Essential Oil Derived from Horticultural By-Products of Artemisa dracunculus L.: A Sustainable Source of Bioactive Compounds with Multiple Biological Activities

1. Introduction

Among aromatic species of agricultural and food interest, Artemisia dracunculus L. represents a particularly interesting and still little-explored case. Commonly known as tarragon, this plant belongs to the genus Artemisia, one of the largest and most widespread genera of dicotyledonous angiosperms [1] which includes over 500 species, distributed mainly in the temperate zones of Europe, Asia, and North America [2]. A. dracunculus is native to Siberia and Mongolia [3] and is currently present in Central Asia, Mediterranean region, Eastern Europe, and North America [4]. In Europe, this species is a widespread crop, mainly used as an aromatic and edible herb for its characteristic aniseed aroma and flavor [4]. It is widely used in sauces, such as béarnaise, in meat, fish, and egg dishes, and to flavor vinegars and traditional beverages in Russia and the Caucasus region [5]. It is also used in Italy as a spice to flavor various dishes, and in some areas of the Northeast, such as Sappada (Friuli-Venezia Giulia region), it is a traditional ingredient in the production of the local “saurnschotte” cheese [6]. In addition to its use as a food, A. dracunculus is traditionally used in several Asian countries for the treatment of digestive disorders and other ailments, finding wide use in folk medicine [3,5,7,8,9,10,11]. Subsequent studies have confirmed numerous biological properties of the species and its metabolites, including antibacterial and antifungal [12,13], anti-inflammatory and analgesic [14], antioxidant [15,16], immunomodulatory [17,18], hepatoprotective [16], hypoglycemic [19] and anticancer [20] activities. Interest in A. dracunculus is mainly related to its essential oil (EO), characterized by the presence of methylcavicol (estragole) as the predominant component and numerous other compounds belonging to the class of monoterpenes, sesquiterpenes, and phenylpropanoids [3,4,5,21,22].

Due to its complex phytochemical profile, the EO of A. dracunculus has attracted growing interest for its potential biological applications. However, despite the available evidence, several of its potential biological properties remain poorly explored. At the same time, a still little-considered aspect concerns the possibility of obtaining this EO from secondary matrices and by-products of the agri-food chain. The development and affirmation of circular economy principles have in recent years promoted the need to valorize waste and by-products from the agri-food industry as a sustainable source of high-value bioactive compounds [23,24]. In the aromatic herbs supply chain, for example, the harvesting, selection, and packaging phases produce non-marketable fractions consisting of damaged or discarded plant material that does not comply with sales standards [25]. These residues, generally destined for disposal and considered of low commercial value, may, however, still contain secondary metabolites of interest and therefore represent a resource for the recovery of high-value products [26,27]. In this context, the use of EOs represents a particularly interesting strategy, as they constitute one of the main forms of chemical interaction between plants and the environment and can be recovered even from plant material considered waste [23,24,28]. Their natural origin, combined with their biodegradability and reduced environmental persistence, makes them promising candidates for sustainable applications in various fields [29]. However, considering the case of A. dracunculus, alongside this potential application, a twofold gap is evident: on the one hand, the limited knowledge of the biological activities of its EO outside the most studied contexts; on the other, the almost total absence of studies focused on EOs obtained from its processing waste.

In light of these considerations, this study was conceived with a dual objective: first, to verify whether the EO obtained from A. dracunculus waste has a chemical profile comparable to that obtained from conventional plant material; second, to evaluate its biological potential through an integrated approach including antioxidant, enzyme inhibitory, phytotoxic, antibacterial, and antibiofilm activities. These investigations are relevant for both crop protections and food safety applications, particularly in relation to natural alternatives to synthetic agrochemicals and antimicrobial preservatives. Thus, the work aims not only to expand knowledge on the biological activities of A. dracunculus EO but also to highlight how the recovery of waste matrices can represent a concrete and unconventional resource for obtaining bioactive compounds, thus contributing to their valorization within the framework of the circular economy.

