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Article

Phytochemical Analysis and In Vitro Antibiofilm Activity of Ethanolic Leaf Extract from Quercus alnifolia Poech Against Staphylococcus aureus

by
Ender Volkan
1,*,†,
Chad Schou
1,†,
Jandirk Sendker
2 and
Panagiotis Karanis
1,3,*
1
Department of Basic and Clinical Sciences, University of Nicosia Medical School, Nicosia 2414, Cyprus
2
Institute of Pharmaceutical Biology and Phytochemistry (IPBP), PharmaCampus, University of Münster, Corrensstrasse 48, 48149 Münster, Germany
3
Faculty of Medicine and University Hospital, University of Cologne, 50923 Cologne, Germany
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sci. Pharm. 2025, 93(3), 39; https://doi.org/10.3390/scipharm93030039
Submission received: 24 June 2025 / Revised: 25 July 2025 / Accepted: 20 August 2025 / Published: 25 August 2025
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Abstract

Antibiotic resistance is on the rise, rendering discovery of new antibacterial sources essential. Biofilms drive resistance and cause complications in healthcare settings, emphasizing that preventing pathogenic biofilms is vital. Quercus species, with medicinal potential, might provide novel approaches against pathogens. Cyprus hosts four understudied Quercus species—Q. alnifolia Poech, Q. × campitica Hadjik. & Hand, Q. coccifera var. calliprinos (Webb) Boiss., and Q. infectoria subsp. veneris (A.Kern.) Meikle—where Q. alnifolia and Q. × campitica are endemic. This study assessed the antibacterial, antibiofilm, and preformed biofilm reduction effects of their ethanolic leaf extracts on Staphylococcus aureus (ATCC 6538) and performed phytochemical analysis. Because biofilm formation often drives recalcitrance, sub-minimum inhibitory concentrations (sub-MIC) of Quercus extracts were tested on planktonic and biofilm S. aureus. At a sub-MIC of 0.156 mg/mL, Q. alnifolia and Q. × campitica extracts displayed notable antibiofilm activity. Liquid chromatography–mass spectrometry of Q. alnifolia revealed several bioactive compounds where these compounds may support wider antibacterial effects. This is the first report of Q. alnifolia and Q. × campitica ethanolic leaf extracts with antibiofilm activity against S. aureus and associated phytochemical analyses. These results support further practical research into the potential applications of these Quercus extracts as antibacterial materials.

1. Introduction

Antimicrobials have revolutionized healthcare, yet their overuse and microbial adaptations have led to many pathogenic species becoming resistant, resulting in treatment failures. Resistance now spreads rapidly in healthcare and community settings, creating a serious global threat for patients and healthcare systems [1]. Without newly developed antibiotics, resistant infections could exceed 10 million cases by 2050, potentially outpacing cancer deaths [2]. Overreliance on antibiotics, in clinical and environmental contexts, has facilitated the emergence of resistant strains that move beyond human settings through wastewater and agricultural runoff [3,4,5,6]. Together, these pollutants and resistant microbes highlight the need for novel treatments and a reduction in chemical contamination.
Biofilms continue to pose significant challenges to antimicrobial control, yet comprehensive strategies to prevent and eradicate them remain limited. Plant-derived products are a promising option, as they often disrupt biofilm development through interference with quorum sensing and can break down established biofilms of multidrug-resistant bacteria by targeting their extracellular matrix [7]. Among these, flavonoids and phenolic compounds are especially effective and are often studied in vitro [8,9].
Staphylococcus aureus is a widespread pathogen affecting both human and veterinary practice of medicine. Its virulence factors include toxins, superantigens, and adhesins [10]. Through biofilm formation, S. aureus can colonize living and inanimate surfaces, complicating infections due to its protected bacterial community within the extracellular polymeric substance (EPS) [11]. Beyond skin and soft tissue infections, it may cause respiratory, bone, joint, endocardial, urinary tract, and device-related infections [12,13]. Methicillin-resistant S. aureus (MRSA) is especially problematic, showing resistance to ampicillin, cefoxitin, ciprofloxacin, erythromycin, gentamicin, chloramphenicol, and, in some hospital settings, vancomycin [14]. While various natural approaches are being tested against MRSA [15,16], management strategies are difficult as S. aureus biofilms increase the risk of contamination [17] in water distribution systems as biofilms can effectively withstand shear stress in the environment and bodily fluids [18].
Developing new antimicrobials is essential for preventing, treating, or supporting treatments of infections, improving patient outcomes, preventing contamination, and mitigating antimicrobial resistance. We have begun exploring extracts of endemic and indigenous plants from Cyprus, the third-largest Mediterranean island with an exceptional biodiversity, to reduce reliance on traditional antibiotics and minimize environmental pollutants in water. Identifying safe, low-toxicity plant extracts may offer additional strategies for managing pathogens without introducing further chemical contamination. Several Cypriot extracts have demonstrated in vitro antimicrobial activities, such as the ethanolic leaf extracts of Odontites linkii subsp. cyprius (Boiss.) Bolliger and Ptilostemon chamaepeuce subsp. cyprius (Greuter) Chrtek & B. Slavík against Blastocystis hominis subtypes 1 and 3 [19] and Leishmania infantum (MCAN/CY/2005/CD57) [20]. Schou et al. [21] also noted the efficacy of O. linkii subsp. cyprius against Acanthamoeba castellanii (American Type Culture Collection (ATCC) 30010). Furthermore, studies by Jafaar et al. [22] and Ozbil et al. [23] highlighted antibacterial and antibiofilm activities of various plant extracts both against laboratory strains and clinical strains of antibiotic-resistant bacteria. Quercus species, known for their anti-inflammatory, anticancer, and antibacterial potential [24], were further investigated using ethanolic leaf extracts from four native Quercus species in this study. Q. alnifolia and Q. × campitica showed notable in vitro antimicrobial, antibiofilm, and preformed biofilm inhibition activity against Staphylococcus aureus. These findings suggest that Cypriot Quercus species, especially Q. alnifolia, could serve as promising candidates for new antibacterial agents.

