latter aspect of their action has not been thoroughly investigated to date. Only a few reports on this topic can be found in the literature regarding individual compounds found in herbal extracts. Regarding antimicrobial activities, the majority of studies are concentrated on terpenoids (Fontanay et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and (Francis et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). They are proved to adsorb to the membrane and induce cells lysis by membrane fluidization, causing cells lysis (Korchowiec et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) (Orczyk et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Broniatowski et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Although a large body of literature, as mentioned above, describes the antimicrobial activity of the local plants and herbs, the aim of our work was to investigate the antimicrobial properties of two herbal extracts with a wide distribution worldwide: dandelion (\u003cem\u003eTaraxacum officinale\u003c/em\u003e F.H. Wigg) and celandine (\u003cem\u003eChelidonium majus\u003c/em\u003e L.). A review of the literature in recent decades confirms that the largest number of publications related to medicinal antibacterial plants concerns dandelion (which is in second place in the cited literature, right after the most frequently studied Baikal skullcap (\u003cem\u003eScutellaria baicalensis\u003c/em\u003e) (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We therefore focused our attention on an in-depth study of celandine, comparing its antibacterial activity with that of dandelion. One reason celandine has been less studied may be its hepatotoxicity (F Pantano 1 2017) when administered orally. However, this does not rule out its potential as an effective antibacterial agent for external (e.g. skin) infections.\u003c/p\u003e \u003cp\u003eCelandine, formerly known as the \u003cem\u003eGift of Heaven\u003c/em\u003e (\u003cem\u003eCoeli donum\u003c/em\u003e, hence the generic name \u003cem\u003eChelidonium\u003c/em\u003e) has been reported to possess antioxidant and antimicrobial activities (Hong et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) due to high contents of phenolic and flavonoid compounds (Zielińska et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, it has been less frequently studied compared to dandelion, which has long been known for its antimicrobial effects due its extracts rich in triterpenes and their saponins, sterols and their glycosides, organic acids, saccharides, volatile oils in addition to phenolic acid and flavonoids (Yan et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we conducted \u003cem\u003ein vitro\u003c/em\u003e experiments, using both living bacterial cells and model lipid membrane systems. The selected pathogenic strains comprised Gram-positive bacteria\u0026mdash;\u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e\u0026mdash;as well as Gram-negative bacteria\u0026mdash;\u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa.\u003c/em\u003e The model investigations included floating Langmuir monolayers and adsorption experiments. Furthermore, lipid monolayers mimicking Gram-positive and Gram-negative membrane were transferred onto solid substrates, and their structures were examined by atomic force microscopy (AFM). Notably, effects of the studied extracts on artificial bacterial membranes have not been previously investigated. Results from both microbiological and monolayer studies confirmed the antibacterial activity of the tested herbal extracts, with celandine exhibiting a more pronounced effect. AFM analysis of Langmuir\u0026ndash;Blodgett (LB) monolayers revealed distinct differences in the extracts\u0026rsquo; impact on Gram-positive \u003cem\u003eversus\u003c/em\u003e Gram-negative membrane models, as well as differences between the two extracts in their destructive action on Gram-positive bacteria lipid membranes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eFor the preparation of extracts, dried herbs from the company Herbapol (Krak\u0026oacute;w, Poland) were used. According to the manufacturer, the herbs were grown in a pollution-free region of northern Poland and air-dried at a temperature not exceeding 35\u0026deg;C. The dandelion (\u003cem\u003eTaraxacum officinale\u003c/em\u003e F.H. Wigg\u003cem\u003e)\u003c/em\u003e root, and celandine (\u003cem\u003eChelidonium majus\u003c/em\u003e L.) aerial part (including the stem, leaves, and flowers, without the root) were used in our experiments. The dried herbs were ground into fine dust in a mortar, then 10 g portions were prepared. The portions were filled with 500 mL of 96% ethanol. They were left for 24 hours, filtered, and the residue was again filled with 300 mL of solvent. Again, it was left for 24 hours, filtered and the residue on the filter was covered with 200 mL of the given solvent. After another 24 hours, filtration was carried out and the filtrate from three days was combined. The solvent was evaporated on a vacuum evaporator to dryness. The dandelion root extract (TOE) or celadine herb extract (CME) were stored at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe following lipids, as components of bacterial membranes, were employed in the monolayer studies: 1\u0026prime;,3\u0026prime;-bis[1,2-dimyristoyl-sn-glycero-3-phospho]-glycerol (14:0 CL), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were purchased from Sigma\u0026ndash;Aldrich in purity\u0026thinsp;\u0026gt;\u0026thinsp;99% and used without further purification. Chloroform of spectroscopic purity stabilized by ethanol was provided by Sigma\u0026ndash;Aldrich. The aqueous solution of NaCl in concentration of 0.1 mol/L was prepared in ultra-pure water of the resistivity of 18.2 MΩ\u0026middot;cm (from HLP system) and was used as a subphase.\u003c/p\u003e \u003cp\u003eAs a model of bacterial membranes, the two-component lipid systems: CL/DPPG (in mole proportion of 1:1) for Gram-positive membrane model (Hąc-Wydro et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Barbosa et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and POPE/DPPG (in mole proportion of 8:2)࿼ membrane model were applied applied (Singleton et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial Activity Testing\u003c/h2\u003e \u003cp\u003eFour strains of pathogenic bacteria were selected for the study. Two of them were Gram-positive bacteria: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC\u0026reg; 25923 and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e ATCC\u0026reg; 12384. The selected Gram-negative bacteria were: \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC\u0026reg; 25922 and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC\u0026reg; 27853. The evaluation of antibacterial activity of isolated plant extracts i\u003cem\u003en vitro\u003c/em\u003e was conducted following the methodology outlined in the EUCAST Broth Microdilution \u0026ndash; Reading Guide v 5.0 (1 January 2024). Initially, bacteria were cultured on Columbia Agar (Oxoid) with 5% Sheep Blood for 24 h in 37\u0026deg;C. Microbial inoculum for antibacterial activity testing were prepared by suspending a few colonies in sterile TSB (Tryptic Soya Broth) (Sigma). The densities of the inoculum were adjusted to 0.5 McF with a densitometer (Biosan, Poland). Three bacterial strains: \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were subsequently diluted 1:100 to concentration 1,5 x 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in Miller Hinton Broth II (MHB) (Merk, Germany) and \u003cem\u003eS. pyogenes\u003c/em\u003e strain were diluted 1:100 in TSB and used for testing immediately after preparation. Plant extracts were dissolved in TSB or MHB to obtain stock solution of 200 mg/mL from which series dilutions were made giving intermediate concentrations ranging from 0.2 mg/mL to 200 mg/mL. The studies were performed in flat-bottom, polystyrene, 96-well microdilution plates (Nest, China). A specific concentration of tested substances diluted in culture medium was placed in the volume of 100 \u0026micro;L in the wells, and 10 \u0026micro;L of bacterial inoculum was added. As a control, pure medium was used to which 10 \u0026micro;L of microbial inoculum was added. Gentamicin (Pol-Aura), a broad-spectrum antibiotic with a concentration range of 0.08 \u0026micro;g/mL to 40 \u0026micro;g/mL was used. All plates were incubated for 24 hours at 36\u0026deg;C. To check the Minimal bactericidal concentrations (MBC) value, each well containing