Verbascoside

All reagents used in this work were at least of analytical reagent grade and used as received. SFE experiments were performed using CO₂ and ethanol as modifier. Solvents were purchased from Air Product and Chemicals (Allentown, PA, USA) and VWR chemicals (Radnor, PA, USA). Ottawa sand was provided by Fisher Scientific (Leicestershire, UK). Glass wool was acquired from Sigma-Aldrich (Steinhemin, Germany). LC-MS-grade acetonitrile was purchased from Fisher chemicals (Waltham, MA, USA) and double-deionized water (conductivity of <18.0 MΩ) was obtained using a Milli-Q system acquired from Millipore (Bedford, MA, USA). Formic acid was purchased from Sigma-Aldrich (Steinheim, Germany). Apigenin, loganic acid, kaempferol 3-glucoside, quercetin, and verbascoside were purchased either from Fluka, Sigma-Aldrich (Steinheim, Germany) or Extrasynthese (Genay Cedex, France).

Verbascoside content of each extract was quantified by UPLC (ACQUITY H-Class system) equipped with an ACQUITY UPLC PDA eλ detector using a BEH C18 1.7 µm column (2.1 × 100 mm). The standard Verbascoside (Sigma-Aldrich, ≥ 99% HPLC grade) at various concentrations in AcCN was used to prepare calibration curves (r > 0.9995). The analytical condition was made in a gradient mobile phase consisting of AcCN (A) and 3% aq. AcOH (B). The eluent was programmed as follows: 0 min 100% B, 1.5 min 95% B, 3 min 85% B, 5 min 80% B and 8 min 70% B at a flow rate of 0.6 mL/min for a 10 min completing an injection^{28 (link)}. The maximum absorption wavelength of Verbascoside was determined under this analytical condition at 330 nm, at which this wavelength was set for its quantification in the extracts in addition to the determined retention time. The analysis was performed in three cycles.

Reagents for extraction and other measurements were supplied by Sigma-Aldrich (St. Louis, MO). Oleuropein, verbascoside, luteolin-7glucoside, apigenin-7glucoside, rutin, apigenin, luteolin, tyrosol, hydroxytyrosol, vanillic acid, vainillin, p-coumaric acid, and ferulic acid were obtained from Sigma Chemical (St. Louis, MO, USA) and Extrasynthese (Genay, France). Non-commercial phenolic standards such as ligstroside, hydroxytyrosol-1-glucoside, or the main secoiridoids derivatives were obtained from olive leaves, fruits and oils using a high performance liquid chromatography (HPLC) preparative system.

Luteolin (≥98%; CAS number 491-70-3); apigenin (≥95%; CAS number 520-36-5); trans resveratrol (CAS number 501-36-0); oleuropein (≥98%; CAS number 36619-42-4); verbascoside (≥99%; CAS number 61276-17-3); isoverbascoside (≥95%; CAS number 61303-13-7); tyrosol (≥95%; CAS number 501-94-0); vanillic acid (≥97%; CAS number 121-34-6); cinnamic acid (≥99%; CAS number 140-10-3); ferulic acid (≥99%; CAS number 537-98-4); p-coumaric acid (≥98%; CAS number 501-98-4); 4-hydroxybenzoic acid (≥99%; CAS number 99-96-7); 3-hydroxybenzoic acid (≥99%; CAS number 99-06-9) were purchased from Sigma Aldrich (St. Louis, MO, USA); secologanoside (>95%) were bought from ChemFaces Biochemical Co., Ltd. (Hubei, China); hydroxytyrosol (>95%) were obtained by Indofine (Hillsborough, NJ, USA); oleuropein-aglycone monoaldehyde (>95%) were purchased from Extrasynthese (Genay, France). Ligstroside were purchased from Wuhan Golden Wing Industry & Trade Co., Ltd. (Wuhan, China), the ligstroside-aglycone monoaldehyde (>95%) was isolated by chromatographic method and identity by nuclear magnetic resonance (NMR). The other chemicals were the analytical (Sigma Aldrich, St. Louis, MO, USA).

The following standards for iridoid glycosides and phenylpropanoid glycosides were purchased from APTBio (Shanghai, China): catalpol, rehmannioside A, rehmannioside D, leonuride, aucubin, and verbascoside, n-alkanes (C8–C20; Sigma-Aldrich, USA). The structures of these standards are shown in Figure 1. Methanol, acetonitrile, and formic acid (HPLC grade) were supplied by Fisher Scientific (Fairlawn, NJ, USA). Ethyl acetate and petroleum ether (HPLC grade) were purchased from Shanghai Chemical Reagent Co. (Shanghai, China). Water was purified by a Milli-Q system (Millipore, Milford, MA, USA).

Folin–Ciocalteu’s reagent, potassium persulfate (K₂S₂O₈) and standards of gallic acid were purchased from Merck (Darmstadt, Germany). Anhydrous sodium carbonate (Na₂CO₃) was acquired from Panreac (Castellar del Vallès, Barcelona, Spain). ABTS diammonium salt (2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), acetic acid and standards of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were obtained from Sigma-Aldrich (Madrid, Spain). Glycerol was acquired from Labbox (Barcelona, Spain), ultrapure water was obtained using a Milli-Q water system (Millipore, Milford, MA, USA) and ethanol was purchased from Romil Ltd. (Waterbeach, UK). High performance liquid chromatography (HPLC) grade acetonitrile was acquired from Honeywell Research Chemicals (Seelze, Germany), whereas the HPLC standards of hydroxytyrosol, protocatechuic acid, verbascoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, oleuropein and apigenin were purchased from Sigma-Aldrich (Madrid, Spain).