2. Materials and Methods

2.1. Reagents

Gentamicin sulfate, Mueller–Hinton broth, Mueller–Hinton agar, gallic acid, quercetin, 1× PBS, and Brain Heart Infusion broth were purchased from Millipore Merck, (Darmstadt, Germany) at the highest quality. Staphylococcus aureus (ATCC 6538) was purchased from American Type Culture Collection (Manassas, VA, USA). Ethanol for extract preparation was of HPLC grade, ensuring its purity. The mobile phase solvents consisted of water, acetonitrile, and 0.1% formic acid, each of HPLC purity grade.

2.2. Plant Collection and Identification

The Quercus species leaves were collected from the Paphos Forest near Kampos, Cyprus (Figure 1) between June–October 2023. The species’ identities were confirmed by Dr. Eleftherios Hadjisterkotis of the Cyprus Agricultural Research Institute, affiliated with the Ministry of Agriculture. Voucher specimens were kept at the University of Nicosia Medical School under suitable conditions (vouchers MS11, MS12, MS13, MS19). To prepare the leaves for extraction, they were first rinsed with clean water to remove any soil and debris, then thoroughly dried. The Quercus spp. branches with the leaves used during the extraction were hung upside down in a dark room with a relative humidity of 65% for three weeks at room temperature. During this period, no fungal growth was detected. After drying, only the leaves were finely ground using a clean electric coffee grinder. The branches or bark were not used in this study. The resulting leaf powder was stored in double-sealed plastic bags from which air was removed and kept refrigerated at 4 °C for later use. Figure 2 shows pictures of the Cyprus Quercus species used in this study.

2.3. Extraction of Plant Compounds

To carry out the extraction, 1 g of finely powdered plant leaves was mixed with 20 mL of ethanol in a clean 50 mL plastic centrifuge tube. The mixture underwent ultrasonication in a water bath (Grant XUB10, 200 W, 32–38 kHz) at 40 °C for 20 min. This extraction procedure was repeated twice. Following this, the supernatants from both extractions were combined and filtered under vacuum using Whatman #1 paper. This entire process was repeated until 4–5 g of plant material had been processed. The resulting ethanolic extracts were then concentrated to dryness with a rotary evaporator in a tared glass vial under reduced pressure at a temperature of 40 °C according to a screening method previously described by Schou et al. [20]. Once dried, the extracts were kept at 4 °C in pre-weighed dark glass vials until further analysis. The yield percentage of the extract was determined by dividing the weight of the dry extract by the weight of the dry plant material and multiplying the result by 100.
% Yield = recovered dry extract (g)/dry leaf material (g) × 100.
This research focused on polar plant compounds present in the leaves to circumvent challenges associated with solubility in bioassays and the requirement for extra solvents, like dimethyl sulfoxide (DMSO), to dissolve non-polar compounds. As a result, only ethanolic extracts from the leaves were used in the assays.

2.4. Antibacterial and Biofilm-Inhibitory Activity Testing

2.4.1. Antibacterial-Susceptibility Test (AST)—Broth Microdilution Assay

The antibacterial properties of the Quercus ethanolic leaf extracts from Cyprus were assessed using the broth microdilution method against Staphylococcus aureus (American Type Culture Collection 6538). The ethanolic leaf extracts were carefully evaluated for their minimum inhibitory concentration (MIC) following the protocol by Eloff [25] followed by plating for minimum bactericidal concentration (MBC) determination. Each plant extract underwent 2-fold serial dilution with sterile saline in a 96-well plate, followed by the addition of 100 µL of the reference bacteria to create a final concentration range of 2.5 to 0.02 mg/mL. The bacteria suspension was freshly diluted from a 0.5 McFarland standard to obtain a final inoculum of 5 × 105 CFU/mL in sterile Mueller–Hinton broth. The 96-well plates were then incubated at 37 °C for 18 h. Using a sterile culture loop, approximately 10 µL of planktonic culture from the same microtiter plate with the MICs was transferred and streaked onto the surface of Mueller–Hinton (MH) agar plate for each Quercus species used in the experiment. The final assay concentrations of the extracts that were assessed for the MBC of each extract were 0.3125 mg/mL to 2.5 mg/mL. The MH plates were incubated for 18 h at 37 °C. The MBC of the ethanolic leaf extracts were determined by visually identifying the concentration at which bacterial growth did not occur.