the tested strain was plated on solid medium and incubated for 24 hours at 36\u0026deg;C. After this time, bacteria growth on the medium was checked. The tests were performed in triplicate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe Adsorption and Penetration Experiments\u003c/h3\u003e\n\u003cp\u003eThe adsorption and penetration experiments were performed in a Teflon cuvette with cylindrical cavity (75 mL capacity) placed above the magnetic stirrer. For adsorption experiments the cuvette was filled with NaCl solution in concentration of 0.1 mol/L in ultrapure water and after several minutes of equilibration the selected volume of ethanolic plant extract was injected into subphase using microsyringe (Hamilton) through a side slot of cuvette. Surface pressure was monitored for 30 minutes with the Wilhelmy method (accuracy of \u0026plusmn;\u0026thinsp;0.01 mN/m). For penetration experiments the chosen lipid mixture mimicking cellular membrane of Gram-positive or Gram-negative bacteria was deposited dropwise on NaCl solution in concentration of 0.1 mol/L in ultrapure water with microsyringe (Hamilton) and stabilized for 5 minutes. The initial surface pressure of spread monolayer was approximately 32 mN/m. Then, the selected volume of ethanolic plant extract was injected into subphase using microsyringe (Hamilton) through a side slot of cuvette and the surface pressure was measured for 40 minutes.\u003c/p\u003e\n\u003ch3\u003eLangmuir Monolayer Experiments\u003c/h3\u003e\n\u003cp\u003eThe experiments were performed employing the Langmuir trough (KSV) with two movable barriers and a total area of 700 cm\u003csup\u003e2\u003c/sup\u003e placed on an anti-vibration table. The surface pressure (π) \u0026ndash; molecular area (A) isotherms for lipid films mimicking bacterial membranes were recorded on 0.1 mol/L NaCl solution and 0.1 mol/L NaCl aq. with addition of TOE or CME solutions. The volumes of TOE added to 1 L of subphase were as follows: 25 \u0026micro;L, 50 \u0026micro;L, 100 \u0026micro;L and 400 \u0026micro;L, while CME was added in volumes of: 3.1 \u0026micro;L, 6.3 \u0026micro;L, 12.5 \u0026micro;L and 50 \u0026micro;L. Every time, appropriate volume of chloroform solution of the investigated lipid mixtures was deposited at the air/water interface with Hamilton analytical syringe. 5 min was left for the spreading solvent evaporation and film stabilization after which the monolayers were compressed with constant compression rate of 20 cm\u003csup\u003e2\u003c/sup\u003e\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Surface pressure was measured with the accuracy of 0.1 mN/m with the Wilhelmy electrobalance (KSV). As the surface pressure sensor, a rectangular plate (1.0 cm x 2.5 cm) of ashless filtration paper (Whatman) was applied. All experiments were performed at a constant temperature (20\u0026deg;C), which was controlled (\u0026plusmn;\u0026thinsp;0.1\u0026deg;C) by a circulating water system. Each π-A isotherm was repeated at least three times to ensure reproducibility of the curves to 2 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e/molecule and of surface pressure of 0.5 mN/m.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFilm Deposition and AFM Measurements\u003c/h2\u003e \u003cp\u003eSamples for atomic force microscopy (AFM) measurements were prepared by transferring Langmuir monolayers of mixed lipid systems mimicking Gram-positive and Gram-negative bacterial membranes from the gas\u0026ndash;aqueous interface onto solid support. The transfers were performed using a double-barrier Langmuir\u0026ndash;Blodgett trough (NIMA 612D; total trough area: 600 cm\u0026sup2;). For deposition, freshly cleaved mica substrates were immersed in the aqueous subphase, and the monolayers were compressed to the target surface pressure of 30 mN/m. After a stabilization period of 10 min at constant pressure, the monolayers were transferred onto mica at a dipping speed of 3 mm/min. For reference systems (bacterial membranes without extracts) the subphase consisted of an aqueous 0.1 mol/L NaCl solution. Modified subphases were prepared by supplementing the 0.1 mol/L NaCl solution with TOE at concentrations of 50 and 400 \u0026micro;L/L, or with CME at concentrations of 3.6, and 50 \u0026micro;L/L (hereinafter referred to as TOE 50, TOE 400, CME 3.6 and CME 50, respectively).\u003c/p\u003e \u003cp\u003eTopography of bacterial membranes transferred on mica surface was visualized using Bruker Multimode 8 AFM instrument equipped with Nanoscope V controller. Imaging was performed in Peak-Force Tapping mode using ScanAsyst Air cantilevers (spring constant 0.4 N/m). To minimalize the influence of tip-sample interaction, scanning was performed using relatively low Peak Force values (~\u0026thinsp;300 pN). Typical scanning conditions were as follow: Scan rate 0.7\u0026ndash;1.0 Hz, Tapping Frequency 4 kHz, Tapping amplitude 30 nm and Lift Height 60 nm.\u003c/p\u003e \u003cp\u003eRaw AFM data was using Gwyddion Software. Typically, images were flattened by subtracting the 2nd order XY polynomial, followed by line levelling using median method. For images with relatively large differences in height, additional step consisting on masking procedure and 2nd order polynomial line levelling was applied. If needed, horizontal scars were removed from particular image using \u003cem\u003eRemove Scars\u003c/em\u003e function. Images were then exported as 1024 x 1024 pixel .tiff files.\u003c/p\u003e \u003cp\u003eGwyddion software was also used to derive the quantitative topographical information from processed AFM scans. This was done by manually setting the mask on Height-Channel, followed by use of the \u003cem\u003eStatistical Quantities\u003c/em\u003e built-in function. In this way, average height and roughness of topographical features and surrounding, continuous phase were derived.\u003c/p\u003e \u003cp\u003eExported tiff images were analyzed using ImageJ software. First, the images were binarized by setting they type to 8-bit image (\u003cem\u003eImage \u0026ndash; Type \u0026ndash; 8-bit\u003c/em\u003e). Then, threshold was set manually (\u003cem\u003eImage \u0026ndash; Adjust - Threshold\u003c/em\u003e) to ensure that topographical features of interest are marked appropriately. Binarized images were then analyzed using \u003cem\u003eAnalyze Particles\u003c/em\u003e (\u003cem\u003eAnalyze \u0026ndash; Analyze Particles\u003c/em\u003e) function, with \u003cem\u003eExclude on edges\u003c/em\u003e option enabled. This allowed to derive the distribution dimensional (area, perimeter) of and dimensionless (aspect ratio, circularity, solidity) topographical quantities.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFluorescence Microscopy Images\u003c/h3\u003e\n\u003cp\u003eFour strains of pathogenic bacteria: \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eS. pyogenes\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were cultured on Columbia Agar (Oxoid) with 5% Sheep Blood for 24 h in 37\u0026deg;C. Microbial inoculum were prepared by suspending a few colonies in sterile TSB (Tryptic Soya Broth) (Sigma). The densities of the inoculum were adjusted to 0.5 McF with a densitometer (Biosan, Poland). Three bacterial strains: \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e were subsequently diluted 1:100 in Miller Hinton Broth II (MHB) (Merck, Germany) and \u003cem\u003eS. pyogenes\u003c/em\u003e strain were diluted in TSB and used for testing immediately after preparation. Plant extracts were dissolved in TSB or MHB to obtain stock solution of 200 mg/mL from which series dilutions were made giving intermediate concentrations ranging from 0.2 mg/mL to 200 mg/mL. A specific concentration of tested substances diluted in culture medium was placed in the test tube, and 100 \u0026micro;L of bacterial inoculum was added. As a control, pure medium was used to which 100 \u0026micro;L of microbial inoculum was added. All test tubes were incubated for 24 hours at 36\u0026deg;C. After 24 hours, the test tubes were centrifuged at 4000 g for 5 minutes. The supernatant was then poured off, and the pellet was resuspended in 1 mL of physiological saline. The sample was centrifuged again, the pellet was resuspended in 100 \u0026micro;L of physiological saline, and then 3 \u0026micro;L of SYTO 9 and 3 ul propidium iodide solution prepared from the LIVE/DEAD BacLight (Thermofisher) kit. The samples were mixed thoroughly and incubated at room temperature in the dark for 15 minutes. 