2. Results

2.1. Yield and Chemical Composition of the Essential Oil

The steam distillation of 2188.30 g of fresh plant material produced 10.23 g of EO, with a yield of 0.47%. The composition of the EO is shown in Table 1, where the components are listed according to their elution order on a HP-5MS column. The GC-MS analysis led to the identification of six compounds, representing 100% of the total EO. Phenylpropanoids were the predominant class (72.79%), followed by monoterpene hydrocarbons (27.21%). Estragole was the major constituent (71.88%), followed by trans-β-ocimene (12.57%) and cis-β-ocimene (10.14%).

2.2. Antioxidant Activity

The EO showed antioxidant activity in all three tests performed: the IC₅₀ value obtained through the DPPH assay was 2.15 ± 0.05 mg/mL, the Fe²⁺ equivalents obtained through the FRAP assay were equal to 305.23 ± 10.56 μmol while the TEAC values obtained from the ABTS assay were equal to 396.42 ± 11.71 μmol. However, the antioxidant power of the EO was in all cases much lower than that found for the substances used as reference.

2.3. Inhibitory Activity of α-Amylase and α-Glucosidase

The EO inhibited both enzymes, with IC₅₀ values of 1.53 ± 0.21 mg/mL for α-amylase and of 7.34 ± 0.69 mg/mL for α-glucosidase. These values demonstrated a good inhibitory activity, especially in the case of α-glucosidase, even if this activity is lower than the reference substance used, acarbose, which showed lower IC₅₀ values: respectively 1.53 ± 0.21 µg/mL for α-amylase and 876.54 ± 48.22 for α-glucosidase

2.4. Phytotoxic Activity

Table 2 shows the phytotoxic activity of EO on the germination and radical elongation of four selected seeds: the two crops, H. vulgare and R. sativus and the two weeds L. multiflorum and S. alba. The table shows the results as percentage inhibition (%) compared to the treatment carried out with the control solution consisting of water and acetone (99.5-0.5 v/v), which was assigned an inhibition of 0.0%. In the table, the results were presented using green for positive inhibitions and red for negative inhibitions (i.e., a process-stimulating effect). White was used when the activity was 0.0%. The more intense the color, the greater the activity.

Figure 1 and Figure 2 show bar graphs used to describe the same effects of the EO solutions on the germination and radical elongation processes of the seeds. The data were constructed using measurements taken directly on the seeds grown: number of germinated seeds and cm of root length.

The EO is poorly effective in inhibiting the germination of all the seeds considered. The greatest inhibitory activity was recorded against H. vulgare, at a 250 µg/mL (20.88%) and L. multiflorum at 125 µg/mL (13.64%). In all other cases the inhibition does not exceed 10% and in many others the EO even promotes the germination processes. The EO was found to be more active in inhibiting root elongation. The greatest inhibitory activity was against S. alba and R. sativus at all concentrations tested. In the case of S. alba, the inhibition varies from 14.73% at 500 µg/mL to 37.39% at 125 µg/mL. In the case of R. sativus, the inhibition ranged from 11.50% (125 µg/mL) to 44.50% (500 µg/mL). In the other cases, the EO promoted an increase in radical elongation for L. multiflorum, while for H. vulgare the EO poorly inhibited the radical elongation at 500 µg/mL (3.23%), promoted radical elongation at 250 µg/mL (-7.74%) and caused an inhibition at 125 µg/mL (30.32%).

