Nova pak

The concentrations of PAs were measured according to the method of Gao [34 (link)] with slight modification. Briefly, 0.2 g of fresh seeds with treatments was homogenized with 2 mL of 5% (w/v) cold HClO₄. Then the seeds were incubated in ice for 1 h, centrifuged at 12,000 rpm for 30 min at 4 °C, and then the supernatant was stored at −80 °C for PAs measurements. Thereafter, 1 mL of 2 mM NaOH and 10 μL of benzoyl chloride were added to 0.5 mL of the obtained supernatant and the mixture was then vortex-mixed vigorously and incubated in 37 °C water for 20 min. Then, 2 mL of saturated NaCl solution and 2 mL of diethylether were added to the mixture. After 1500× g centrifugation for 5 min at 4 °C, 1 mL of the ether phase was obtained and evaporated with a warm air-stream. The dried materials were dissolved in 100 μL methanol and its filtration through a 0.22 μm filter was subjected to reading on an HPLC, which included a 45 mm × 250 mm, 5 μm particle size reverse-phase (C18) column (Waters Nova-Pak), and a Waters 2487 dual λ absorbance detector. The mobile phases consisted of methanol-water (64:36, v/v) at a flow rate of 1.0 mL/min. Three PAs standard samples of Put, Spd, and Spm were prepared at different concentrations for the development of standard curves.

The extraction of LMWOAs was carried out based on the method described by Cayún et al. [18 (link)] with minor modifications; 0.5 g of rhizosphere soil was crushed and dissolved in 1 mL of 0.2 M calcium chloride, shaken, and centrifuged (Centurion Scientific Ltd, Bosham, UK) at 4000× g at 4 °C for 15 min. Afterward, the supernatant was filtered through 0.45 μm filters. The extraction procedure was repeated three times.
Chromatographic separation was performed using high-performance liquid chromatography with diode array detection (HPLC-DAD) (Shimadzu, Tokyo, Japan) equipped with a quaternary pump (LC-20AD), a degassing unit (DGU-20A5R), a column oven (CTO-20A), an autosampler (SIL-20A), and a UV–Vis diode array detector (SPD-M20A) using a C₁₈ column (Eclipse, 250 × 4.6 mm, 5 µm) and a C₁₈ precolumn (NovaPak, Waters, 22 × 3.9 mm, 4 µm). The mobile phase was 0.2 N phosphoric acid (pH 2.1) at a flow rate of 1.0 mL min⁻¹ with isocratic elution at 30 °C. Detection was performed at 210 nm with an analysis time of 15 min using oxalic acid (y = 12,295x + 11,179, detection limit (DL): 1.90 mg L⁻¹, quantification limit (QL): 6.34 mg L⁻¹, linear range (LR): 6.34 to 100 mg L⁻¹) and citric acid (y = 1274.2x + 3763.9, DL: 0.20 mg L⁻¹, QL: 0.66 mg L⁻¹, LR: 0.66 to 500 mg L⁻¹) as external calibration standards.

Individual polyphenols were determined by using a reversed-phase HPLC (Waters 2695, Milford, MA, USA), which was equipped with a C18 column (Nova-Pak, 150 × 3.9 mm, 4 μm, Waters), a 600 E multi-solvent delivery system, a Waters U6K sampler and a Waters 2996 photodiode array detector (DAD) [22 (link)]. Briefly, a gradient elution of polyphenols was performed using a binary solvent system (acetonitrile and acidified distilled water (pH 2 with orthophosphoric acid)) under isothermal conditions (25 °C). A detection wavelength range of 210–550 nm was used. The identification of the single compounds was confirmed by comparing their retention times and UV-DAD spectra with those of the corresponding standards, as well as with those reported in the literature [25 (link)]. The amount of each compound (expressed as area unit (AU) × 10⁶), was calculated before and after digestion.

Liquid chromatography-mass spectrometry (LC/MS) was developed on a Waters Alliance model 2690 system and model 996 photodiode array detector (Waters; Eschborn, Germany) controlled by a Micromass model ZMD mass detector (Micromass; Altrincham, UK). Peptide samples were applied to a 150 × 2.1 mm C18 column, 3.5 µm particle size, 60 Å pore size (Nova-Pak, Waters, Milford, MA, USA). Elution was performed with 0.1% TFA:H₂O as solvent A and 60% acetonitrile:0.1% TFA:H₂O as solvent B, at a flow rate of 0.4 mL·min⁻¹, using a linear gradient from 5% to 95% for solvent B for 30 min at 220 nm and a mass range of 500–3930 Da.

Kiwifruit samples [5 g fresh weight] from S1 to S7 were ground in liquid nitrogen before anthocyanin extraction using 5:1 (v/w) ethanol/H₂O/acetic acid (80:20:1 v/v/v) in an Ultra-Turrax homogenizer for 30 minutes. Homogenates were held for 24 hours at 4°C in the dark. The supernatant was then collected, filtered with 0.45 μm filter paper, and retained for HPLC analysis [39 (link)]. Extracts were analyzed by reversed-phase HPLC using an Alliance 2695 instrument equipped with a 996 photodiode array detector (Waters, USA). Separation was achieved with a 150 ×3.9 mm i.d., 4 μm, C18 Aqua column (Nova-pak, Waters), and a binary solvent system of (A) 1% aqueous formic acid (v/v) and (B) acetonitrile. Anthocyanin was monitored at 530nm, and chromatographic peaks were identified by comparison with authentic standards of cyaniding 3-O-galactoside (Chromadex USA, CAS No. 27661-36-5) and cyaniding 3-O-glucoside (WAKO, Jap, CAS No. 7084-24-4).