5 \u0026micro;L of the stained bacterial suspensions were trapped between a slide and an 18 mm square coverslip. The samples were examined using a fluorescence microscope (Olympus BX63, Japan) equipped with a 20\u0026times; objective lens (UPLXAPO 20\u0026times;/0.8, dry) to evaluate the effects of the plant extracts. The excitation/emission maxima for these dyes was about 480/500 nm for SYTO 9 stain and 490/635 nm for propidium iodide.\u003c/p\u003e\n\u003ch3\u003eChromatographic Analysis with Detection by a High-resolution Mass Spectrometric System (UHPLC-QTOF)\u003c/h3\u003e\n\u003cp\u003eAll high-resolution mass spectra were analyzed using a UHPLC-QTOF system consisting of Waters Synapt XS mass spectrometer (electrospray ionization mode ESI-QTOF) coupled to a Waters Acquity I-Class Plus chromatographic separation system (Waters Corporation, Milford, MA, USA). The samples were eluted through a 2.1 \u0026times; 100 mm and 1.7 \u0026micro;m particle size chromatographic column (Phenomenex, Torrance, CA, USA) preceded by a guard column of the same material (2.1 \u0026times; 5 mm and 1.7 \u0026micro;m particle size). The injection volume was 10 \u0026micro;L, and the flow rate was 0.3 mL/min. The column was thermostated at 40\u0026deg;C. The separation of the analytes was performed with binary gradient elution. The mobile phases were: A\u0026thinsp;\u0026minus;\u0026thinsp;0.1% formic acid in water and B\u0026thinsp;\u0026minus;\u0026thinsp;0.1% formic acid in acetonitrile. The gradient profile was: (t (min), % B), (0, 5), (6, 60), (8, 90). The full range of Photodiode Array (PDA) detector spectra were recorded in the 200\u0026ndash;700 nm range with a 1.2 nm resolution and a sampling rate of 20 points/s. The conditions for MS detection settings of Waters Synapt XS mass spectrometer were as follows: source temperature 150\u0026deg;C, desolvation temperature 250\u0026deg;C, desolvation gas flow rate 600 L/h, cone gas flow 100 L/h, capillary potential 3.00 kV, and cone potential 30 V. Nitrogen was used for both nebulizing and drying gas. The full scan data ranging from \u003cem\u003em/z\u003c/em\u003e 50 to 1000 were obtained in a resolution MSE scan mode in positive or negative polarity. Leu-enkephalin was used as a mass reference. Data acquisition and analysis software were MassLynx v4.2 and MSe Data Viewer v2.0 (Waters Corporation, Milford, MA, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the Total Phenolic Content (TPC) by the Folin-Ciocalteu Spectrophotometric Method\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe total phenolic content (TPC) of different plant samples was determined using the Folin-Ciocalteu spectrophotometric method (Singleton et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) with the absorbance of the mixtures measured in a microplate reader Tecan Infinite 200 (Tecan Austria GmbH, Gr\u0026ouml;dig/Salzburg, Austria) at a wavelength of 740 nm. Three repetitions were done for each of the samples. The ethanolic extract of a sample (1 mL) was mixed with 0.5 mL of Folin-Ciocalteu reagent and 2.5 mL of 7.5% Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. A blank was prepared in the same way, using ethanol instead of the sample. The prepared mixtures were incubated in a dark place for 120 min, after which the absorbance was measured. The content of total phenols was determined using a calibration curve of standard gallic acid and the results were expressed as milligrams of gallic acid equivalents (mg GAE/g DW) per gram of dried plant sample.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eTests on Bacterial Strains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe antimicrobial potential of celandine extract is mainly due to the alkaloids (Hong et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (Zuo et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), while sesquiterpene lactones are mostly responsible for dandelion root antimicrobial efficacy (Gonz\u0026aacute;lez-Castej\u0026oacute;n et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Futhermore, phenolic compounds (especially flavonoids) present in both extracts, known for their antioxidant properties, may mediate their antimicrobial activity (Gonz\u0026aacute;lez-Castej\u0026oacute;n et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) (Sengul M et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn literature the authors report the results of the composition of celandine and dandelion extracts using different solvents (Tettey et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), mostly alcohol (ethanol, methanol), hexane, methylene chloride, ethyl acetate, butanol or water. For our investigations, we chose ethanol extracts based on literature reports proving their high content of bioactive components of antimicrobial effects, contrary to water extracts (Kenny et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). The presence of polysaccharides in the water extracts is the most probable cause for the lack of significant (Kenny et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e) or even no (Shittu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) antimicrobial activity of these extracts as sugars may act as bacteria feed source, and in this way promote their proliferation.\u003c/p\u003e \u003cp\u003eMinimal bactericidal concentrations (MBC) values were determined for two different extracts (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the case of greater celandine extract, for Gram-negative bacteria; \u003cem\u003eE. coli\u003c/em\u003e MBC was 160 mg/ml, and for \u003cem\u003eP. aeruginosa\u003c/em\u003e MBC was lower and amounted to 128 mg/mL. For Gram-positive bacteria incubated with celandine extract, the MBC was 64 mg/mL for \u003cem\u003eS. aureus\u003c/em\u003e and 1.75 mg/mL for \u003cem\u003eS. pyogenes.\u003c/em\u003e Dandelion extract exhibited generally weaker bactericidal activity, with the highest value for \u003cem\u003eS. aureus\u003c/em\u003e of 160 mg/mL and 60 mg/mL for \u003cem\u003eS. pyogenes\u003c/em\u003e. For Gram-negative bacteria: \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, the MBC value for dandelion extract was 128 mg/mL. Celandine extract has been shown to have greater bactericidal activity, especially against Gram-positive bacteria. Stronger antimicrobial activity of dandelion \u003cem\u003eversus\u003c/em\u003e celandine extracts (TOE \u003cem\u003eversus\u003c/em\u003e CME, respectively) may be related to a lower amount of bactericidal components present in the latter herb.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMBC value for celandine and dandelion extracts acting on selected pathogenic bacterial strains.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ebacteria strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003edandelion extract (TOE)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ecelandine extract (CME)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli ATCC\u0026reg; 25922\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e160 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa ATCC\u0026reg; 27853\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus ATCC\u0026reg; 25923\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus pyogenes ATCC\u0026reg; 12384\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.75 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll four strains of bacteria tested were found to be sensitive to alcohol extracts from the plants used. Gram-positive bacteria were found to be more sensitive to the compounds contained in the extracts, particularly the CME herb extract. The differences resulting from the action of the extracts on the two groups of bacteria may result from the composition of the cell membrane structure. Gram-negative bacteria possess different cell envelope components than Gram-positive. Namely, Gram-negative bacteria have two lipid bilayer membranes in their structure: an outer and an inner one, which is separated by a periplasmic space and thin layer of peptidoglycan. In the Gram-negative cell wall, the outer membrane may act as a barrier against the diffusion of many compounds, making the cell more resistant to toxic substances. Gram-positive bacteria have thick layer of peptidoglycan but they do not have an outer membrane. The lack of outer lipid bilayer membranes makes them more sensitive to external factors, especially chemical compounds that can penetrate more easily through peptidoglycan, destroying the cell membrane more quickly and penetrating the interior of the bacterial cell (Silhavy