2.5. Antibiofilm Activity

The antibiofilm activity of the EO was evaluated against 24 h preformed biofilms of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus using CV and MTT assays at concentrations of 2, 4, and 6 µL/mL (Table 3). The tested concentrations were selected based on the MIC values, which were relatively homogeneous among the six pathogens, ranging from 10 to 12 µL/mL: 10 ± 1 for A. baumannii, E. coli and S. aureus, 11 ± 1 for K. Pneumoniae and L. monocytogenes, and 12 ± 1 for P. aeruginosa. The MTT assay showed an evident concentration-dependent reduction in sessile-cell metabolic activity. Notably, measurable inhibitory effects were already observed at the lowest tested concentration (2 µL/mL), corresponding to approximately one-fifth of the MIC values. At this concentration, metabolic inhibition ranged from 10.12% in P. aeruginosa to 40.00% in L. monocytogenes. Increasing the EO concentration to 4 µL/mL significantly enhanced the activity, with inhibition values ranging from 29.16% (S. aureus) to 61.42% (L. monocytogenes). The strongest effects were observed at 6 µL/mL, where metabolic inhibition reached 55.23, 75.79, 69.28, 79.91, 75.40, and 74.80% against A. baumannii, E. coli, K. pneumoniae, L. monocytogenes, P. aeruginosa, and S. aureus, respectively. The CV assay revealed a more moderate but still concentration-dependent effect on total biofilm biomass. At 2 µL/mL, biomass reduction was evident for most tested strains, exceeding 20% for A. baumannii, K. pneumoniae, P. aeruginosa, and S. aureus. At the highest concentration tested (6 µL/mL), biomass reduction reached 45.00% for A. baumannii, 34.47% for E. coli, 22.56% for K. pneumoniae, 29.39% for L. monocytogenes, 51.25% for P. aeruginosa, and 41.04% for S. aureus. Overall, the EO exhibited a substantially stronger effect on the metabolic activity of sessile cells than on biofilm biomass removal (Table 4). According to the MTT assay, L. monocytogenes, P. aeruginosa, E. coli, and S. aureus were the most susceptible strains, whereas K. pneumoniae exhibited the lowest reduction in biofilm biomass according to the CV assay. Despite relatively similar MIC values among the tested microorganisms, marked differences were observed in their biofilm responses, suggesting that the anti-biofilm activity of the EO is influenced by biofilm-specific traits rather than by planktonic susceptibility alone.

3. Discussion

The yield of A. dracunculus EO depends on environmental and phenological differences. Recent studies show that geographic variability (climate, soil, and pedoclimatic conditions) can significantly influence the biosynthesis of volatile metabolites, and therefore the percentage yield [4,30]. Likewise, the plant’s developmental stage is a key factor: early vegetative growth typically results in lower yields than pre-flowering or early flowering, when terpene accumulation is generally highest [30]. In this context, the yield of 0.47% on fresh weight obtained from material grown in Capaccio in May is consistent with recent literature, which reports a range from 0.4 to 1.0% for the percentage yield and above all lower values for spring harvests and in Mediterranean conditions compared to summer ones or at full phenological maturity, where yields tend to be higher [4,5,30]. Chemical composition is also influenced by various factors, such as habitat, soil salinity, and plant age, which affect both the qualitative and quantitative profile [3]. However, it is possible to identify recurring main components in several studies. Estragole is generally indicated as the predominant compound, with amounts that can vary between 40 and 85%. Other frequently reported constituents among the main ones include elemicin (up to 57%), methyleugenol (up to 25%), terpinen-4-ol, and sabinene (up to 40%). Other recurring compounds, which reach amounts up to 20%, are terpinolene, limonene, and cis- and trans-ocimene, trans-anethole, α-phellandrene, β-phellandrene, and (Z)-artemidine [3,5,12,31,32,33,34,35,36,37]. The EO obtained from A. dracucunculus waste was characterized by a clear predominance of estragole (71.88%) and smaller amounts of cis- and trans-β-ocimene, limonene, α-pinene, and eugenol. This compositional profile is fully consistent with the estragole-rich chemotype frequently reported for A. dracunculus EO, in which this compound generally represents the predominant constituent and can represent over 70% of the volatile fraction [4,5,38]. Interestingly, the recovery of EO from waste matrices did not result in substantial changes in the qualitative composition, which is comparable to that described for EOs obtained from conventional plant material. Similarly, the quantitative profile also shows a high comparability with the Italian samples reported in the literature, in which estragole generally represents the main constituent (up to 73.3-82%), while the observed differences mainly concern the relative abundance of secondary metabolites. As also reported in studies conducted on samples from other geographical areas, these quantitative variations reflect the known chemotypic variability of the species, without modifying the general estragole compositional pattern [31,35,39,40,41].

