Oleuropein

The liquid–liquid extraction of phenolic compounds was performed with the method proposed by Capriotti et al. [67 (link)]. 1 g of EVOO was dissolved in hexane (oil/hexane 1:1, w/v) in a 10 mL centrifuge tube and shaken for 30 s. The polyphenols were extracted with 2 mL of MeOH and stirred for 30 s; the emulsion was then centrifuged at 3000 rpm and 4 °C for 3 min. The supernatant (methanolic extract) was subjected to a second cleaning with hexane, and the hexane extract was subjected to a second extraction of polyphenols with MeOH. All extracts were shaken for 30 s and centrifuged at 3000 rpm and 4 °C for 3 min. The methanolic extracts were recovered and cleaned up by dispersing 50 mg of C18. The samples were evaporated and reconstituted with 800 μL of MeOH:H₂O (80:20 v/v), filtered with (Polytetrafluoroethylene) PTFE syringe filters (0.2 µm), transferred to an amber glass vial and stored at −80 °C until analysis. The internal standard was added to the EVOO to obtain a final concentration of 5 ppm after the reconstitution. The experiment was done in triplicate.
The identification and quantification of phenolic compounds was performed using an Acquity^TM UPLC (Waters; Milford, MA, EUA) coupled to an API 3000 triple-quadruple mass spectrometer (PE Sciex) with a turbo ion spray source. Separation of compounds was achieved using an Acquity UPLC^® BEH C₁₈ Column (2.1 × 50 mm, i.d., 1.7 µm particle size) and Acquity UPLC^® BEH C₁₈ Pre-Column (2.1 × 5 mm, i.d., 1.7 µm particle size) (Waters Corporation^®, Ireland) (See supporting information). The mobile phases were H₂O with 0.2% acetic acid (A) and ACN (B). An increasing linear gradient (v/v) of B was used (t (min), %B), as follows: (0, 5); (2.5, 5); (12.5, 40); (12.6, 100); (13.5, 100); (13.6,5); (15,5), at a constant flow rate of 0.4 mL/min. The injection volume was 10 µL and the column temperature 40 °C.
The quantification of OLC was performed using a methodology proposed by Sánchez de Medina et al. with some modifications. Separation was achieved using an Acquity UPLC^® BEH C₁₈ Column (2.1 × 50 mm, i.d., 1.7 µm particle size) and Acquity UPLC^® BEH C₁₈ Pre-Column (2.1 × 5 mm, i.d., 1.7 µm particle size) (Waters Corporation^®, Ireland). The mobile phases were MeOH (A) and H₂O (B), both with 0.1% of formic acid. An increasing linear gradient (v/v) of B was used (t (min), %B), as follows: (0, 100); (2, 100); (4.75, 46.4); (4.9, 0); (5.9, 0); (6.100); (6.5, 100), at a constant flow rate of 0.6 mL·min⁻¹. The injection volume was 5 µL and the column temperature 50 °C. The MS potentials were optimized for the compound (Supporting Table S2). Method suitability was evaluated by submitting random samples to a comparative NMR study.
Ionization was achieved using an electrospray interface operating in the negative mode [M–H] and all the compounds were monitored in the multiple monitoring mode (MRM) with the following settings: capillary voltage, −3500 V; nebuliser gas (N₂), 10 (arbitrary units); curtain gas (N₂), 12 (arbitrary units); and drying gas (N₂) heated to 450 °C. The declustering potential, focusing potential, collision energy and entrance potential were optimized to detect phenolic compounds with the highest signals, following the method described by Suárez et al. [39 (link)]. The system was controlled by Analyst version 1.4.2 software supplied by Applied Biosystems.
The calibration curves were prepared in refined oil and were linear over the concentration ranges 0–20 mg·mL⁻¹ using oleuropein, hydroxytyrosol, p-coumaric acid, m-coumaric acid, vanillic acid, ferulic acid, apigenin, luteolin, pinoresinol, lariciresinol, isolariciresinol, secoisolariciresinol, verbascoside and OLC.

The RT- qPCR method was used to investigate the inhibitory effect of oleuropein on gene expression in the biofilm of C. albicans (Hwp1, Als3), C. glabrata (Epa1, Epa6) and E. coli (LuxS, Pfs). The primers were designed based on sequences that have been reported in previous studies (Table 1). RNA extraction and cDNA synthesis were performed using the method described in the next section.
Table 1

Forward (FW) and reverse (RV) primers used in study [45 (link), 46 (link)]

Primer sequence 5′ to 3′	Gene
TGCTGAACGTATGCAAAAGG	ACT1_alb FWa
TGAACAATGGATGGACCAGA	ACT1_alb RVa
TTGCCACACGCTATTTTGAG	ACT1_gla FWa
ACCATCTGGCAATTCGTAGG	ACT1_gla RVa
TCTACTGCTCCAGCCACTGA	Hwp1 FW
CCAGCAGGAATTGTTTCCAT	Hwp1 RV
CTGGACCACCAGGAAACACT	Als3 FW
GGTGGAGCGGTGACAGTAGT	Als3 RV
ATGTGGCTCTGGGTTTTACG	Epa1 FW
TGGTCCGTATGGGCTAGGTA	Epa1 RV
TTATGCCGTATGGGGTTCTC	Epa6 FW
GAGTCAACTGAGGCACACGA	Epa6 RV
ATACCGCATAACGTCGCAAG	rrsD FWa
ATATTCCCCACTGCTGCCTC	rrsD RV a
AATCACCGTGTTCGATCTGC	LuxS FW
GCTCATCTGGCGTACCAATC	luxS RV
ATCGTTGTCTCGGACGAAGC	Pfs FW
GGACAGCCTGGTAACTGACCG	Pfs RV

^aThe housekeeping genes

Selected chitosan nanoparticles encapsulating oleuropein were centrifuged at 15,000× g rpm, 4 °C for 30 min (Hermle Labortechnik GmbH, Model Z216 MK, Wehingen, Germany). The free drug in the supernatant of the selected formulation was determined using a UV spectrophotometer (Shimadzu, Kyoto, Japan) at 280 nm [19 (link)]. The entrapment efficiency (EE%) was calculated using the following equations: $$EE\%=\frac{W_t-W_f}{W_t}\times 100$$
where W_t is the total weight of oleuropein used and W_f is the weight of free oleuropein.