et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although the cytoplasmic membrane of both kinds of bacteria is primarily composed of phosphatidylethanolamines (PE) and negative phospholipids (phosphatidylglycerols, PG and cardiolipin, Cl) (L\u0026oacute;pez-Lara \u0026amp; Geiger, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) (Sohlenkamp \u0026amp; Geiger, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)(Gunstone et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), their mutual proportions are different (Epand et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In Gram-negative bacteria, PE predominates over anionic phospholipids while in Gram-positive inner membrane anionic phospholipids dominate over PE. Interestingly, some Gram-positive species are even completely devoid of PE (Trombe et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1979\u003c/span\u003e) and references therein. This different proportion of anionic-to-zwitterionic phospholipids, resulting in different charge of bacterial cells, contribute to their different interactions with various molecules, including antimicrobial agents (Breijyeh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence Microscopy Images\u003c/h2\u003e \u003cp\u003eThe LIVE/DEAD BacLight Bacterial Viability Kits utilize mixtures of SYTO\u0026reg; 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide. These stains differ both in their spectral characteristics and in their ability to penetrate healthy bacterial cells. When used alone, the SYTO 9 stain generally labels all bacteria in a population \u0026mdash; those with intact membranes and those with damaged membranes. In contrast, propidium iodide penetrates only bacteria with damaged membranes, causing a reduction in the SYTO 9 stain fluorescence when both dyes are present. Bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. The staining results of bacterial suspensions incubated in plant extract solutions are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe control sample consisted of bacterial cells grown in medium. The photos showing the control cells of the four bacterial strains tested show a green coloration of the bacteria, mainly indicating live cells. Bacterial cultures treated with different, appropriate bactericidal doses of extract concentrations after staining show a significant decrease in the number of bacteria and indicate dead or membrane-compromised cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdsorption of TOE and CME at the Air/Water Interface and their Penetration to the Model Membranes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on chromatographic results (Supplementary Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S6) it can be seen that some compounds present in the analyzed extracts are insoluble in the aqueous phase, but their amphiphilic structure enables their surface activity. This suggests that the components of TOE and CME can penetrate the membranes. To confirm this, in the first step we performed adsorption experiments. In a typical experiment, the chosen volume of herb extract was injected below the interface of NaCl solution of concentration of 0.1 mol/L, and then the change in surface pressure was monitored overtime. As can be seen based on the course of π \u0026ndash; time plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), both TOE and CME form stable insoluble films at the surface of water already a few minutes after injection. The difference between extracts becomes visible when the amount of extract necessary to induce similar increase of surface pressure is considered. Namely, in comparison to the TOE, the CME extract seems to be enriched in surface active compounds; therefore the smaller amount of extract is needed to induce comparable raise of surface pressure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the next stage of our experiments, we aimed to verify if compounds of TOE and CME are able to penetrate the cell membranes of Gram-positive and Gram-negative bacteria in physiological conditions (Marsh \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). For this purpose on the surface of NaCl solution films of lipid composition mimicking bacterial membranes were equilibrated at the surface pressure of 32 mN/m (biologically relevant conditions (Marsh \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) (Dynarowicz-Latka et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Then the chosen volume of extract was injected below the film, and the change in surface pressure was monitored overtime. The doses of each extract for those experiments were selected based on the adsorption experiments i.e. the smallest dose causing change in surface pressure in adsorption experiments, and this value multiplied by two, four and sixteen. The measured π \u0026ndash; time plots were presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe obtained π - time curves confirm incorporation of extract components to membrane models; however, they show different characteristics depending on the investigated membrane model. In case of Gram-negative membrane model, the incorporation of extract in higher doses occurs immediately after its injection into the subphase and the film remains stable overtime. For smaller doses, after injection, the surface pressure rises rapidly and then a gradual penetration of the extract components into the model membrane is still progressing. The film stabilizes after about 15 minutes after extract injection, which is demonstrated by achieving a constant surface pressure values. In the case of Gram-positive membrane model, the penetration kinetics is diverse, depending on the kind of extract investigated and its dose. However, in all cases it can be noticed that the value of surface pressure of the membrane after the extract injection stabilizes overtime. To compare the scale of the effect of the tested plant extracts on the models of Gram-negative and Gram-positive bacterial membranes, based on the data from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the dependence of the change in surface pressure (Δπ) on the applied extract dose is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The Δπ values were calculated based on experimental curves by subtracting of the initial surface pressure value (before the extract injection) from surface pressure value of membrane after 20 minutes of equilibration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor CME, the increase in the surface pressure of bacterial membrane models with the quantity of injected extract is more steep in comparison with the results for TOE. This suggests that both types of membranes are more sensitive to components of CME extract or - more likely - CME is enriched in a larger quantity of surface active compounds in comparison to TOE. Furthermore, the Gram-positive membrane model seems to be more susceptible to incorporation of plant extracts components, whereas analogical TOE or CME cause greater increase of surface pressure in comparison to Gram-negative. This phenomenon can be explained using additional experimental methods such as Langmuir monolayers technique and AFM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eLangmuir Monolayer Study\u003c/h2\u003e \u003cp\u003eTo get insight into the effect of TOE and CME on model lipid bacterial membranes, the π-A isotherms were measured on 0.1 mol/L NaCl subphase with different amount of studied extracts. π-A isotherms and compressibility modulus plots are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Figures\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e present the collective graphs showing the changes in the compressibility moduli at 32 mN/m and the shift of the mean molecular area in relation to the value measured on pure 0.1 mol/L subphase (without extracts).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe π-A isotherm for Gram-positive membrane registered during the compression of the film on 0.1 mol/L NaCl subphase has a characteristic course (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The surface pressure starts to rise at ca. 195 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e /molecule and at ca. 7 mN/m and surface area of ca. 138 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e /molecule a plateau occurs. Further increase in surface pressure is not monotonous, namely the slope of the curve changes twice: at pressures of approx. 