2.4.2. Antibiofilm Assay

The biofilm inhibition assay was conducted for each Quercus ethanolic leaf extract using a 96-well microdilution method based on crystal violet (CV) staining. The bacterial strain S. aureus (ATCC 6538) was grown in 5 mL of Brain Heart Infusion (BHI) media at 37 °C for 24 h under shaking conditions. For the biofilm assay, the bacterial inoculum was adjusted to a density of approximately 1 × 108 CFU/mL.
A stock solution of each Quercus ethanolic leaf extract (10 mg/mL) was prepared in sterile 1× phosphate-buffered saline (PBS). Each stock solution was serially diluted 2-fold with sterile 1× PBS to prepare a range of concentrations. A 100 µL volume of these serially diluted extracts was added to the wells of a 96-well plate. Following this, 100 µL of the bacterial inoculum was added to each well, resulting in final extract concentrations ranging from 0.078 mg/mL to 1.25 mg/mL, which included sub-minimum inhibitory concentrations (sub-MICs) previously determined for the extracts. Sterile 1× PBS, gentamicin, gallic acid, and quercetin served as the controls.
The microplates were incubated at 37 °C for 18 h under shaking conditions at 30 rpm. After incubation, the wells were washed with sterile dH2O to remove planktonic cells and loaded with 0.1% CV solution for 10 min followed by the application of 33% acetic acid solution [13]. Absorbance readings were recorded after at least 6 h of incubation via Victor X3 spectrophotometer plate reader (PerkinElmer, Waltham, MA, USA) at OD595. The percentage of biofilm inhibition was calculated with Microsoft Excel using the following formula:
% Biofilm Inhibition = [(OD Control − OD Sample) / OD Control] × 100

2.4.3. Preformed Biofilm Reduction Assay

S. aureus biofilms were established using the reagents and techniques detailed in Section 2.4.2, however the biofilms were incubated and grown to maturity for 24 h without any extract/antibiotic addition. Once the biofilm incubation was over, the liquid media was removed and replaced with 2-fold serially diluted extracts (0.078 mg/mL to 1.25 mg/mL) in sterile 1× PBS and fresh media then incubated over night at 37 °C under shaking conditions at 30 rpm. After incubation, the wells were washed with sterile dH2O to remove planktonic cells and loaded with 0.1% CV solution for 10 min followed by the application of 33% acetic acid. Gentamicin (0.02 mg/mL) was used as the control. Absorbance readings were measured after at least 6 h using a Victor X3 spectrophotometer plate reader (PerkinElmer, Waltham, MA, USA) OD595. The percentage of biofilm inhibition was calculated with Microsoft Excel using the formula above.

2.5. Liquid Chromatography–Mass Spectroscopy of Ethanolic Leaf Extracts of Q. alnifolia, Q. × campitica, Q. coccifera, and Q. infectoria

Chromatographic separations were conducted using a Dionex Ultimate 3000 RS Liquid Chromatography system with a Thermo Acclaim RSLC 120, C18 column (2.1 × 100 mm, 2.2 µm) employing a binary gradient. The mobile phase consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid), with a flow rate set at 0.4 mL/min. The gradient program was as follows: isocratic at 5% B from 0 to 0.4 min, a linear increase from 5% B to 100% B from 0.4 to 9.9 min, isocratic at 100% B from 9.9 to 15.0 min, a linear decrease from 100% B to 5% B from 15.0 to 15.1 min, and isocratic at 5% B from 15.1 to 20.0 min. The volume of sample injected was 2 µL. Detection of eluted compounds was performed using a Dionex Ultimate DAD−3000 RS, covering a wavelength range from 200 to 400 nm, and a Bruker Daltonics micrOTOF-QII time-of-flight mass spectrometer with an Apollo electrospray ionization source operating in a positive mode at 3 Hz across a mass range of m/z 50 to 1500, using the following settings: nitrogen nebulizer gas at 2 bar, dry gas nitrogen at 9 L/min and 200 °C, a capillary voltage of 4500 V, end plate offset at −500 V, transfer time of 100 µs, prepulse storage of 6 µs, nitrogen as collision gas, collision energy of 25 eV (AutoMS/MS only), and collision RF set to 130 Vpp. For each analysis, internal dataset calibration was carried out using the mass spectrum of a 10 mM sodium formate solution in 50% isopropanol, which was infused during LC re-equilibration via a divert valve equipped with a 20 µL sample loop. Data analysis was undertaken using Bruker DataAnalysis 4.1 SP1 software.

2.6. Statistics

Antibiofilm and preformed biofilm inhibition data were reported as the mean ± standard error of the mean (SEM) of triplicates subjected to one-way analysis of variance (ANOVA). The experiments were repeated twice. The data were calculated with Microsoft Excel and analyzed in GraphPad Prism for Windows, Version 10 (GraphPad Software, San Diego, CA, USA). The significant differences between the mean results of the various treatments and the control were determined by Dunnett’s multiple comparison test. A p-value < 0.05 was considered significant.

