Antimicrobial, Antibiofilm, and Antioxidant Properties of Essential Oil of Foeniculum vulgare Mill. Leaves

Foeniculum vulgare (Apiaceae) is an aromatic fennel with important practices in medicinal and traditional fields, used in the treatment of digestive complications, and gastrointestinal and respiratory disorders. Its leaves and stems, tender and fresh, are used in the production of pasta dressing and main courses, while its seeds, with a strong smell of anise, are excellent flavoring for baked goods, meat dishes, fish, and alcoholic beverages. The aim of this work is concerning the extraction of essential oil (EO) from the leaves of F. vulgare subsp. vulgare var. vulgare, investigating antimicrobial, antibiofilm, and antioxidant efficacy. In particular, GC-MS analysis showed how the chemical composition of EO was influenced by the massive presence of monoterpene hydrocarbons (α-pinene 33.75%) and phenylpropanoids (estragole 25.06%). F. vulgare subsp. vulgare var. vulgare EO shows excellent antimicrobial activity against both Gram-positive and Gram-negative strains. This EO can inhibit biofilm formation at very low concentrations and has a good ability to scavenge oxygen radicals in vitro. F. vulgare subsp. vulgare var. vulgare EO also has an increased activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes and decreased ROS levels in zymosan opsonized PMNs (OZ).


Introduction
Apiaceae is a family of flowering plants, comprising 444 genera. This family has a wide distribution: from northern temperate regions to mountainous landscapes, up to tropical areas [1]. This geographical diversity is not accompanied by the structural diversity of plants. In fact, all the genera of this family are characterized by strong flavors and smells due to the presence of schizogonic ducts containing oil [2], mucilage, and resins, typical of both the aerial parts (leaves, stems, and fruits) and the roots [3]. The presence of different metabolites (coumarins, flavonoids, saponins, and terpenoids) allows the use of these plants in different sectors: food use (nutrition, drinks, and spices), pharmaceutical and cosmetic areas. Furthermore, many of them are used in traditional medicine for the treatment of gastrointestinal, reproductive, and respiratory diseases [2,4,5].
Moreover, from these plants it is possible to distil essential oils (EOs) with high yields and with great chemical variability [6,7]. Very different biological properties have been investigated and confirmed over the years; in addition to the antibacterial [8], antifungal [9,10], antioxidant [11], anti-inflammatory [12], and antitumor activities [13], their insecticidal potential has been very promising [14,15].
Belonging to the Apiaceae family, F. vulgare subsp. vulgare var. vulgare is a perennial with soft, feathery, almost hair-like foliage growing up to 6.6 ft (2 m) tall. This plant can 2.2. Antimicrobial Activity of F. Vulgare subsp. Vulgare var. Vulgare EO F. vulgare subsp. vulgare var. vulgare has been used as an ethnic remedy for the cure of numerous infectious disorders of bacterial, fungal, viral, and mycobacterial origin. In the past, several studies have been carried out to assess its antimicrobial activity [24,28].  The second most abundant chemical class was that of phenylpropanoids (30.36%), with the presence of estragole (25.06%) and (E)-anethole (5.30%). On the other hand, the oxygenated monoterpene compounds were present in minimal quantities (2.29%). The entire chemical composition is reported in Table S1. Based on these observations, the antimicrobial, antibiofilm, and antioxidant potential of F. vulgare subsp. vulgare var. vulgare EO was explored.
2.2. Antimicrobial Activity of F. vulgare subsp. vulgare var. vulgare EO F. vulgare subsp. vulgare var. vulgare has been used as an ethnic remedy for the cure of numerous infectious disorders of bacterial, fungal, viral, and mycobacterial origin. In the past, several studies have been carried out to assess its antimicrobial activity [24,28]. The compounds presented in the EO distilled from fennel leaves were used in different interesting studies [29][30][31][32]. To test so, we performed an inhibition halo assay, which provides a qualitative approach to understanding whether a particular compound possesses antibacterial activity. Figure 2 (panel 1A) displays a bacterial plate with the E. coli indicator strain, a model bacterium for Gram-negative, showing sensitivity to essential oil. Panel 1B of the same figure shows S. aureus, a model of a Gram-positive indicator strain, which also manifests sensitivity towards F. vulgare subsp. vulgare var. vulgare EO. The graph in Figure 2 (panel 2), having arbitrary units in mL, allows us to have a more precise idea of the antibacterial activity of our EO. The bigger the amount of EO used in the experiment, the greater the inhibition halo obtained, suggesting a proportionality between the quantity of used EO and its antimicrobial activity. To learn more about the data on antibacterial activity, a viable cell count quantitativetype of test was performed. As shown in Figure 3 (  To learn more about the data on antibacterial activity, a viable cell count quantitativetype of test was performed. As shown in Figure 3 (panel A) the Gram-negative strains E. coli, P. aeruginosa, and S. Typhimurium manifest mortality against F. vulgare subsp. vulgare var. vulgare EO, E. coli being the most sensitive, having 100% mortality at the highest concentration (200 µg/mL). The same figure (panel B) shows the dose-response curves for Gram-positive bacteria: S. aureus, M. smegmatis, and B. cereus. F. vulgare subsp. vulgare var. vulgare EO is effective against all strains, causing 100% mortality at the maximum concentration (200 µg/mL) for both S. aureus and B. cereus. The antimicrobial activity of EOs is assigned to several small terpenoids and phenylpropanoids compounds, which, in pure form, also demonstrate high antibacterial activity [33].