16 mN/m and 30 mN/m. The monolayer collapses at a surface pressure of 64 mN/m. The plateau region and the slope changes are reflected as minima on the graphs C\u003csub\u003es\u003c/sub\u003e\u003csup\u003e\u0026minus;1\u003c/sup\u003e \u003cem\u003eversus\u003c/em\u003e π (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The addition of both extracts (TOE and CME) causes the shift of isotherms towards larger surfaces area per molecule and a slight increase in surface pressure at which a plateau occurs. In the presence of extracts, no inflection is observed at 16 mN/m. The most visible change in the course of the π-A isotherm is observed for the highest content of extracts in the subphase (namely, 400 \u0026micro;L for TOE and 50 \u0026micro;L for CME). The isotherms are more inclined, and the collapse of the films occurs at a significantly lower pressure (ca. 53 mN/m in both cases). On the other hand, the π-A isotherm for Gram-negative model membrane has a different course. The surface pressure starts to rise at ca. 125 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e/molecule and it increases monotonically up to ca. 45 mN/m, where a characteristic bend in the curve is observed. The collapse occurs at a surface pressure of 48 mN/m. Interestingly, in the C\u003csub\u003es\u003c/sub\u003e\u003csup\u003e\u0026minus;1\u003c/sup\u003e \u003cem\u003eversus\u003c/em\u003e π dependency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), an additional minimum at about 37 mN/m is observed, suggesting a change in the slope of the π-A curve (not visible on the isotherm). In the case of the Gram-negative membrane, a shift of the isotherms towards larger surface areas per molecule is also observed with an increase in the amount of extracts in the subphase, as well as a decrease in the value of collapse pressure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs can be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the addition of the studied herb extracts causes fluidization of the model membrane. In the case of TOE and Gram-positive model, strong fluidization occurs already for the smallest amounts of the extract, i.e. 25 \u0026micro;L and 50 \u0026micro;L. The increase of TOE amount from 50 \u0026micro;L to 100 \u0026micro;L does not affect film packing. For the highest TOE content (400 \u0026micro;L), a decrease in C\u003csub\u003es\u003c/sub\u003e\u003csup\u003e\u0026minus;1\u003c/sup\u003e value is observed pointing on film physical state change from liquid condensed (LC) to the expanded state (LE). In the case of the Gram-negative membrane, the fluidization effect appears for the volume of 50 \u0026micro;L and occurs linearly with the increase of TOE concentration. For the second of the studied extracts - CME - some differences in the fluidization of the Gram-positive \u003cem\u003eversus\u003c/em\u003e Gram-negative model membranes are observed. The decrease in molecular packing is stronger for the Gram-positive membrane. In contrast, in the case of Gram-negative membrane, the strongest fluidization occurs when the CME concentration is changed from 50 \u0026micro;L to 100 \u0026micro;L. Interestingly, further increase in the amount of the extract does not affect the film packing. The observed fluidization of the films in all the studied cases can be related to the increase in the mean molecular area (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In summary, the investigated extracts exert a comparable influence on the packing of bacterial films, leading to a reduction in membrane condensation in both models. However, for CME the effect is achieved at significantly lower doses, which indicates its stronger activity. On the other hand, comparing the activity towards Gram-positive \u003cem\u003eversus\u003c/em\u003e Gram-negative strains, the effect of molecular packing loosening is slightly stronger for Gram-positive bacteria.\u003c/p\u003e \u003cp\u003eTo better investigate the fluidization effect, as well as morphological changes caused by both extracts the selected systems were deposited on a solid surface (mica) and imaged using AFM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe Effect of Extract Addition on the Topography of Model Lipid Membranes\u003c/h2\u003e \u003cp\u003eTo gain visual insight into micro- and nanoscale topographical changes in model bacterial membranes upon the addition of TOE or CME, AFM imaging was performed on Langmuir monolayers transferred onto atomically flat mica substrates. Topographical AFM images of Gram-negative and Gram-positive model membranes treated with different amounts of TOE (50 \u0026micro;L and 400 \u0026micro;L) and CME (3.6 \u0026micro;L and 50 \u0026micro;L) (referred to as TOE 50, TOE 400, CME 3.6 and CME 50, respectively) are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e and \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFor the Gram-negative model membrane control sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA\u0026ndash;C), two distinct types of domains protruding above the general membrane plane are clearly observed. AFM height analysis shows that, independent of their lateral size, the domains protrude on average by approximately 0.9 nm. The bimodal nature of the domains is reflected in the area distribution shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. This distribution is relatively broad, extending up to ~\u0026thinsp;0.2 \u0026micro;m\u0026sup2;, which is primarily associated with the presence of large, nearly circular domains. A similar trend is observed in the distribution of domain perimeters. In contrast, smaller domains exhibit significantly less regular shapes, as indicated by the dimensionless shape descriptors presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. The aspect ratio (AR) within the interquartile range (Q1\u0026ndash;Q3) is approximately 1.5, although more elongated structures with AR values up to 4 are also present. This behavior is consistent with the circularity distribution, which is centered around 0.8 but extends to values as low as 0.4 or lower. In addition, the domains are not fully compact, as the presence of internal voids (\u0026ldquo;holes\u0026rdquo;) is clearly visible in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC.\u003c/p\u003e \u003cp\u003eUpon the addition of TOE and CME, the AFM topography is preserved and no qualitative changes in the overall morphology are observed. However, AFM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD-F and G-I for TOE 50 and 400, respectively) and the data presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e reveal a significant increase in domain size. This effect is observed for both large (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, first row) and small domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, second row), as evidenced by shifts in the area and perimeter distributions. Simultaneously, the number of domains per unit area decreases by factors of 13.5 and 7.6 for TOE 50 and TOE 400, respectively (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for exact values).\u003c/p\u003e \u003cp\u003eIn addition to size changes, domain shape is modified upon extract addition. Domain boundaries become more irregular and jagged. While the AR remains largely unchanged at the lower TOE concentration, it increases at the higher concentration. In contrast, circularity decreases significantly in both cases. This apparent discrepancy arises from the definition of AR, which is based on a best-fitting ellipse and is therefore less sensitive to boundary roughness, whereas circularity depends explicitly on the perimeter and is strongly affected by edge irregularities. Together, these observations indicate that the addition of TOE extract promotes domain fusion. Analysis of domain area and perimeter suggests that the fusion mechanism is at least twofold: small domains are able to merge with one another, as directly observed in AFM images (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, panels C, F, and I), while larger domains can undergo self-fusion and/or incorporate smaller domains. Fusion is accompanied by a decrease in solidity, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. Despite substantial changes in lateral dimensions and shape descriptors, the height difference between domains and the surrounding continuous phase remains relatively constant at approximately 1.1 nm, further supporting a fusion-driven mechanism. It should be noted that AFM is chemically blind; therefore, the chemical origin of the fusion process cannot be resolved solely from topographical imaging.