3. Results

3.1. Q. alnifolia, Q. × campitica, Q. coccifera, and Q. infectoria Inhibit S. aureus Biofilm Formation

Our prior research [20] demonstrated that Cypriot Quercus species exhibit antibacterial activity against S. aureus, with Q. alnifolia and Q. × campitica effective at 0.3125 mg/mL, and Q. coccifera and Q. infectoria at 1.25 mg/mL. In the present study, the minimum bactericidal concentration (MBC) experiments further revealed bactericidal activity associated with Q. alnifolia at 1.25 mg/mL followed by Q. × campitica at 2.5 mg/mL where no minimum bactericidal concentration was observed for Q. × coccifera and Q. infectoria up to 2.5 mg/mL (Table S1). Since biofilm formation is a crucial mechanism utilized by S. aureus to mediate infection and display antibiotic recalcitrance, we investigated if ethanolic leaf extracts obtained from Quercus leaves could inhibit the formation of S. aureus biofilms. Concentrations at or below MBC were utilized to specifically assess antibiofilm activity of Quercus species using a CV-based microdilution assay [13]. The antibiofilm activities from the Quercus ethanolic leaf extracts were compared to activities of 0.02 mg/mL gentamicin, 0.05 mg/mL gallic acid and 0.05 mg/mL quercetin. The ethanolic leaf extracts obtained from Q. alnifolia, Q. × campitica, Q. coccifera and Q. infectoria displayed notable antibiofilm activity at concentrations as low as 0.078 mg/mL and 0.156 mg/mL (Figure 3). Specifically, the two endemic species Q. alnifolia and Q. × campitica displayed the highest inhibition of biofilm formation. At concentrations ranging from 0.078 mg/mL to 1.25 mg/mL, Q. alnifolia ethanolic leaf extract inhibited biofilm formation by 17–89%, and Q. × campitica inhibited formation by 54–100% (Table 1). Under the same concentrations, the ethanolic leaf extracts of the indigenous oak species Q. coccifera and Q. infectoria were only able to inhibit biofilm formation by 8–37% and 17–46%, respectively (Table 1). Gallic acid (32%), quercetin (55%), and gentamicin (49%) provided moderate biofilm inhibition at the tested concentrations. These results reveal Cyprus Quercus species as promising candidates for further antibiofilm research, especially Q. alnifolia and Q. × campitica.

3.2. Q. alnifolia Extract Displays Activity Against Preformed S. aureus Biofilm

Since the Q. alnifolia ethanolic leaf extract displayed both antibacterial potency and efficiency in inhibiting biofilm formation, this endemic oak species was selected to test its impact on preformed biofilms. A multi-well plate of previously established S. aureus biofilms was treated with differing concentrations of the Q. alnifolia ethanolic leaf extract and the remaining biofilm was assessed upon incubation and compared with the controls. The Q. alnifolia extract was able to slightly reduce the biomass of preformed biofilms at the tested concentrations (Figure 4). However, concentrations up to 1.25 mg/mL were not sufficient to eliminate the preformed biofilm. Similarly, the antibiotic, gentamicin, was unable to completely eradicate the preformed biofilm at the tested concentration, though it did result in a modest reduction in biofilm biomass (Figure 4).

3.3. Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis of Quercus alnifolia with Potent Antibiofilm Properties

The preliminary LC-MS analysis of Q. alnifolia ethanolic leaf extracts revealed a complex mixture of phenolic compounds (Figure 5 and Figure 6), including gallotannins, ellagitannins, procyanidins, and derivatives of the flavonols kaempferol, quercetin, and isorhamnetin. Peaks corresponding to hydrolyzed tannins, flavonol glycosides, and megastigmane hexosides were characterized, with structures proposed based on MS/MS spectra and database searches (Figure 7). Monomeric (epi)catechin was the only condensed tannin identified, though procyanidin presence was indicated in the chromatogram (Figure 6, Figures S1 and S2 and Table 2). Several flavonol glycosides featured acylations with coumaric, acetic, or gallic acids, consistent with previously reported compounds in Quercus species, though some, such as peaks 14 and 16, demonstrated unique compositions possibly specific to Q. alnifolia. These ester compounds, provisionally named quercetin-(O-acetyl-di-O-coumaroyl hexoside) and quercetin-(di-O-acetyl-di-O-coumaroyl hexoside), exhibit a previously unreported combination of acyl moieties that has not been described in the literature or documented in existing databases. The group of acylated flavonols was further examined using extracted ion chromatograms that indicate the presence of different phenylpropenoyl fragments. This highlighted a significant excess of coumaric acid esters in the extract when compared to those of caffeic and ferulic acid (Figure S3). The caffeic acid derivatives were observed in low abundancy only but appear to represent a new class of substances not previously described for the genus Quercus. In addition, the MS/MS spectra of the two quercetin derivatives showed small fragment ions at m/z 163.04 (a caffeoyl fragment), m/z 287, and a neutral loss of 286 u (m/z 513 for 14 and m/z 555 for 16), indicating the presence of coeluting, isomeric kaempferol derivatives differing from peaks 14 and 16 by the position of a phenolic hydroxyl group. The molecular formula of peak 14 (C41H34O17) is absent from major databases, including LOTUS and PubChem. Comparisons with LC-MS data from other Quercus species suggest that peaks 14 and 16 (Figure 5) are likely unique to Q. alnifolia or are highly prevalent in this species. Further phytochemical studies are needed to confirm their uniqueness and significance.