10%.
To learn more about the data on antibacterial activity, a viable cell count quantitativetype of test was performed. As shown in Figure 3 (panel A) the Gram-negative strains E. coli, P. aeruginosa, and S. Typhimurium manifest mortality against F. vulgare subsp. vulgare var. vulgare EO, E. coli being the most sensitive, having 100% mortality at the highest concentration (200 μg/mL). The same figure (panel B) shows the dose-response curves for Gram-positive bacteria: S. aureus, M. smegmatis, and B. cereus. F. vulgare subsp. vulgare var. vulgare EO is effective against all strains, causing 100% mortality at the maximum concentration (200 μg/mL) for both S. aureus and B. cereus. The antimicrobial activity of EOs is assigned to several small terpenoids and phenylpropanoids compounds, which, in pure form, also demonstrate high antibacterial activity [33].  We performed MIC experiments using the microdilution method. Table 1 shows the results. As expected, the lowest MIC values were calculated for E. coli, S. aureus, and B. cereus (250 µg/mL). Through fluorescence microscopy, we tried to obtain some information about the F. vulgare subsp. vulgare var. vulgare EO mechanism of action, using E. coli and S. aureus indicator strains. The bacteria were treated with the EO at the maximum concentration used in the other tests, and two dyes were added: DAPI, a live cell DNA intercalator that gives blue coloration, and propidium iodide, a dead cell DNA intercalator that gives a red coloration through the damaged membrane. As shown in Figure 4, under optical microscopy conditions, the E. coli treated cells (panel C) show the same shape and color as the control (panel A). E. coli treated cells appear in blue (panel D), thus indicating no damage to the cell membranes, as well as those of the control (panel B). The same experiments on S. aureus confirmed the previously obtained results. Via optical microscopy, the treated cocci ( Figure 4, panel G) are no different from the control ones (panel E), and the S. aureus cells appear in blue even after the treatment (panel H), similar to the control in fluorescence microscopy (panel F). Even for the Gram-positive strains, we can state that the bacterial membrane was intact after treatment with the EO. Essential oils and their components are known to be active against a wide variety of Gram-negative and Grampositive bacteria. However, Gram-negative bacteria are more resistant to their antagonistic effects than Gram-positive ones, because of the lipopolysaccharide present in the outer membrane [34].
conditions, the E. coli treated cells (panel C) show the same shape and color as the control (panel A). E. coli treated cells appear in blue (panel D), thus indicating no damage to the cell membranes, as well as those of the control (panel B). The same experiments on S. aureus confirmed the previously obtained results. Via optical microscopy, the treated cocci ( Figure 4, panel G) are no different from the control ones (panel E), and the S. aureus cells appear in blue even after the treatment (panel H), similar to the control in fluorescence microscopy (panel F). Even for the Gram-positive strains, we can state that the bacterial membrane was intact after treatment with the EO. Essential oils and their components are known to be active against a wide variety of Gram-negative and Gram-positive bacteria. However, Gram-negative bacteria are more resistant to their antagonistic effects than Gram-positive ones, because of the lipopolysaccharide present in the outer membrane [34].