\u003c/p\u003e \u003cp\u003eSimilar overall behavior is observed upon the addition of CME extract; however, the fusion process is less pronounced. This is reflected in the domain area and perimeter distributions shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e. As summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, for the lower CME amount (6.3 \u0026micro;l/1L), the number of domains per unit area is comparable to that observed for TOE 50, while the area of small domains is significantly reduced. Notably, the AR remains at a similar level for both investigated CME concentrations. Domain fusion also leads to changes in circularity (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e): for CME 6.3, the interquartile range (Q1-Q3) shifts toward lower values and is centered around ~\u0026thinsp;0.3, whereas for CME 50 the distribution, although broad, is centered near 0.7.\u003c/p\u003e \u003cp\u003eA similar trend is observed for solidity. As with TOE, the addition of CME extract does not significantly affect the height difference between domains and the continuous phase (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that the concentration of active compounds in both extracts plays a crucial role in domain fusion in Gram-negative model membranes. At higher concentrations, smaller domains are observed, which can be rationalized in terms of nucleation-controlled kinetics. Assuming that both extracts contain active components capable of modifying domain surface charge, increased concentration may enhance nucleation, thereby suppressing domain coalescence and stabilizing smaller clusters. Alternatively, higher concentrations may reduce lateral domain mobility. Although the precise mechanism responsible for the suppression of domain fusion cannot be unambiguously determined, the results clearly indicate that the system is kinetically limited, despite sufficient equilibration prior to transfer onto the mica substrate.\u003c/p\u003e \u003cp\u003eFor Gram-positive model membranes, AFM imaging revealed the presence of circular and elliptical domains depressed, on average, 1.5 nm below the general plane. Clearly, these are not discontinuities in layer, as this difference is significantly smaller than thickness of a lipid monolayer. Similar to Gram-negative membranes, these domains exhibit bimodal characteristics, with a relatively broad area distribution, as determined from 5 \u0026micro;m \u0026times; 5 \u0026micro;m AFM scans. Smaller domains are semi-elliptical, as visible in high-resolution AFM scans (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC) and indicated by the distributions of their AR and circularity (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). The continuous phase surrounding these topographical features is smooth, with a RMS roughness of 90 pm. Despite the concentrations used, the addition of TOE extract preserves the overall sample topography (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, A\u0026ndash;I).\u003c/p\u003e \u003cp\u003eHowever, as deducted from AFM images and changes in \u003cem\u003eN\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), upon addition of TOE extract at a concentration of 50 \u0026micro;l/1L, the number of domains decreases, whereas almost no changes are observed for samples treated with TOE extract at a concentration of 400 \u0026micro;l/1L. Surprisingly, at the lower concentration, the decrease in the number of domains per unit area is not associated with their fusion, as the area of smaller domains also decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). An adverse effect is observed for the TOE 400 sample, where both area and perimeter distributions are not only significantly broader but also shifted toward higher values. Simultaneously, no changes in dimensionless shape descriptors are observed for TOE 50. For TOE 400, the AR remains unchanged, but circularity decreases significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Compared to the control sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-C), the domains exhibit increased boundary complexity and attained a more lobed shape. Considering the lack of changes in \u003cem\u003eN\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e alongside a simultaneous increase in domain area, it can be postulated that the active ingredients of TOE interact with components of the continuous phase. This interaction results in the formation of clusters that are deposited within the membrane, for example near the edges of domains formed in this way, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eH and I.\u003c/p\u003e \u003cp\u003eFor samples treated with CME extract, the influence is highly concentration-dependent. Comparison of AFM images obtained for the control sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA\u0026ndash;C) and CME 6.3 reveals no detectable effect. However, quantitative data presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figs.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e clearly show that upon addition of CME extract, both the number of domains and their size increase, without significant changes in their dimensionless shape descriptors. The most probable explanation for this observation is that the active ingredients of CME extracts interact with membrane components, altering the delicate interplay between domain composition and line tension within the membrane, which leads to the formation of a higher number of larger domains. This effect may be responsible for the topography observed for Gram-positive membranes treated with CME 50 extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eM-O), where the nanoscale domain character is no longer preserved and characteristic interconnected patches are visible. Due to the continuous nature of these patches, determination of their shape descriptors was not feasible. The height difference between these patches and the continuous phase is approximately 3 nm, which corresponds to the thickness of a single lipid monolayer.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTopographical characteristics of lipid bacterial membranes treated with different amounts of TOE and CME derived from AFM imaging: number of domains per unit area (\u003cem\u003eN\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e), average difference between height of domains and surrounding, continuous phase (Δ\u003cem\u003ez\u003c/em\u003e) and roughness, expressed as Root Mean Square (RMS). All data is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (if applicable).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003emembrane model\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e [um\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔ\u003cem\u003ez\u003c/em\u003e [nm]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRMS [pm], continuous layer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRMS [pm], features*\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(-)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e166\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e264\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(-) with 50 \u0026micro;L TOE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(-) with 400 \u0026micro;L TOE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(-) with 6.3 \u0026micro;L CME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(-) with 50 \u0026micro;L CME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e110\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e216\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(+)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(+) with 50 \u0026micro;L TOE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e251\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(+) with 400 \u0026micro;L TOE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(+) with 6.3 \u0026micro;L CME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-1.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram(+) with 50 \u0026micro;L CME\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en. a.