4. Discussion

4.1. In Vitro Antibacterial and Antibiofilm Activities of the Quercus Ethanolic Leaf Extracts from Cyprus Against S. aureus

Biofilms have complex extracellular matrix structures, overexpressed efflux pumps, and multiple layers of cells that impede antimicrobial penetration while creating anaerobic conditions in deeper layers [26]. These features, coupled with lowered bacterial metabolism, significantly increase resistance. Quorum sensing prompts the release of extracellular polymeric substances (EPS), generating a network-like matrix that further hinders antimicrobials [27,28]. Such barriers underscore the need for agents capable of preventing or disrupting biofilms in healthcare settings including device-related infections on catheters, stents, and implants [12,13,29,30,31].
This study presents a preliminary screening of antibiofilm activity of ethanolic leaf extracts from four Quercus species native to Cyprus and to conduct preliminary LC-MS phytochemical analysis. Building on our earlier work comparing the antibacterial activity of Q. alnifolia, Q. × campitica, Q. coccifera, and Q. infectoria ethanolic leaf extracts against Staphylococcus aureus, in vitro [16], we found in this study that Q. alnifolia (1.25 mg/mL) and Q. × campitica (2.5 mg/mL) also displayed bactericidal activity, whereas Q. coccifera and Q. infectoria were bacteriostatic, requiring high concentrations for any effect (Table S1). Importantly, biofilm formation observed in the study was significantly reduced at lower concentrations, suggesting a mechanism distinct from direct bacterial killing for these Quercus ethanolic leaf extracts. Among the species tested, Q. alnifolia showed strong antibacterial, bactericidal, and antibiofilm activity in vitro. It modestly reduced preformed biofilm biomass without fully eliminating it, aligning with the partial effectiveness of many plant-derived compounds [7,32]. Reducing preformed biofilm biomass has been notoriously difficult, as demonstrated in our assays and others. For instance, previous studies revealed that only about 28% reduction in S. aureus ATCC25923 preformed biofilms was observed when 1 mg/mL of a seaweed, Posidonia oceanica ethanolic extract has been utilized and much higher concentrations (2–4 mg/mL) were needed for more robust results [23]. However, even a small reduction in biofilm biomass can play a pivotal role in disassembling the strong, matrix-encased biofilm, allowing immune factors or antibiotics to have easier access. To this end, such extracts may still enhance conventional antibiotics through additive or synergistic means, which could be particularly beneficial in nosocomial settings or water sources prone to biofilm formation [27,33,34]. Long-term catheter use, a major contributor to catheter-associated urinary tract infections (CA-UTIs), might be mitigated by coating or irrigating with Q. alnifolia phytochemicals [29]. Combining these extracts with standard antibiotics could offer new methods of managing complex infections [35].
Nonetheless, S. aureus may still cause localized issues if it reaches suitable environments, with biofilms impairing standard disinfection measures [36]. Both Q. alnifolia and Q. × campitica ethanolic leaf extracts inhibited S. aureus biofilm formation in this study at concentrations as low as 0.156 mg/mL. This is in line with previous studies where ethanolic extracts from natural products such as P. oceanica displayed S. aureus ATCC 25923 antibiofilm activity at 0.06–0.5 mg/mL range [23]. This outcome suggests Cypriot Quercus species may be promising candidates for trials aimed at preventing biofilm establishment in clinical settings particularly considering the widespread means by which resistance can spread [36].
In a recent study by Coyotl-Martinez et al. [37], researchers investigated the antimicrobial and antioxidant properties of Quercus sartorii and Quercus rysophylla leaf and bark extracts, identifying phenolic compounds and flavonoids as key contributors to their efficacy against bacteria (including Staphylococcus aureus and Escherichia coli) and fungi. Our preliminary study on Cypriot Quercus species (e.g., Q. alnifolia, Q. × campitica) also found antibacterial and antibiofilm activity against S. aureus, similarly attributing this to phenolic and flavonoid content, as supported by our LC-MS analysis of Q. alnifolia extracts. While both studies underscore the antimicrobial potential of Quercus species, our research specifically focused on the antibiofilm properties against S. aureus, observing biofilm inhibition at lower concentrations, suggesting a distinct mechanism. Additionally, our preliminary phytochemical analysis of Q. alnifolia identified potentially novel quercetin derivatives, offering more specific insights than the broader compound class characterization in the Coyotl-Martinez et al. [37] study. Interestingly, Q. × campitica, an uncommon hybrid of Q. alnifolia and Q. coccifera, showed similarity to Q. alnifolia based on the phytochemical and antibacterial analyses conducted in this study. Our prior work [20] demonstrated variability in radical scavenging capacity, total phenolic content, and total flavonoid content among ethanolic leaf extracts of Quercus species native to Cyprus. This variability has been further explored in the current study using LC-MS analysis (Figure 5). Among the extracts, Q. alnifolia previously exhibited the highest radical scavenging capacity and total flavonoid content, which may account for its observed antibiofilm activity. The ethanolic leaf extract of Q. × campitica displayed similar results, consistent with its hybrid status, suggesting overlapping phytochemical properties with Q. alnifolia. As a result, the LC-MS analysis in this study focused primarily on the ethanolic leaf extract of Q. alnifolia. This collectively extends the understanding of Quercus species as potential sources of bioactive compounds.
The preliminary LC-MS analysis in this study suggest that the flavonoid and phenolic compounds identified in the ethanolic leaf extracts may play a role in the observed in vitro antibacterial and antibiofilm activities against S. aureus. This aligns with a prior review by Górniak et al. [32] which highlights the antibacterial properties of flavonoids, particularly against Gram-positive bacteria. Studies have also shown that quercetin can enhance the effectiveness of aminoglycoside antibiotics, such as gentamicin and tobramycin, in vitro [38,39]. Notably, several quercetin derivatives were identified in the Q. alnifolia ethanolic leaf extract in this study. Additionally, other compounds such as (epi)catechins, quercetin, kaempferol, and their associated derivatives—which have been shown in previous studies to exhibit antibacterial activity and quorum sensing disruption [40]—were also detected in the Q. alnifolia ethanolic leaf extract. These findings warrant further investigation to better understand their individual or combined contributions to the observed antibacterial and antibiofilm activities.
One limitation of this study is the lack of microscopic evaluation of biofilms, which could provide valuable insights into the effects of the ethanolic leaf extracts on biofilm structure at different concentrations. Future research will incorporate techniques such as scanning electron microscopy (SEM) and confocal microscopy to examine the three-dimensional impacts of Quercus ethanolic extracts on biofilm formation, structural integrity, and reduction in established biofilms. The extraction procedure should also be optimized to improve phytochemical recovery, which can then be reassessed in bioassays, while future studies should focus on isolating specific phytochemicals and flavonoids identified through LC-MS analysis and evaluating their individual or combined antibiofilm activities to determine the compounds responsible for the observed bioactivity.