Antibiofilm Activity of F. vulgare subsp. vulgare var. vulgare EO
A crystal violet-based colorimetric assay was used to test the antibiofilm activity of F. vulgare subsp. vulgare var. vulgare EO. Figure 5 graph shows the M. smagmatis bacterial biofilm formation percentage, depending on the added oil concentration. The mycobacterium used is a non-pathogenic strain, a model for the microbial biofilms' formation [35]. In this type of experiment, very low concentrations of EO were used (from 0 to 5 µg/mL), which have no effect on microbial growth, in such a way that the effect of reduction in the formation of the biofilm is linked only to the compound used and not to a decrease in cell vitality. The light gray curve in Figure 5 shows a good biofilm inhibition capacity (over 50%) at the highest concentration used. It is remarkable to use small quantities of a compound, in our case EO, to inhibit the formation of bacterial biofilms. This aspect has an essential impact on the potential use of the EO both in a natural environment to preserve the plants against pathogens [36] and in a medical one [34].
which have no effect on microbial growth, in such a way that the effect of reduction in the formation of the biofilm is linked only to the compound used and not to a decrease in cell vitality. The light gray curve in Figure 5 shows a good biofilm inhibition capacity (over 50%) at the highest concentration used. It is remarkable to use small quantities of a compound, in our case EO, to inhibit the formation of bacterial biofilms. This aspect has an essential impact on the potential use of the EO both in a natural environment to preserve the plants against pathogens [36] and in a medical one [34].

Antioxidant Activity of F. Vulgare subsp. Vulgare var. Vulgare EO
The EO of F. vulgare subsp. vulgare var. vulgare is rich in hydrocarbon monoterpene, which has an antioxidant activity [37]. Figure 6 shows the increasing percentage of scavenging activities of ABTS and H2O2 radicals, as the concentration (1-1000 μg/mL) of EO increases. The data shown in Figure 6 are expressed in Table 2 as IC50 values, representing the EO concentration that causes a 50% reduction in ABTS and H2O2 radicals. The F. vulgare subsp. vulgare var. vulgare EO shows anti-H2O2 activity with IC50 values of 100 μg/mL and the lowest anti-radical effect (IC50 value > 100 μg/mL) for ABTS. Cell survival experiments were performed against HaCat cells (immortalized human keratinocytes) at different concentrations up to 250 μg/mL. By MTT assay after 24 and 48 h of incubation with the F. vulgare subsp. vulgare var. vulgare EO, the treated cells were comparable to the control ones. From these experiments we can conclude that the EO of F. vulgare subsp. vulgare var. vulgare is not toxic for this cell line under the experimental conditions used. The EO of F. vulgare subsp. vulgare var. vulgare is rich in hydrocarbon monoterpene, which has an antioxidant activity [37]. Figure 6 shows the increasing percentage of scavenging activities of ABTS and H 2 O 2 radicals, as the concentration (1-1000 µg/mL) of EO increases. The data shown in Figure 6 are expressed in Table 2    IC50 of ABTS IC50 of H2O2

ROS Generation and Antioxidant Enzymes Activity on Polymorphonuclear Leukocytes (PMN)
The antioxidant activity was investigated by testing the EO extract of F. vulgare subsp. vulgare var. vulgare on OZ-stressed PMNs. Both ROS levels and the activity of SOD, CAT, and GPx enzymes were evaluated (Figure 7). Following the stress induced by OZ, there is a significant increase in ROS, but by treating PMN with EO, a gradual reduction proportional to the increase in concentration was observed. Indeed, already in the PMNs treated with 1 µg of EO, a significant reduction of the ROS levels was observed, and moreover, in the PMNs treated with 100 µg and 200 µg of EO, the ROS levels show levels comparable to the control (PMN not stressed). Regarding the activity of antioxidant enzymes in PMNs treated with EO, they show the same trend. Indeed, the activity of CAT, SOD, and GPx increases statistically with increasing EO concentration.

ROS Generation and Antioxidant Enzymes Activity on Polymorphonuclear Leukocytes (PMN)
The antioxidant activity was investigated by testing the EO extract of F. vulgare subs vulgare var. vulgare on OZ-stressed PMNs. Both ROS levels and the activity of SOD, CA and GPx enzymes were evaluated (Figure 7). Following the stress induced by OZ, there a significant increase in ROS, but by treating PMN with EO, a gradual reduction propo tional to the increase in concentration was observed. Indeed, already in the PMNs treat with 1 μg of EO, a significant reduction of the ROS levels was observed, and moreover, the PMNs treated with 100 μg and 200 μg of EO, the ROS levels show levels comparab to the control (PMN not stressed). Regarding the activity of antioxidant enzymes in PMN treated with EO, they show the same trend. Indeed, the activity of CAT, SOD, and G increases statistically with increasing EO concentration. A decrease in ROS is probably due to the increased activity of antioxidant enzymes; in fact, in the ROS detoxification cascade, SODs are the first antioxidant defense enzymes, catalyzing the dismutation of superoxide anions. The H 2 O 2 generated by, e.g., O 2 − dismutation by SODs, is further detoxified by the action of catalases.
The observed antioxidant activity has correlated with the chemical composition of the EO. It seems plausible that their main constituents, such as estragole (25.06%), γ-terpinene (9.45%), and α-pinene (33.75%), may play a significant role in the antioxidant action of EO. This is confirmed by a different study on the antioxidant activity of several EO components, in which many of the compounds present in these EOs show antioxidant effectiveness [38].