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e241\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e*Protruding domains (Gram-negative: or depressed domains (Gram-positive:\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eChromatographic Profiles of the\u003c/b\u003e \u003cb\u003eChelidonium majus\u003c/b\u003e \u003cb\u003eand Extractives Detected by a High-resolution Mass Spectrometric System (UHPLC-QTOF)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe identified compounds in extracts of \u003cem\u003eChelidonium majus\u003c/em\u003e and \u003cem\u003eTaraxacum officinale\u003c/em\u003e by the UHPLC-QTOF system with high-resolution mass spectrometric detection are reported in Supplementary Tables S1-S6, based on previous reports and representative chromatograms, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe most prominent were isoquinoline alkaloids identified in \u003cem\u003eChelidonium majus\u003c/em\u003e extract in positive electrospray ionization mode. Mass spectrometric data were used to determine elemental composition of the alkaloids (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), which can be characterized by their the protonated adducts ([M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e) (\u003cb\u003eA1\u003c/b\u003e, \u003cb\u003eA2\u003c/b\u003e, \u003cb\u003eA3\u003c/b\u003e, and \u003cb\u003eA10\u003c/b\u003e) as well as molecular ions [M]\u003csup\u003e+\u003c/sup\u003e (\u003cb\u003eA5\u003c/b\u003e, \u003cb\u003eA6\u003c/b\u003e, \u003cb\u003eA9\u003c/b\u003e, \u003cb\u003eA11\u003c/b\u003e, and \u003cb\u003eA12\u003c/b\u003e) which are already charged in solution. Unknown compounds \u003cb\u003eA4\u003c/b\u003e, \u003cb\u003eA7\u003c/b\u003e and \u003cb\u003eA8\u003c/b\u003e where tentatively assigned as Na\u003csup\u003e+\u003c/sup\u003e adducts. The highest MS signal in \u003cem\u003eChelidonium majus\u003c/em\u003e extract was observed for coptisine \u003cb\u003eA5\u003c/b\u003e and the other more abundant peaks were detected for \u003cb\u003eA1\u003c/b\u003e, \u003cb\u003eA2\u003c/b\u003e, \u003cb\u003eA4\u003c/b\u003e, and \u003cb\u003eA6\u003c/b\u003e (Supplementary Table S4).\u003c/p\u003e \u003cp\u003eSeven isoquinoline alkaloids (Qiao et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zielińska et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), two protopine derivatives (protopine \u003cb\u003eA1\u003c/b\u003e, allocryptopine \u003cb\u003eA3\u003c/b\u003e), two protoberberine derivatives (coptisine \u003cb\u003eA5\u003c/b\u003e and berberine \u003cb\u003eA11\u003c/b\u003e), and three phenanthridine derivatives (chelidonine \u003cb\u003eA2\u003c/b\u003e, sanguinarine \u003cb\u003eA9\u003c/b\u003e, chelerythrine \u003cb\u003eA12\u003c/b\u003e) were readily detected (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe other rare compounds were tentatively identified as demethyleneberberine \u003cb\u003eA6\u003c/b\u003e (Bahadur and Shukla \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1983\u003c/span\u003e), and Pessoine \u003cb\u003eA10\u003c/b\u003e (Farrow et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eTaraxacum officinale\u003c/em\u003e root extract, the alkaloid MS signals were much lower but still with the highest peak detected for coptisine \u003cb\u003eA5\u003c/b\u003e. In the case of allocryptopine \u003cb\u003eA3\u003c/b\u003e, unknown \u003cb\u003eA7\u003c/b\u003e, sanguinarine \u003cb\u003eA9\u003c/b\u003e, and chelerythrine \u003cb\u003eA12\u003c/b\u003e, the signals were completely not observed (Tables S1 and S4, Supplementary material).\u003c/p\u003e \u003cp\u003eSeveral flavonoids were found in extract of \u003cem\u003eChelidonium majus\u003c/em\u003e at low levels. Two monoglycosides of quercetin and kaempferol (\u003cb\u003eB10\u003c/b\u003e and \u003cb\u003eB13\u003c/b\u003e) and three diglycosides (rutinosides; \u003cb\u003eB8\u003c/b\u003e, \u003cb\u003eB11\u003c/b\u003e and \u003cb\u003eB12\u003c/b\u003e) were assigned according to (Grosso et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and one triglycoside of kaempferol \u003cb\u003eB4\u003c/b\u003e (Krizhanovska et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) were identified by HRMS measurements and comparison with previous reports. The highest signals were observed for quercetin 3-\u003cem\u003eO\u003c/em\u003e-rutinoside \u003cb\u003eB8\u003c/b\u003e and isorhamnetin 3-\u003cem\u003eO\u003c/em\u003e-rutinoside \u003cb\u003eB12\u003c/b\u003e (Supplementary Table S5). Another group of three isomeric kaempferol triglycosides was tentatively identified as robinin and its possible isomers (\u003cb\u003eB5\u003c/b\u003e, \u003cb\u003eB6\u003c/b\u003e and \u003cb\u003eB7\u003c/b\u003e) according to (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A presence of two structures of rare iridoids (\u003cb\u003eB2\u003c/b\u003e and \u003cb\u003eB3\u003c/b\u003e) was indicated, presumably isomeric to barlerine (8-\u003cem\u003eO\u003c/em\u003e-acetyl shanzhiside methyl ester), based on characteristic molecular formula (C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e) inferred from \u003cem\u003em/z\u003c/em\u003e value of the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e ion close to 447.1503 determined by HRMS (Alipieva et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The presence of quinic \u003cb\u003eB1\u003c/b\u003e and \u003cem\u003ep\u003c/em\u003e-coumaric \u003cb\u003eB9\u003c/b\u003e acids was also acknowledged.\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003eC1\u003c/b\u003e ([M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e at \u003cem\u003em/z\u003c/em\u003e 191), \u003cb\u003eC2\u003c/b\u003e and \u003cb\u003eC3\u003c/b\u003e ([M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e at \u003cem\u003em/z\u003c/em\u003e 353) detected in \u003cem\u003eTaraxacum officinale\u003c/em\u003e root extract corresponded to quinic acid, 3-\u003cem\u003eO\u003c/em\u003e-caffeoylquinic acid and 5-\u003cem\u003eO\u003c/em\u003e-caffeoylquinic acid, respectively, based on the elution order described in previous studies (Garcia-Perez et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Similarly, compounds \u003cb\u003eC9\u003c/b\u003e, \u003cb\u003eC10\u003c/b\u003e and \u003cb\u003eC12\u003c/b\u003e ([M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e at \u003cem\u003em/z\u003c/em\u003e 515) were attributed to 3,4-\u003cem\u003eO\u003c/em\u003e-dicaffeoylquinic acid, 3,5-\u003cem\u003eO\u003c/em\u003e-dicaffeoylquinic acid and 4,5-\u003cem\u003eO\u003c/em\u003e-dicaffeoylquinic acid, respectively. The following peaks of quinic derivatives, \u003cb\u003eC2\u003c/b\u003e, \u003cb\u003eC3\u003c/b\u003e and \u003cb\u003eC12\u003c/b\u003e, were highly abundant in the chromatograms after the MS and PDA detection.\u003c/p\u003e \u003cp\u003eFurther analysis of the MS results at negative ionization revealed several chromatographic peaks (\u003cb\u003eC4\u003c/b\u003e-\u003cb\u003eC8, C11\u003c/b\u003e and \u003cb\u003eC14\u003c/b\u003e) detected at nominal \u003cem\u003em/z\u003c/em\u003e 447 Da (PDA absorption: λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;222 and 275 nm) with \u003cb\u003eC4\u003c/b\u003e and \u003cb\u003eC5\u003c/b\u003e showing the highest signals (Supplementary Tables S3 and S6). The HRMS analysis gave an elemental composition of C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e observed at \u003cem\u003em/z\u003c/em\u003e 447.1265 (calculated \u003cem\u003em/z\u003c/em\u003e 447.1286) for all the peaks. This indicated a presence of di-4-hydroxyphenylacetic acid inositols identified previously in \u003cem\u003eTaraxacum officinale\u003c/em\u003e (Kenny et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e). Likewise, peaks \u003cb\u003eC16-C18\u003c/b\u003e were detected at nominal \u003cem\u003em/z\u003c/em\u003e 581 Da (λ\u003csub\u003emax\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;222 and 275 nm) giving rise to tentative identification of isomeric tri-4-hydroxyphenylacetic acid inositols (Kenny et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e) of ionic molecular formula C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e observed at \u003cem\u003em/z\u003c/em\u003e 581.1672 (calculated \u003cem\u003em/z\u003c/em\u003e 581.1654).