Si gel 60

UV spectra measurements were obtained with a Shimadzu UV-1650PC spectrophotometer; IR spectra were determined with a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). The ¹H and ¹³C-NMR measurements were carried out on Bruker Avance DRX-850 MHz (for ¹H) and 212.5 MHz (for ¹³C) (Bruker BioSpin, Billerica, MA, USA) in CDCl₃ solution, and chemical shifts were expressed in δ (ppm) with reference to TMS, and coupling constant (J.) in Hertz. ESIMS was carried out on Advion compact mass spectrometer (CMS, Ithaca, NY, USA). EIMS was carried on Scan EIMS-TIC, VG-ZAB-HF, X-mass (158.64, 800.00) mass spectrometer (VG Analytical, Inc. Manchester, UK). Open column chromatography was performed on normal-phase Si gel (Si gel 60, Merck, Darmstadt, Germany). Normal-phase and reversed-phase (SPE-C₁₈) cartridges (Strata columns) was used for solid phase extraction. TLC was carried out on precoated silica gel 60 F254 (Merck, Darmstadt, Germany) plates. Developed chromatograms were visualized by spraying with 1% vanillin-H₂SO₄, followed by heating at 100 for 5 min.

¹H and ¹³C NMR spectra were collected using a JEOL JNM ECP-400 spectrometer (Tokyo, Japan) at 600, 500, and 400 MHz for ¹H NMR and 150, 125, and 100 MHz for ¹³C NMR in deuterated solvent (methanol-d₄ (CD₃OD), dimethyl-sulfoxide-d₆ (CD₃)₂SO), and pyridine-d₅ (C₅D₅N)). Column chromatography was carried out using Sephadex LH-20 (20–100 µM, Sigma, St. Louis, MO, USA), silica (Si) gel 60 (70–230 mesh, Merck, Darmstadt, Germany), and Lichroprep^® RP-18 (40–63 µm, Merck, Darmstadt, Germany). Thin-layer chromatography (TLC) was conducted on pre-coated Merck Kieselgel 60 F₂₅₄ plates (20 × 20 cm, 0.25 mm, Merck) and RP-18 F₂₅₄S plates (5 × 10 cm, Merck) using 10% H₂SO₄ (sulfuric acid dissolved in methanol) as a spray reagent. All the solvents used for column chromatography were of reagent grade and obtained from commercial sources.

To obtain a mixture of portoamides A and B in the proportions found inside PHO cells, we carried out a slightly modified version of a previously reported isolation (Leão et al., 2010 (link)). Briefly, biomass from exponentially growing cultures of PHO was harvested by centrifugation, lyophilized and repeatedly extracted with a warm (<40°C) mixture of CH₂Cl₂/MeOH (2:1). The vacuum liquid chromatography (VLC) fractionation of this crude extract was performed on a normal phase (Si gel 60, 0.015–0.040 mm, Merck KGaA) column with an elution gradient of increasing polarity, from 3:2 EtOAc/n-hexane to EtOAc to MeOH. The fraction eluting with 100% MeOH contained the portoamides (from HPLC analysis) and was further fractionated by analytical-scale HPLC. A gradient from 50% MeOH (aq.) to 100% MeOH (1 mL min^-1) was used for both the analysis and isolation of the portoamides A and B, whose mixture eluted and was collected between t_R = 13.0–15.0 min over multiple runs. The purity of the portoamide mixture was confirmed by ¹H NMR, namely by integration and comparison with previously reported portoamides spectral data (Leão et al., 2010 (link)); the proportion of the mixture was found to be 2.7:1 on the basis of integration of LC-HRESIMS peaks for each compound (Figure 1).

Oleacein was isolated from appropriately selected olive oil after screening by NMR of 500 samples collected from Greece the harvest season 2015–2016. The selected oil contained the highest amount of oleacein among the studied samples. Olive oil from Olea europaea cv Koroneiki (500 g) from northern Peloponnese, Greece, was mixed with cyclohexane (2 L) and extracted with acetonitrile (2.5 L). The acetonitrile phase was collected and evaporated using a rotary evaporator under reduced pressure. The residue was submitted to column chromatography using Si gel 60 Merck (40–63 μm) as stationary phase and mobile phase, as shown in Table 1:

The melting points were determined on a Stuart Melting Point Apparatus SMP3 (Bibby Sterilin, Stone, Staffordshire, UK) and are uncorrected. Optical rotations were measured on an ADP410 Polarimeter (Bellingham + Stanley Ltd., Tunbridge Wells, Kent, UK). The ¹H and ¹³C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA) operating at 300 and 75 MHz, respectively. High-resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Aquity UPLC system. A Merck (Darmstadt, Germany) silica gel GF₂₅₄ was used for preparative TLC, and Merck Si gel 60 (0.2–0.5 mm) was used for column chromatography. LiChroprep silica gel and Sephadex LH 20 were used for column chromatography.

The specific rotations were operated on a JASCO DIP-370 digital polarimeter. The ¹H- and ¹³C-NMR spectra were recorded in methanol-d₄ and chloroform-d on a JEOL JNM ECP-400 spectrometer (Tokyo, Japan) at 400 MHz and 100 MHz, respectively. The infrared (IR) spectra were measured on a Mattson Polaris FT/IR-300E spectrophotometer. Mass spectra were recorded using a Quattro II mass spectrometer. Column chromatography was conducted using Diaion HP-20, Sephadex LH-20 (20–100 µM, Sigma, St. Louis, MO, USA), silica (Si) gel 60 (70–230 mesh, Merck, Darmstadt, Germany), and LiChroprep RP-18 (40–63 µM, Merck). All TLC was performed on precoated Merck Kiesel gel 60 F₂₅₄ plates (20 × 20 cm, 0.25 mm) and RP-18 F_254S plates (5 × 10 cm, Merck). The spray reagent was 25% H₂SO₄.