Plant Material and Isolation of Essential Oil
Leaves on the stems of F. vulgare subsp. vulgare var. vulgare were collected in July 2020 in Rocca Busambra, Palermo (Italy) and identified by Prof. Vincenzo Ilardi (3750 51.60 N; ). An herbarium sample is present in the Herbarium Mediterraneum Panormitanum, Palermo, Italy. Using the Clevenger's apparatus, 120 g of fresh leaves were hydrodistilled according to the indications reported by the European Pharmacopoeia [39]. The EO, obtained with a yield equal to 0.68% (v/w), once dried with sodium sulphate, showing an intense yellow color, was stored in the freezer at 20 • C.

GC-MS Analysis
Analysis of EO was performed according to the procedure reported by Rigano et al. [40].

Antimicrobial Activity Assay
The presence of antimicrobial molecules in EO of F. vulgare subsp. vulgare var. vulgare was detected using the method of Kirby-Bauer with modifications [41]. Three different volumes (1, 10, and 50 µL) of EO concentrated 22 mg/mL were placed on Luria bertani agar plates that were overlaid with 10 mL of soft agar (0.7%) and pre-mixed with 10 µL of E. coli DH5α and S. aureus ATCC6538P grown for 24 h at 37 • C. The negative control was 50 µL dimethylsulfoxide (DMSO) 80% used to resuspend the F. vulgare subsp. vulgare var. vulgare EO; the positive control was represented by the antibiotic ampicillin (1 µL) concentrated 22 mg/mL. Plates were incubated overnight at 37 • C and the antimicrobial activity was calculated according to the relation cited below [42].
A/mL = Diameter clearance zone (mm) × 1000 Volume taken in the well (µL) Another method to evaluate the antimicrobial activity involved the cell viability counting of the Gram-positive and Gram-negative strains. Bacterial cells were incubated with both essential oils at 1, 10, 100, and 200 µg/mL concentration. Bacterial cells without essential oils represented the positive control and instead cells with DMSO at 80% were used as the negative control. The following day, the surviving percent of bacterial cells was estimated by counting the number of colonies [43]. Each experiment was carried out in triplicate and the reported result was an average of three independent experiments. (p value was <0.05).

Determination of Minimal Inhibitory Concentration
Minimal inhibitory concentrations (MICs) of F. vulgare subsp. vulgare var. vulgare EO against the Gram-positive and Gram-negative strains were determined according to the microdilution method established by the Clinical and Laboratory Standards Institute (CLSI). A total of~5 × 10 5 CFU/mL were added to 95 µL of Mueller-Hinton broth (CAM-HB; Difco) supplemented or not with various concentrations (1-250 µg/mL) of F. vulgare subsp. vulgare var. vulgare EO [44]. After overnight incubation at 37 • C, MIC 100 values were determined as the lowest concentration responsible for no visible bacterial growth. Each experiment was performed in triplicate and the reported result was an average of three independent experiments.

Antibiofilm Activity Assay
Crystal violet dye was used to evaluate the biofilm formation of M. Smegmatis mc 2 155. A 24 wells plate was prepared in which each well contained a final volume of 1 mL; the negative control was represented by only bacterial cells and medium, the positive control was represented by bacterial cells with antibiotic kanamycin 2 µg/mL, the other samples contained cells and EO [1, 2.5, and 5 µg/mL]. The plate was incubated at 37 • C for 36 h. The OD of the crystal violet present in the distaining solution was measured at 570 nm by spectrophotometric reading, carried out with a Multiskan microplate reader (Thermo Electron Corporation, Waltham, MA, USA) [45]. The biofilm formation percentage was calculated by dividing the OD values of samples treated with EO and untreated samples.

