Diode array detector

For TEM analysis, Rg3/Lipos (0.3 μg) were placed on a 300-mesh copper grid and air-dried. After washing the grid three times with distilled water, the samples were stained with 2% uranyl acetate solution. Excess uranyl acetate was removed by washing and the grids were observed using a Bio-TEM instrument (Tecnai G 2 Spirit Twin; FEO, USA). For aqueous stability assay, Rg3/Lipos (10 μg) in PBS solution were stored at 4°C and -18°C for predetermined time intervals (1, 4, 7, 10, and 14 days). The sizes of the incubated Rg3/Lipos were measured using DLS.
To determine the encapsulation efficiency, Rg3/Lipos were purified by dialysis to remove free Rg3(S) and the solvent. After freeze-drying, the resulting powder was dissolved in 500 μl methanol and the amount of Rg3(S) was analyzed using RP-HPLC (Agilent Technologies, USA) equipped with a diode-array detector (Agilent Technologies) on a Varian polar C18 reverse-phase column (4.60 × 250 mm, 0.45 μm; Varian, USA) to calculate Rg3(S) and lecithin [7 (link)]. The mobile phase consisted of 0.1% formic acid in water and acetonitrile [14 (link)]. The flow rate was set at 1 ml/min. The eluent was analyzed at a wavelength of 205 nm. The encapsulation efficiency was calculated as follows: Rg3(S) encapsulation efficiency (%) = encapsulated amount of Rg3(S)/initial amount of Rg3(S) × 100.

Anthocyanin extraction and quantification were performed as previously described^{28 (link)}. Briefly, the extraction buffer was 70% methanol containing 2% formic acid. Extracted anthocyanin was filtered through a 0.45-μm syringe filter prior to HPLC analysis. The anthocyanin concentration was determined by the absorbance at 520 nm on an HP 1200 liquid chromatograph equipped with a diode array detector (Agilent, CA, USA). The anthocyanin in three biological replicates was quantified based on the calibration curve for a cyanidin 3-galactoside standard (Sigma-Aldrich, MO, USA).

The phenolic characterization and quantification were performed using an Agilent 1200 liquid chromatography system (Agilent Technologies, Palo Alto, CA, USA) equipped with a standard autosampler and analytical column Agilent Zorbax extended C18 (5 × 2.1 cm, 1.8 µm), as reported by Nicolì et al. [54 (link)] and Vergine et al. [55 (link)]. The HPLC system was coupled to an Agilent diode-array detector. The detection wavelength was 280 nm and an Agilent 6320 TOF mass spectrometer was equipped with a dual ESI interface (Agilent Technologies) operating in a negative ion mode. Detection was carried out within a mass range of 50–1700 m/z. Accurate mass measurements of each peak from the total ion chromatograms (TICs) were obtained by using an ISO pump (Agilent G1310B) using a dual nebulizer ESI source that introduced a low flow (20 μL min⁻¹) of a calibration solution containing the internal reference masses at m/z 112.9856, 301.9981, 601.9790, 1033.9881, in negative ion mode. The accurate mass data of the molecular ions were processed using Mass Hunter software (Agilent Technologies).
The compounds were quantified using calibration curves of authentic standards (quinic acid, hydroxytyrosol, oleuropein, luteolin 7-O glucoside, luteolin, and verbascoside) and the regression equation and the correlation coefficient (r²) were calculated, as reported by Luvisi et al. [35 (link)].