South Tyrol (Italy) Pastinaca sativa L. subsp. sativa Essential Oil: GC-MS Composition, Antimicrobial, Anti-Biofilm, and Antioxidant Properties

Abstract

Pastinaca L. is a small genus belonging to the Apiaceae family, traditionally used for both nutritional and medicinal purposes. Pastinaca sativa L. subsp. sativa is a biennial plant widely distributed in Europe and Asia, with recognized ethnopharmacological relevance. In this study, the essential oil (EO) obtained from the aerial parts of P. sativa subsp. sativa, collected in Alto Adige (Italy)-a previously unstudied accession-was analyzed by GC-MS, and the volatile profile has been compared with that of EOs previously studied in Bulgaria and Serbia. The EO was found to be rich in octyl acetate (38.7%) and octyl butanoate (26.7%), confirming that this species biosynthesizes these natural esters. The EO and its main constituents were tested to evaluate their antimicrobial properties. Furthermore, their biological potential was evaluated through antimicrobial, antibiofilm and antioxidant assays. This research work, in addition to evaluating possible chemotaxonomic differences at the geographical level of EOs of Pastinaca sativa subsp. sativa, has been extended to the determination of the biological properties of this accession never investigated before, with the aim of acquiring a broader vision of biofilm and antibacterial properties.

Keywords: Apiaceae; Pastinaca sativa subsp. sativa; antibiofilm property; antioxidant activity; natural antimicrobial; octyl acetate; octyl butanoate.

Figure 1

Kirby–Bauer Antimicrobial Assay. Panel (A): activity against E. coli; Panel (B): activity against B. cereus. Images 1, 2, and 3 in each panel correspond to inhibition zone plates: 1 = halo with PSS, 2 = halo with octyl butanoate, 3 = halo with octyl acetate. Image 4 represents the corresponding quantitative data in arbitrary units/mL. Treatments were tested with 2.5, 5, and 10 µL. Ampicillin and 50% DMSO served as positive and negative controls, respectively. Statistical analysis was performed using a two-tailed paired t-test vs. DMSO 50%, data = mean ± SD (n = 3); ns = not significant; p < 0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).

Figure 2

Outer Membrane Damage Assessed by NPN Assay. NPN fluorescence was used to evaluate outer membrane permeability in E. coli (Panel (A)) and B. cereus (Panel (B)) after treatment with MIC and 2 × MIC concentrations of PSS. Data represent the mean of three independent experiments.

Figure 3

Fluorescence microscopy of E. coli (panel (1)) and B. cereus (panel (2)) cells after 30 min of treatment with PSS (B,F,1-2,2-2), octyl butanoate (C,G,1-3,2-3), and octyl acetate (D,H,1-4,2-4), stained with DAPI (blue) and propidium iodide (red). Untreated control cells are shown in (A,E,1-1,2-1). Scale bars: 5 µm.

Figure 4

Inhibition of biofilm formation by PSS on M. smegmatis (Panel (A)) and P. aeruginosa (Panel (B)). Biofilm biomass was quantified after treatment with concentrations included between 0 and 2500 µg/mL of PSS. The results show a dose-dependent inhibition. Data represent the mean ± SD of three independent experiments.

Figure 5

Inhibition of biofilm formation by octyl acetate and octyl butanoate on M. smegmatis (Panel (A)) and P. aeruginosa (Panel (B)). Biofilm biomass was quantified after treatment with concentrations included of between 0 and 2500 µg/mL of these compounds. The results show a dose-dependent inhibition. Data represent the mean ± SD of three independent experiments.

Figure 6

Cytotoxicity of PSS, octyl acetate and octyl butanoate on Caco-2 cells assessed by MTT assay. Panel (A) shows cell viability after 4 h of treatment; Panel (B) shows results after 24 h. Cells were treated with 10,000 and 25,000 µg/mL. Data are presented as mean ± SD of three independent experiments. Statistical analysis was performed by two-tailed paired t-test relative to untreated cells: ns = not significant; p < 0.01 (**), p < 0.0001 (****).

Figure 7

Effect of PSS, octyl acetate and octyl butanoate on cell proliferation in Caco-2 cells assessed by EdU incorporation. (A) Caco-2 cells were pulsed with 10 mM EdU 3 h before fixation and stained 24 h after PSS treatment. Detection by immunofluorescence analysis of proliferating EdU positive cells (purple) and total nuclei (Hoechst in blue). (B) Percentage of EdU+ nuclei/total number of nuclei in Caco-2 cells treated with 10,000 µg/mL of PSS octyl acetate and octyl butanoate for 24 h Data are presented as mean ± SD of three independent experiments. Statistical analysis was performed by two-tailed paired t-test relative to untreated cells: ns = not significant.

Figure 8

Effect of PSS, octyl acetate and octyl butanoate on H₂O₂-induced ROS generation in Caco-2 cells. Oxidative stress determination using CellROX fluorescent dye. Caco-2 cells were treated with 800 μM H₂O₂ 3 h before ROS detection. 5 μM of CellROX was added to Caco-2 cells for 30 min and cells were analysed 24 h after pre-treatment. Data are presented as mean ± SD of three independent experiments. Statistical analysis was performed by two-tailed paired t-test relative to negative control DMSO: p < 0.05 (*); p < 0.01 (**).