4.2. LC-MS Analysis of the Ethanolic Leaf Extract of Q. alnifolia

The preliminary LC-MS analysis of Q. alnifolia leaf extract identified a wide range of phenolic compounds, such as derivatives of (epi)catechin, quercitol, quercetin, kaempferol, isorhamnetin, and byzantioside B, which may contribute to the antibacterial and antibiofilm properties observed in the study. Previous research supports the antimicrobial efficacy of flavonoids like (epi)catechin, quercetin, and kaempferol [9,38,39,40,41,42,43]. The structures in Figure 7 were assigned by searching their MS/MS spectra in the mzCloud mass spectral library [44]. In addition, the LC-MS analysis revealed a complex mixture of other phenolic constituents in the Q. alnifolia ethanolic leaf extract, including gallotannins, ellagitannins, procyanidins, and diverse flavonol glycosides. Many flavonol glycosides contained sugar groups acylated with coumaric or acetic acid, consistent with Quercus species, but notably, caffeoyl moieties were relatively scarce. Such findings expand the catalog of known flavonoid compounds in the genus [45,46,47,48,49,50]. Moreover, to the best of our knowledge, the two unreported quercetin derivatives from peaks 14 and 16 appear exclusive to Q. alnifolia (Figure 5 and Figure 6).
These findings mark the first observation of these compounds, provisionally named quercetin-(O-acetyl-di-O-coumaroyl hexoside) (14, Figure 6) and quercetin-(di-O-acetyl-di-O-coumaroyl hexoside) (16, Figure 6). Further phytochemical research with adequate biological replicates and structural confirmation via NMR is required to validate these results and fully characterize their significance, as they were not purified and retested for biological activity against S. aureus in this study.
Continued exploration of plant-based antibacterial and antibiofilm compounds may bolster efforts to develop novel approaches against successful human pathogens such as S. aureus. Further work on optimizing plant extraction methods, refining toxicity evaluations, and identifying potential synergy or additive effects with standard antibiotics is fundamental to developing effective antibiofilm strategies. By integrating progress in phytochemistry and pathogenesis, targeted methods for controlling multidrug-resistant microorganisms can be developed, ultimately reducing healthcare costs and preserving lives.

5. Conclusions

This preliminary study investigated the in vitro antibiofilm properties of ethanolic leaf extracts from four Cypriot Quercus species against Staphylococcus aureus. Endemic Q. alnifolia and Q. × campitica showed the highest antibacterial and antibiofilm activity, with Q. alnifolia exhibiting moderate bactericidal effects. Preliminary LC-MS analysis of Q. alnifolia suggested that phenolic compounds, including (epi)catechin, quercetin, and kaempferol, may contribute to its antimicrobial activity. Notably, two novel quercetin derivatives were observed in Q. alnifolia. These findings suggest that Q. alnifolia constituents hold potential as innovative strategies for preventing surface contamination and biofilm-mediated infections, particularly in medical devices. Further research should explore their synergistic efficacy with conventional antibiotics.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/scipharm93030039/s1, Figure S1: +ESI-LCMS of ethanolic leaf extract from Quercus alnifolia; Figure S2: +ESI-LCMS of ethanolic leaf extract from Quercus alnifolia; Figure S3: +ESI-LCMS of ethanolic leaf extract from Quercus alnifolia; Table S1: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration (MBC) of Quercus ethanolic leaf extracts against Staphylococcus aureus (ATCC 6538).

Author Contributions

Conception and design of the work: E.V., C.S. and P.K.; data collection: E.V., C.S., J.S. and P.K.; analysis and interpretation of the data: E.V., C.S., J.S. and P.K.; Statistical analysis: C.S.; drafting the manuscript: E.V., C.S. and J.S.; critical revision of the manuscript: E.V., J.S. and P.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data available upon request.

Acknowledgments

This work was supported by the University of Nicosia Medical School. The cooperation between the University of Nicosia Medical School and the University of Münster, Institute of Pharmaceutical Biology and Phytochemistry, was initiated within the framework of COST action CA21111 (One Health drugs against parasitic vector borne diseases in Europe and beyond (OneHealthdrugs); see https://onehealthdrugs.com/. This research is part of the PhD thesis of Chad Schou. We would like to thank Eleftherios Hadjisterkotis for identifying the plants used in this study. We would also like to thank Justus Mukavi for his assistance with the LC-MS preparation. We would like to thank Catherine O’Dowd-Phanis for her assistance in the laboratory.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