In recent years, the growing demand for crops to satisfy the ever-expanding world population and the need to achieve ever-higher yields have led to the widespread and sometimes uncontrolled use of synthetic herbicides for weed control [42]. Despite the undoubted effectiveness of synthetic products, they are unfortunately known to be a cause of environmental pollution (water and soil contamination) and a risk to human health due to their accumulation in the body following the consumption of treated products [43]. For these reasons, and to prevent the emergence of resistance, the search for environmentally friendly and safer alternatives to these products is ongoing. One such alternative could be the use of substances often involved in the interaction of plants with the environment, such as EOs, characterized by a strong allelopathic and phytotoxic component [44,45]. The phytotoxic activity observed for A. dracunculus EO could result from the combined action of several mechanisms involved in seed germination and early seedling development. In particular, the inhibition of α-amylase and α-glucosidase could limit the mobilization of starch reserves necessary for embryo growth [46,47,48], while the antioxidant activity could interfere with the physiological balance of reactive oxygen species (ROS), now recognized as essential signaling molecules regulating seed dormancy breaking, germination, and radicle elongation [49,50]. Therefore, the phytotoxic effects observed in the present study are likely the result of a multi-target mode of action rather than the modulation of a single biological process.

The limited data available in the literature show discordant results regarding the antioxidant activity of A. dracunculus EO. Some studies report IC₅₀ values, obtained through the DPPH assay, between 0.070 [51] and 3,19 mg/mL. An EO studied by Mrbati and collaborators [52] showed instead a powerful antioxidant activity, much higher than that found in this work, for all three assays (DPPH, FRAP and ABTS), with IC₅₀ values of 84.44 ± 5.98 µg/mL, 160.38 ± 8.56 µg/mL and 96.71 ± 1.52 µg/mL respectively. Such variability probably reflects differences in the chemical composition of the EO related to genotype and environmental conditions. To the best of our knowledge, however, no studies have investigated the antioxidant activity of EOs obtained from A. dracunculus processing waste. Likewise, no studies have evaluated the inhibitory activity of A. dracunculus EO against α-amylase and α-glucosidase, either from conventional plant material or processing waste. Previous investigation only considered different plant extracts, reporting heterogeneous inhibitory activities against human enzymes, with IC₅₀ values lower than those in this study, ranging between 1.41-13.59 mg/ml for α-amylase and 0.20-5.45 mg/ml for α-glucosidase [53,54,55]. The activity could be attributable to the massive presence of estragole for which, although studies are still few, a certain inhibitory activity on α-amylase [33,34,35], and α-glucosidase [56,57] has been highlighted, whereas no evidence is currently available for trans- and cis-β-ocimene, the other main components found in our EO. Only one study investigated the phytotoxic properties of an EO of A. dracunculus [39], demonstrating a marked inhibition of the germination of Papaver rhoeas L. and Avena fatua L., as well as radicle elongation in A. fatua, P. rhoeas and Lepidium sativum L., with only little effects on Raphanus sativus L. Also in this case, the activity could be linked to the massive presence of estragole, for which inhibitory properties have been reported in various species (Allium cepa L., Lactuca sativa L., R. sativus and Lepidium sativum [58,59,60]. Components such as trans- and cis-β-ocimene may also contribute to the phytotoxic potential of the EO, as numerous publications demonstrate their activity on various plant species [L. sativa, Phalaris minor Retz., Triticum aestivum L., Cassia occidentalis (L.) Link] [59,61,62,63].

Subsequently, considering the widespread use of A. dracunculus as a culinary herb, the evaluation of its antimicrobial and antibiofilm potential may be of particular interest for both food preservation and public health. EOs have attracted increasing attention as natural antimicrobial agents since they can inhibit both planktonic bacterial growth and biofilm formation through multiple mechanisms of action, thus representing promising alternatives to synthetic preservatives and complementary tools against antibiotic-resistant pathogens [64,65,66].