ABTS Scavenging Capacity Assay
This assay was performed according to the reported method [47], with some modifications, which are based on ABTS radical cation scavenging. Then, 1 mL ABTS solution was added to 100 µL of EO (1; 10; 100; 200, and 250 µg/mL concentrations). The absorbance was measured at 734 nm against a blank, and the percentage inhibition of ABTS radical was determined from the following equation: ABTS •+ radical scavenging activity (%) = (1 − AS/AC) × 100, where AC is the absorbance of the ABTS solution and AS is the absorbance of the sample at 734 nm. The concentration required for 50% inhibition was determined and represented as IC 50 . Each experiment was performed in triplicate and the reported result was an average of three independent experiments.

Hydrogen Peroxide Scavenging Assay
Quantitative determination of H 2 O 2 scavenging activity was measured by the loss of absorbance at 240 nm, as previously described by Beers and Sizer [48]. Different concentrations of EO (1; 10; 100; 200, and 250 µg/mL) were incubated at 20 • C in 1 mL of hydrogen peroxide solution [50 mM Potassium Phosphate Buffer, pH 7.0; 0.036% (w/w) H 2 O 2 ]. After 30 min, the hydrogen peroxide concentration was determined by measuring the absorbance at 240 nm. The percentage of peroxide removed was calculated as follows: peroxide removed (%) = (1 − AS/AC) × 100, where AC is the absorbance of 1 mL of hydrogen peroxide solution and AS is the absorbance of the sample at 240 nm.

Eukaryotic Cell Culture
HaCat (human keratinocytes) cells are spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin, widely used in scientific research [45,46]. These cells were maintained in Dulbecco Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37 • C in a humidified atmosphere of 5% CO 2 . The EO of F. vulgare subsp. vulgare var. vulgare was added in a complete growth medium for the cytotoxicity assay [49,50].

ROS Generation and Antioxidant Enzymes Activity on Polymorphonuclear Leukocytes (PMN)
Whole blood was obtained with informed consent from healthy volunteers. Six healthy fasting donors were subjected to peripheral blood sampling with K3EDTA vacutainers (Becton Dickinson, Plymouth, UK). The PMN were isolated following the protocol described by Harbeck et al. [51]. The isolated PMNs were measured in the presence or absence of various concentrations of EO of F. vulgare subsp. vulgare var. vulgare, without or with opsonized zymosan (OZ).
Dichlorofluorescein (DCF) assay was performed to quantify ROS generation according to Manna et al. [52]. The PMN were treated with EO of F. vulgare at different concentrations (1; 10; 100; 200; µg/mL) without or with OZ (500 µg/mL) for 6 h and then incubated with the non-polar and non-fluorescent 2 ,7 -dichlorodihydrofluorescin diacetate (DCFH-DA), at 10 µM final concentration, for 15 min at 37 • C. The ROS quantity was monitored by fluorescence on a microplate reader. Results were expressed as fluorescence intensity.
A commercial kit (BioAssay System, San Diego, CA, USA) was used to determine superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzymatic activity in PMN cells according to the manufacturer's recommendations. The activity of enzymes was expressed as U/L [53]. The EO of F. vulgare subsp. vulgare var. vulgare was tested at the concentration of 1; 10; 100; 200; µg/mL. The experiments were performed in the presence and absence of OZ (500 µg/mL).

Statistical Analysis
The data were examined by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison post-hoc test. In Figures 6 and 7, values are presented as mean st. err; numbers not accompanied by the same letter are significantly different at a p value < 0.05.

Conclusions
Most of the pharmacological studies have been conducted using uncharacterized crude fennel extracts. It is difficult to reproduce their results or identify the bioactive compounds. Therefore, chemical standardization and bioactivity-driven identification of bioactive compounds is required. However, fennel's extensive traditional use and proven pharmacological activities indicate that there is still immense scope for its chemical exploration. In this study, the oil of F. vulgare subsp. vulgare var. vulgare was characterized by the occurrence of a high amount of phenylpropanoids (30.36%), such as estragole (25.06%) and (E)-anethole (5.30%), and of the monoterpene hydrocarbon α-pinene (33.75%). Its antimicrobial, and antioxidant properties have been investigated and the foundations have been laid for future studies. We have discovered remarkable antibiofilm properties at very low concentrations, which may represent a pioneering study for the use of the essential oil of the aerial parts of fennel to avoid the formation of biofilms and combat a scientific and public health problem of great importance. They should focus on validating the mechanism of action responsible for the various beneficial effects, and on understanding which plant-based compounds are responsible for such effects. The information requested, when available, will enhance our knowledge and appreciation for the use of fennel in our daily diets. Furthermore, the result of such chemical studies could further expand its existing therapeutic potential.