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Figure 1. Quercus species collection site in the Paphos Forest near Kampos, Cyprus.
Figure 1. Quercus species collection site in the Paphos Forest near Kampos, Cyprus.
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Figure 2. Pictures of Cyprus Quercus species used in this study. From left to right: (A) Quercus alnifolia Poech, (B) Quercus campitica Hadjik. & Hand (hybrid Q. alnifolia × Q. coccifera), (C) Quercus coccifera var. calliprinos (Webb) Boiss, and (D) Quercus infectoria subsp. veneris (A.Kern.) Meikle.
Figure 2. Pictures of Cyprus Quercus species used in this study. From left to right: (A) Quercus alnifolia Poech, (B) Quercus campitica Hadjik. & Hand (hybrid Q. alnifolia × Q. coccifera), (C) Quercus coccifera var. calliprinos (Webb) Boiss, and (D) Quercus infectoria subsp. veneris (A.Kern.) Meikle.
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Figure 3. Antibiofilm activity of Cyprus Quercus species ethanolic leaf extracts against Staphylococcus aureus (ATCC 6538). (A) Quercus alnifolia. (B) Quercus × campitica. (C) Quercus coccifera. (D) Quercus infectoria. Results are expressed as mean ± standard error of the mean (n = 6). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Mean values with one or more asterisks above the column denote statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns = not significant.
Figure 3. Antibiofilm activity of Cyprus Quercus species ethanolic leaf extracts against Staphylococcus aureus (ATCC 6538). (A) Quercus alnifolia. (B) Quercus × campitica. (C) Quercus coccifera. (D) Quercus infectoria. Results are expressed as mean ± standard error of the mean (n = 6). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Mean values with one or more asterisks above the column denote statistical significance: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ns = not significant.
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Figure 4. Preformed biofilm reduction by Quercus alnifolia ethanolic leaf extract against Staphylococcus aureus (ATCC 6538). Results are expressed as mean ± standard error of the mean (n = 6). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Mean values with one or more asterisks above the column denote statistical significance: ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 4. Preformed biofilm reduction by Quercus alnifolia ethanolic leaf extract against Staphylococcus aureus (ATCC 6538). Results are expressed as mean ± standard error of the mean (n = 6). One-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. Mean values with one or more asterisks above the column denote statistical significance: ** p < 0.01; *** p < 0.001; **** p < 0.0001.
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Figure 5. Overlay of +ESI-LCMS-BPC of the four Cyprus Quercus ethanolic leaf extracts. Peaks 14 and 16 are significantly prevailing in Q. alnifolia (red overlay). Q. × campitica, Q. coccifera, and Q. infectoria have blue overlays. ESI = electrospray ionization. LC-MS = Liquid Chromatography–Mass Spectrometry. BPC = base peak chromatograph. Peaks 14 and 16 were found specific for Q. alnifolia and were tentatively assigned to quercetin-(O-acetyl-di-O-coumaroyl hexoside) and quercetin-(O-di-acetyl-di-O-coumaroyl hexoside), respectively.
Figure 5. Overlay of +ESI-LCMS-BPC of the four Cyprus Quercus ethanolic leaf extracts. Peaks 14 and 16 are significantly prevailing in Q. alnifolia (red overlay). Q. × campitica, Q. coccifera, and Q. infectoria have blue overlays. ESI = electrospray ionization. LC-MS = Liquid Chromatography–Mass Spectrometry. BPC = base peak chromatograph. Peaks 14 and 16 were found specific for Q. alnifolia and were tentatively assigned to quercetin-(O-acetyl-di-O-coumaroyl hexoside) and quercetin-(O-di-acetyl-di-O-coumaroyl hexoside), respectively.
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Figure 6. +ESI-LCMS base peak chromatogram (black plot and scale) overlaid with Dissect chromatograms of characterized peaks (red plots and scale) of Quercus alnifolia ethanolic leaf extract. The bold, red numbers refer to the compounds listed in Table 2 and shown in Figure 7.
Figure 6. +ESI-LCMS base peak chromatogram (black plot and scale) overlaid with Dissect chromatograms of characterized peaks (red plots and scale) of Quercus alnifolia ethanolic leaf extract. The bold, red numbers refer to the compounds listed in Table 2 and shown in Figure 7.
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Figure 7. Structures tentatively assigned on the basis of the +ESI-LCMS analysis of Quercus alnifolia ethanolic leaf extract. Bold numbers refer to compounds listed in Table 2. The letters in curly brackets for R and R2 denote either a hydrogen atom (H) or the acyl residues acetyl- (A), p-cumaroyl- (C), desoxyhexosyl- (D), hexahydroxydiphenoyl- (E) or galloyl- (G), whose structures are also shown in the figure.