The anti-biofilm assays showed clear concentration-dependent activity against mature biofilms. Notably, anti-biofilm effects were already identifiable at 2 µL/mL, a concentration corresponding to approximately 17–20% of the MIC values determined for the planktonic counterparts. At this sub-inhibitory concentration, reductions in both sessile-cell metabolism and biofilm biomass were observed for most tested microorganisms, indicating that the EO can interfere with biofilm-associated physiology even at concentrations well below those required to inhibit planktonic growth. Similar sub-MIC effects have been reported for several plant EOs and have been associated with alterations in membrane function, cellular communication, and biofilm homeostasis [67,68]. Interestingly, the EO exerted a substantially stronger effect on sessile-cell metabolic activity than on total biofilm biomass. At the highest tested concentration (6 µL/mL), metabolic inhibition exceeded 75% for L. monocytogenes, E. coli, P. aeruginosa, and S. aureus, whereas biomass reduction remained below 55% for all tested species. Similar differences between MTT and CV assays have been reported for several plant EOs and generally indicate that biofilm-embedded cells are more readily inactivated than the extracellular polymeric substance (EPS) matrix is removed [69,70]. The considerable reduction in metabolic activity may be associated with the lipophilic nature of estragole and the other volatile constituents of the oil. Essential oil components are known to interact with bacterial membranes, increasing permeability, disrupting proton gradients, impairing ATP synthesis, and finally reducing cellular viability [71,72]. Such mechanisms are particularly relevant in mature biofilms, where complete matrix disruption is often difficult to achieve, while cellular metabolism remains vulnerable to antimicrobial stress. Among the tested microorganisms, L. monocytogenes exhibited the highest susceptibility, showing approximately 79.91% inhibition of sessile-cell metabolic activity at 6 µL/mL. This finding is particularly relevant considering the persistence of Listeria biofilms in food-processing environments and their involvement in post-processing contamination of ready-to-eat foods [73]. Likewise, the strong activity observed against P. aeruginosa is noteworthy, as this species is widely recognized for its highly structured biofilms and intrinsic resistance to antimicrobial agents [74]). Notably, all tested strains exhibited relatively similar MIC values (10–12 µL/mL), whereas their mature biofilms responded differently to EO treatment. For instance, P. aeruginosa, which displayed the highest MIC value, showed one of the strongest reductions in sessile-cell metabolic activity, whereas A. baumannii, although exhibiting one of the lowest MIC values, was less affected in the MTT assay. These results indicate that the anti-biofilm activity of A. dracunculus EO is not directly associated with planktonic susceptibility but is instead influenced by biofilm-specific characteristics, including EPS composition, biofilm architecture, cell density, and the physiological heterogeneity of sessile populations [74,75]. Although estragole is likely the principal contributor to the observed activity due to its high abundance, synergistic interactions among minor constituents such as ocimenes, limonene, α-pinene, and eugenol cannot be excluded. Numerous studies have demonstrated that the antimicrobial efficacy of EOs often arises from complex interactions among major and minor components rather than from the action of a single constituent [71-72[. Overall, these outcomes indicate that A. dracunculus EO is particularly effective at reducing the viability of cells embedded within mature biofilms, while exerting a more limited effect on biofilm biomass removal. The EO’s ability to exert measurable anti-biofilm effects even at concentrations far below the MIC further reinforces its potential as a promising natural agent for weakening established biofilms and enhancing the susceptibility of sessile cells to subsequent antimicrobial interventions.

4. Materials and Methods

4.1. Plant Material

By-products from aerial parts of A. dracunculus were collected in May 2026 from Aroma Domus O.P. Sc.a.r.l., an organic farm of medicinal and aromatic plants sited in Capaccio Scalo (Salerno, Southern Italy; 40°27’ N, 15°00’ E, 120 m a.s.l.). The A. dracunculus crop was grown in a plastic greenhouse on a previously ploughed and fertilized fine-texture soil. Planting, by using rooted cuttings, took place on March 2025 with a spacing of 40 cm in rows spaced 60 cm apart to obtain a density of 4,2 plants per m². Moreover, the normal agronomic practices (irrigation, fertilization, weed, disease and pest control) of local growers were followed. The harvest was carried out by cutting the plants 30 cm above ground level. By selecting the aerial parts suitable for marketing, waste biomass was obtained, consisting of damaged or discarded plant material which was used for distillation. The plant identification was carried out by Professor Vincenzo De Feo and a voucher specimen, labelled as DF/2026/121, is stored in the herbarium of the Pharmaceutical Botany Chair at the University of Salerno.

4.2. Essential Oil Extraction

Fresh aerial parts were subjected to steam distillation, making the process last for 2 hours, following the procedure reported in the European Pharmacopoeia [76]. The EO obtained was subsequently solubilized in n-hexane and then filtered on anhydrous sodium sulphate and subjected to a flow of N₂ to remove the residual solvent. Storage took place in amber vials, at +4 °C, away from sources of light, heat and humidity, until the analysis.