\u003c/p\u003e \u003cp\u003eA group of known sesquiterpenoids was attributed to compounds \u003cb\u003eC13\u003c/b\u003e, \u003cb\u003eC15\u003c/b\u003e, \u003cb\u003eC20\u003c/b\u003e, and \u003cb\u003eC21 (\u003c/b\u003eMichalska et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Peak \u003cb\u003eC13\u003c/b\u003e was assigned to highly abundant 14-\u003cem\u003eO\u003c/em\u003e-\u003cem\u003eβ\u003c/em\u003e-D-glucosyl-taraxinic acid (Kashiwada et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), based on accurate mass measurement which gave an elemental composition of detected negative ion observed at \u003cem\u003em/z\u003c/em\u003e 423.1642 (calculated \u003cem\u003em/z\u003c/em\u003e 423.1650) as C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e. Similar derivative was attributed to \u003cb\u003eC15\u003c/b\u003e as 14-\u003cem\u003eO\u003c/em\u003e-\u003cem\u003eβ\u003c/em\u003e-D-glucosyl-11,13-dihydro-taraxinic acid with ionic molecular formula C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e detected at \u003cem\u003em/z\u003c/em\u003e 425.1789 (calculated \u003cem\u003em/z\u003c/em\u003e 425.1806). Similarly, the presence of free taraxinic acid \u003cb\u003eC20\u003c/b\u003e (ionic molecular formula C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) and 11,13-dihydro-taraxinic acid \u003cb\u003eC21\u003c/b\u003e (ionic molecular formula C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) was acknowledged with \u003cb\u003eC21\u003c/b\u003e demonstrating much higher signal (Tables S3 and S6, Supplementary material).\u003c/p\u003e \u003cp\u003ePeak \u003cb\u003eC19\u003c/b\u003e was assigned to ferulic acid (obtained ionic molecular formula C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) (Tanasa (Acretei) et al. 2025).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the Total Phenolic Content (TPC) by the Folin-Ciocalteu Spectrophotometric Method\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo support data from UHPLC-QTOF technique, the Total Phenolic Content (Gallic Acid Equivalents (GAE)) was determined for the investigated extracts. The Total Phenolic Content was equal to 0.359 +/- 0.016 (mg GAE/g DW) for \u003cem\u003eChelidonium majus\u003c/em\u003e extract, whereas for \u003cem\u003eTaraxacum officinale\u003c/em\u003e it was equal to 0.0852 +/- 0.0040 (mg GAE/g DW). This four times greater phenolic content corroborates with increased antimicrobic activity of celadine \u003cem\u003eversus\u003c/em\u003e dandelion.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eBoth tested extracts, celandine (CME) and dandelion (TOE), showed antibacterial activity in microbiological tests, with the celandine extract proving more effective. Among the bacterial strains tested, \u003cem\u003eStreptococcus pyogenes ATCC\u0026reg; 12384\u003c/em\u003e was found to be the most sensitive, while \u003cem\u003eEscherichia coli ATCC\u0026reg; 25922\u003c/em\u003e showed the least sensitivity to the extracts used. Complementary experiments performed on bacterial artificial membranes, modeled with the Langmuir monolayer technique, confirmed the stronger fluidization effect exerted by celandine compared to dandelion extract. This can be explained comparing the content of active antimicrobial substances in both extracts. The higher content of phenolic compounds in celadine compared to dandelion extracts confirms the results obtained for monolayers.\u003c/p\u003e \u003cp\u003eAFM imaging combined with quantitative image analysis revealed that the interactions of active compounds from dandelion and celandine extracts differ between Gram-negative and Gram-positive membranes. Although concentration-dependent effects are observed for both extracts, the overall topography of Gram-negative membranes remains conserved, with changes occurring primarily at the domain level, most likely due to domain fusion. In contrast, for Gram-positive membranes, the actions of TOE and CME extracts are markedly different. TOE affects membrane topography under both investigated conditions, leading to the formation of deposits within the monolayer. The action of CME is more complex: at lower concentrations, changes occur mainly in the number and area of domains, whereas at higher concentrations, interactions between active compounds and lipid membrane components result in drastic alterations of membrane topography, such that the overall membrane structure is no longer preserved.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupplementary material\u003c/h2\u003e \u003cp\u003eAdditional tables presenting chromatographic, spectrophotometric and high-resolution mass spectrometric data.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.G-S. and P.D-L. wrote the main manuscript text. A.G-S. , A.W. and A.Ch-B. , D.L. , S.W., Ł.K. , K. D., E.Ł. wrote the experimental parts, did experiments, analysed the results and prepared figures. P.D-L and M.B-W. supervised the mauscript. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe AFM measurements were carried out using research infrastructure funded by the European Union in the framework of the Smart Growth Operational Programme, Measure 4.2; Grant No. POIR.04.02.00-00-D001/20, \u0026ldquo;ATOMIN 2.0 \u0026ndash; Center for materials research on ATOMic scale for the INnovative economy\u0026rdquo;.\u003c/p\u003e \u003cp\u003eThe BX63 fluorescence microscope (Olympus) used in the research was funded from the budget of the qLIFE Priority Research Area under the Strategic Program Excellence Initiative at Jagiellonian University\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAjayi A (2008) Antimicrobial nature and use of some medicinal plants in Nigeria. 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J Pharm Pharm Sci 11:90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18433/J3D30Q\u003c/span\u003e\u003cspan address=\"10.18433/J3D30Q\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"pathogenic bacteria, phenolic compounds, celandine extract, dandelion extract, bacterial lipid model membranes","lastPublishedDoi":"10.21203/rs.3.rs-9356537/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9356537/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe article presents research on the destruction of the bacterial membrane of celandine (CME) and dandelion (TOE) extracts containing phenolic compounds responsible for their antibacterial activity. The \u003cem\u003ein vitro\u003c/em\u003e microbiological tests were done on living cells of different pathogenic bacteria species: Gram-positive (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e) and Gram-negative (\u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e). The obtained results clearly demonstrate the antibacterial effect of both extracts. However, in the case of celandine herb extract, the effect was stronger, especially for Gram-positive bacteria. For a deeper understanding of the mechanism of antibacterial action, both studied extracts were subjected to biophysical studies on artificial bacterial lipid membranes, modeled with the Langmuir monolayer technique. The monolayer investigations were performed using two different methodologies. First was based on preparing Langmuir monolayers of model bacterial lipid membranes on subphases containing the tested extracts and analysis of the biophysical parameters of the recorded pressure-area isotherms compared to those without the presence of the herb's extracts. In the other approach, the extracts were introduced into the aqueous subphase and their penetration to model bacterial lipid membranes was monitored. The results of the monolayer experiments, including AFM analysis of the domain structures of the LB transferred films, are in good agreement with the results of biological tests and analytical analyses of the tested extracts, which confirm that the stronger antibacterial effect of celandine extract, associated with a greater amounts of phenolic compounds, has a more destructive effect on the lipid membranes of bacteria compared to dandelion.\u003c/p\u003e","manuscriptTitle":"Disruption of Bacterial Lipid Membranes by Phenolic Compounds from Plant Extracts:Biophysical Evidence from Microbiological and Langmuir Monolayer Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 08:37:33","doi":"10.21203/rs.3.rs-9356537/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"06e35e82-79f2-4cf5-b91b-7c322af86fb3","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-17T08:58:27+00:00","index":11,"fulltext":""},{"type":"reviewerAgreed","content":"314984365768531246882504109643959939406","date":"2026-05-17T08:56:07+00:00","index":10,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T09:32:40+00:00","index":8,"fulltext":""},{"type":"reviewerAgreed","content":"109371964098506113509082300004014222214","date":"2026-05-03T07:26:41+00:00","index":7,"fulltext":""},{"type":"reviewersInvited","content":"2","date":"2026-04-30T06:56:36+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T08:37:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 08:37:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9356537","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9356537","identity":"rs-9356537","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}