Figure 7. Structures tentatively assigned on the basis of the +ESI-LCMS analysis of Quercus alnifolia ethanolic leaf extract. Bold numbers refer to compounds listed in Table 2. The letters in curly brackets for R and R2 denote either a hydrogen atom (H) or the acyl residues acetyl- (A), p-cumaroyl- (C), desoxyhexosyl- (D), hexahydroxydiphenoyl- (E) or galloyl- (G), whose structures are also shown in the figure.
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Table 1. Percent biofilm inhibition for Quercus ethanolic leaf extracts against Staphylococcus aureus (ATCC 6538).
Table 1. Percent biofilm inhibition for Quercus ethanolic leaf extracts against Staphylococcus aureus (ATCC 6538).
Percent Biofilm Inhibition (%)
0.078 mg/mL0.156 mg/mL0.3125 mg/mL0.625 mg/mL1.25 mg/mL
Q. alnifolia17 ± 8.369 ± 21.274 ± 2.881 ± 9.189 ± 4.4
Q. campitica54 ± 6.170 ± 7.282 ± 2.889 ± 3.1100 ± 0.0
Q. coccifera8 ± 0.318 ± 1.932 ± 11.221 ± 1.937 ± 1.6
Q. infectoria17 ± 3.918 ± 2.816 ± 2.624 ± 3.946 ± 5.8
Gallic acid32 ± 5.6 (0.05 mg/mL)
Quercetin55 ± 11.0 (0.05 mg/mL)
Gentamicin49 ± 1.4 (0.02 mg/mL)
Table 2. +ESI-LCMS data and tentative assignments of characterized peaks of Quercus alnifolia ethanolic leaf extract.
Table 2. +ESI-LCMS data and tentative assignments of characterized peaks of Quercus alnifolia ethanolic leaf extract.
Compound No.tR/minm/z [M + H]+Ion FormulaErr/mDamΣMS2 (25 eV)Tentative Assignment
13.88291.0884[C15H15O6]+−2.123.7207, 189, 179, 165, 161, 147, 139, 123(Epi)catechin
24.60387.2010[C19H31O8]+−0.326.3225, 207Corchoionoside C or optical isomer
34.87769.0907[C34H25O21]+−2.421.9599, 447, 429, 321, 303, 277, 261, 233, 153, 145, 127Bis-hexahydroxydiphenoly- proto-quercitol
44.97619.0950[C27H23O17]+−2.023.9449, 315, 303, 297, 279, 261, 237, 153, 145, 1275-O-galloyl-3,4-(S)-hexahydroxydiphenoyl proto-quercitol
55.03617.1146[C28H25O16]+0.920.5449, 345, 315, 303, 297, 279, 237, 171, 153, 127Quercetin (galloyl-hexoside)
65.05611.1578[C27H31O16]+−2.822.4465, 303Quercetin (desoxyhexosyl- hexoside)
75.15465.1007[C21H21O12]+−2.027.7303Quercetin hexoside
85.19771.1048[C34H27O21]+−0.89.2431, 305, 279, 261, 233, 153Hexahydroxydiphenoyl-digalloyl-proto-quercitol
9a5.51479.1177[C22H23O12]+−0.722.1317, 127Isorhamnetin hexoside
9b5.51371.2048[C19H31O7]+−1.623.5209, 191, 173, 149, 137, 133, 121, 1073-Oxo-α-ionol glucoside or optical isomer
105.66373.2202[C19H33O7]+1.910.1211, 193, 175, 165, 151, 135, 119, 109, 95Byzantioside B (blumenol C glucoside) or optical isomer
116.24595.1444[C30H27O13]+0.335.5309, 287, 147Kaempferol (coumaroyl-hexoside)
126.36595.1434[C30H27O13]+1.214.0309, 287, 147Kaempferol (coumaroyl-hexoside)
137.14741.1864[C39H33O15]+5.037.6455, 437, 287, 273, 255, 147Kaempferol (dicoumaroyl-hexoside)
147.54799.1903[C41H35O17]+3.421.0635, 513, 497, 437, 351, 333, 303, 287, 273, 163, 147Quercetin (diacetyl-dicoumaroyl-hexoside)
157.75783.1965[C41H35O16]+−4.627.0619, 497, 437, 351, 333, 287, 273, 255, 147Kaempferol (acetyl-dicoumaroyl-hexoside)
168.06841.2036[C43H37O18]+6.226.3695, 677, 555, 539, 479, 419, 393, 303, 293, 287, 273, 163, 147Quercetin (diacetyl-dicoumaroyl-hexoside)
178.28825.2070[C43H37O17]+4.525.3765, 679, 661, 539, 479, 419, 393, 375, 333, 315, 293, 287, 273, 255, 147Kaempferol (diacetyl-dicoumaroyl-hexoside)
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Volkan, E.; Schou, C.; Sendker, J.; Karanis, P. Phytochemical Analysis and In Vitro Antibiofilm Activity of Ethanolic Leaf Extract from Quercus alnifolia Poech Against Staphylococcus aureus. Sci. Pharm. 2025, 93, 39. https://doi.org/10.3390/scipharm93030039

AMA Style

Volkan E, Schou C, Sendker J, Karanis P. Phytochemical Analysis and In Vitro Antibiofilm Activity of Ethanolic Leaf Extract from Quercus alnifolia Poech Against Staphylococcus aureus. Scientia Pharmaceutica. 2025; 93(3):39. https://doi.org/10.3390/scipharm93030039

Chicago/Turabian Style

Volkan, Ender, Chad Schou, Jandirk Sendker, and Panagiotis Karanis. 2025. "Phytochemical Analysis and In Vitro Antibiofilm Activity of Ethanolic Leaf Extract from Quercus alnifolia Poech Against Staphylococcus aureus" Scientia Pharmaceutica 93, no. 3: 39. https://doi.org/10.3390/scipharm93030039

APA Style

Volkan, E., Schou, C., Sendker, J., & Karanis, P. (2025). Phytochemical Analysis and In Vitro Antibiofilm Activity of Ethanolic Leaf Extract from Quercus alnifolia Poech Against Staphylococcus aureus. Scientia Pharmaceutica, 93(3), 39. https://doi.org/10.3390/scipharm93030039

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