4.3. GC and GC-MS Analysis

GC-MS analysis was performed using an Agilent 6850 Ser. II Apparatus (Santa Clara, CA, USA) equipped with an HP-5MS fused silica capillary column (30 m× 0.25 mm; 0.25 μm film thickness) and connected to an Agilent Mass Selective Detector (MSD 5973) (Santa Clara, CA, USA) with an ionization voltage of 70 V and an ion multiplier energy of 2000 V. The mass spectra were scanned in the range of 40–500 amu, with 5 scans per second. The analysis was conducted on a schedule basis: 5 min isothermally at 40 °C; subsequently, the temperature was increased by 2 °C/min until 270 °C, and finally it was kept in isotherm for 20 min. The transfer line temperature was 295 °C. The analysis was also performed on an HP Innowax column (50 m × 0.20 mm i.d.; 0.25 μm film thickness). In both cases, helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. Most of the components were identified by comparing their Kovats indices (Ki) with those of the literature [77,78,79,80] and by a careful analysis of the mass spectra compared to those of the pure compounds available in our laboratory or to those present in the NIST 17 and Wiley 257 mass libraries [81]. The Kovats indices were determined in relation to a homologous series of n-alkanes (C₁₀–C₃₅), under the same operating conditions. For some compounds, the identification was confirmed by co-injection with standard compounds. Components relative concentrations were calculated by peak area normalization. Response factors were not considered.

4.4. Antioxidant Activity

4.4.1. DPPH Assay

The free radical scavenging activity was evaluated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) assay, according to the method described by Ud-Daula et al. [82]. In triplicate, an aliquot of the EO solution (dissolved in methanol to obtain different concentrations) was added to a DPPH solution (60 μM) to a final volume of 1 mL. As a control, the same amount of DPPH solution was added to the cuvette and methanol alone was used as a blank. After 45 min, the absorbance at 515 nm was measured with a Multiskan GO spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland). The results were expressed as the IC₅₀ value, which represents the sample concentration required to reduce the absorbance of DPPH by 50% ± the standard deviation (SD).

4.4.2. FRAP Assay

The FRAP (ferric-reducing antioxidant power assay) was performed according to the method of Benzie and Strain [83]. The experiment was performed in triplicate in multiwell. The reaction occurred in each well with a final volume of 272 μL. The reaction mixture, consisting of the FRAP reagent and different concentrations of sample previously dissolved in methanol to obtain different concentrations, was incubated in a dark environment at 37 °C for 30 min. The absorbance of the FRAP blank alone was subtracted from the absorbance of the FRAP with the samples. The results were the means of three experiments ± SD and expressed as μmol of Fe²⁺ equivalents/g of EO. Trolox was used as reference compound.

4.4.3. ABTS Assay

The 2,2-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay was performed following the method of Ud-Daula et al. [82]. In triplicate, 10 μL of the different sample concentrations previously dissolved in methanol to obtain different concentrations and 190 μL of ABTS were added to the wells for analysis. Quantities of 10 ml of PBS and 190 μL of ultrapure water were added to the wells for control. The results were the means of three experiments ± SD and are presented as μmol of Trolox equivalent (TE) per g of EO. Ascorbic acid was used as a reference compound.

4.5. α-Amylase Inhibition Assay

The approach of Jaradat et al. [84], with minor modifications, was used to assess the activity against amylase. The mixture of 100 μL of different concentrations of EO (previously dissolved in methanol), 200 μL of 20 mM sodium phosphate buffer (pH = 6.9) and 100 μL of amylase solution (10 U/mL) was placed at 37 °C for 10 min. Then, 180 μL of 1% substrate solution was added and placed at 37 °C for 20 min. An amount of 180 μl of 3,5-dinitrosalicyclic acid (DNSA) aqueous solution (96 mM) was added to the mixture and boiled in a block heater at 100 °C for 10 min. Then, the absorbance of the solution was read at 540 nm in a UV spectrophotometer (Thermo Fischer Scientific, Vantaa, Finland). All experiments were performed in triplicate and the results were expressed as IC₅₀ ± SD. Enzyme solution was purchased from Sigma-Aldrich (Merck Life Science s.r.l., Milan, Italy).

4.6. α-Glucosidase Inhibition Assay

The inhibitory activity of α-glucosidase was determined as previously described by Nguyen et al. [85]. The assay was performed in multiwell: 150 μL of 0.1 M phosphate buffer pH 7.0, 10 μL of EO dissolved in methanol to obtain different concentrations and 15 μL of the aqueous solution of the α-glucosidase enzyme (1 U/mL) were added to each well and incubated at 37 °C for 5 min. Then, 75 μL of the substrate (2.0 mM) 4-nitrophenyl α-D-glucopyranoside was added and, subsequently, the plate was placed for 10 min at 37 °C. The absorbance was measured at 405 nm in a UV spectrophotometer (Thermo Fischer Scientific, Vantaa, Finland). The positive control was acarbose. The absorbance of the negative control (phosphate buffer instead of sample) was also recorded. The enzyme inhibition was calculated and the results expressed as IC₅₀ ± SD. Enzyme solution was purchased from Sigma-Aldrich (Merck Life Science s.r.l., Milan, Italy).

4.7. Phytotoxic Activity

Phytotoxic effects of the EO were assessed by examining the germination and root elongation of seeds of two plants of agricultural interest, Raphanus sativus L. (radish) and Hordeum vulgare L. (barley), as well as two weeds, Lolium multiflorum Lam. (Italian ryegrass) and Sinapis alba L. (wild mustard). Seeds of R. sativus and H. vulgare were purchased from Blumen group s.r.l., Bologna, Italy, while seeds of L. multiflorum were obtained from Fratelli Ingegnoli s.p.a., Milan, Italy. Seeds of S. alba were collected from wild populations, in June 2025. These seeds are commonly used in phytotoxicity assessments due to their ease of germination and well-documented histological characteristics. Before testing, seeds were sterilized with 95% ethanol for 15 s and then placed in Petri dishes (Ø 90 mm) on three layers of Whatman filter paper, soaked in distilled water (7 mL, for the control group) or in a solution containing the EO (7 mL) at various concentrations. Germination conditions were maintained at 20 ± 1 °C, under a 16 h light and 8 h dark photoperiod. To improve solubility, the EO was dissolved in a mixture of water and acetone (99.5:0.5 v/v) and tested at concentrations of 500, 250 and 125 μg/mL. No differences were observed between the control groups treated with the water-acetone mixture and those treated with water alone. The progress of seed germination was monitored in Petri dishes at 24-h intervals, with a seed considered germinated when radicle protrusion became visible [86]. After 120 h for R. sativus, S. alba and H. vulgare and 168 h for L. multiflorum, germination was observed and radicle lengths were measured in cm. Each measurement was performed in triplicate, using Petri dishes containing 10 seeds each.

4.8. Antimicrobial Activity

4.8.1. Microorganisms and Culture Conditions

The following bacterial strains were utilized: Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa DSM 50079, Escherichia coli DSM 8579, and Klebsiella pneumoniae, clinical isolate (Gram-negative), Staphylococcus aureus subsp. aureus Rosenbach ATCC 25923 and Listeria monocytogenes ATCC 7644 (Gram-positive). The strains were provided by the Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany. Before analysis, the bacteria were cultivated in Luria-Bertani broth at 37 °C for 18 h, except A. baumannii, which was grown at 35 °C under the same conditions.

4.8.2. Minimal Inhibitory Concentration (MIC)

A. dracunculus EO and Dimethyl Sulfoxide (DMSO) were subjected to ultrafiltration before use in the experiments in this study. The Minimum Inhibitory Concentration (MIC) of the EO was assessed using the resazurin method described by Sarker et al. [87], and Khedri et al. [88]. The solution was prepared by dissolving 270 mg of resazurin in 40 mL of sterilized deionized water. For the assay, 96-well microtiter plates were used: the first row was filled with 100 µL of samples in DMSO (1:100 v/v), while the remaining wells were filled with 50 µL of Luria-Bertani broth or a standard sterile solution. Serial dilutions of the EO were performed in descending concentrations. This was followed by adding 10 µL of resazurin indicator solution to each well. Furthermore, 30 µL of 3.3× sensitized broth and 10 µL of bacterial suspension (5 × 10⁶ CFU/mL) were added to each well. The plates were sealed with parafilm to prevent evaporation. A column of the plate contained the broad-spectrum antibiotic tetracycline, which was previously suspended in DMSO and served as a positive control. A negative control consisted of Luria-Bertani broth containing resazurin and bacteria, but no samples. The plates were incubated at 37 °C (35 °C for A. baumannii) for 24 h. Visual observation was used to assess color change. If the solution changed from dark purple to pink or colorless, it was recorded as a positive result. The MIC value was defined as the lowest concentration of EO that could prevent the color change from dark purple to pink.

4.8.3. Biofilm Inhibitory Activity

To assess the inhibitory activity of the EO against mature biofilm, flat-bottomed 96-well microtiter were used [88]. Bacterial cultures were standardized to 0.5 McFarland using fresh culture broth. Each well of the microtiter plate was inoculated with 10 µL of the bacterial culture and incubated for 24 h at 37 °C (35 °C for A. baumannii). Following the removal of the planktonic cells, 2.5 or 5 µL of EO, previously dissolved in sterile DMSO, were added to each well to achieve a final EO concentration of 5–10 µg/mL. The final volume in each well was adjusted to 250 µL with Luria-Bertani broth. Plates were sealed with parafilm tape to prevent evaporation and further incubated at 37 °C (35 °C for A. baumannii) for an additional 24 h. After removal of the planktonic cells, sessile cells were washed twice with sterile Phosphate-Buffered Saline (PBS). Subsequently, the plates were left under a laminar flow hood for 10 min to allow fixation of the sessile cells and then removed after 15 min. Once dried, the sessile cells were stained with 200 µL of a 2% (w/v) crystal violet (CV) solution per well for 20 min. The staining solution was discarded, and the plates were gently washed with sterile PBS. The bound dye was solubilized by adding 200 µL of 20% w/v glacial acetic acid. The absorbance was measured at 540 nm using a Cary Varian spectrophotometer (Cary Varian, Palo Alto, CA, USA). The biofilm inhibitory activity was estimated as a percentage relative to the control (cells grown without EO were considered to have 0% inhibition). Triplicate tests were performed, and average results were calculated to ensure reproducibility.

4.8.4. Effects on Cell Metabolic Activity Within Biofilm

To evaluate the effect of the EO on the metabolic activity of bacterial cells within the mature biofilm, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method was employed [88]. Two concentrations of the EO, previously dissolved in sterile DMSO (to achieve final concentrations of 5 and 10 µg/mL), were added to each well. After 24 h of bacterial incubation, the planktonic cells were removed, and the procedure was followed as described above. After another 24 h of incubation, 150 µL of PBS and 30 µL of 0.3% MTT were added. The microplates were incubated for 2 h at 37 °C (35 °C for A. baumannii) before the MTT solution was removed. Wells were then washed twice with 200 µL of sterile physiological solution, and 200 µL of DMSO were added to dissolve the formazan crystals. Absorbance was measured at 570 nm (Cary Varian, Palo Alto, CA, USA). Experiments were performed in triplicate, and mean values were calculated.

4.5. Statistical Analysis

Statistical analysis of phytotoxic activity was performed by analysis of variance (ANOVA) using GraphPad Prism 6.0 (Software Inc., San Diego, CA, USA). The results were compared to the untreated control and considered statistically significant, by Dunnett’ s test, when p < 0.05.

Statistical analysis was performed separately for each bacterial strain and for each assay (CV and MTT). For each strain, untreated control and EO-treated samples were compared by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. Differences were considered statistically significant at p < 0.05.

5. Conclusions

The EO obtained from horticultural by-products of A. dracunculus showed a broad spectrum of biological activities. It was able to inhibit root elongation of some selective weed species, an effect that may be associated with its moderate antioxidant capacity and its enzymatic inhibitory capacity towards key enzymes involved in seed germination processes. More importantly, the EO showed pronounced antibacterial and antibiofilm activity, proving particularly effective in reducing the metabolic activity of mature cells associated with biofilms. Furthermore, the EO maintained a chemical profile comparable to that commonly reported for EOs obtained from conventional plant material. Overall, the results suggest that the waste biomass generated during the processing of A. dracunculus represents a valuable source of bioactive compounds that can be effectively valorized within the principles of the circular economy, offering promising opportunities for the development of sustainable natural products for agri-food applications.