Sunfiretm c18 column

The HPLC system was comprised of a Waters e2695 HPLC system equipped with a Waters SunFire^TM C₁₈ column (250 mm × 4.6 mm, 5 μm, Waters, Milford, MA, USA) as well as a Diode Array Detector (DAD, Waters 2998, Milford, MA, USA). The mobile phase consisted of 0.1% formic acid aqueous solution (v/v, solution A) and acetonitrile solution (solution B) in the gradient elution at 0.8 mL/min with time-course increasing of solution B to 15% B for 0–5 min, 15–20% B for 5–10 min, 20–25% B for 10–20 min, 25–35% B for 20–30 min, 35–50% B for 30–40 min, 80% B for 40–50 min, and 15% B for 50–55 min. The column temperature was set at 30 °C. The detected wavelength was set at 280 nm. Before HPLC analysis, the samples were filtered through a 0.25-μm membrane filter (Millipore, Billerica, MA, USA). Accurate amounts of standard phenolics were added to GL extracts after enzymatic hydrolysis, and they were extracted as described in Section 4.3, Section 4.4 and Section 4.5. As calculated according to the amount found and amount added, the recovery rate of these phenolics ranged from 95.31% to 101.07% (Table 3). The contents of individual phenolics were expressed as milligram per 100 g DM of GL samples.

High-Performance Liquid Chromatography (HPLC) analysis was performed using an Alliance e2695 system (Waters, Milford, MA, United States) equipped with an autosampler and photodiode array detector (DAD, Waters 2998) and a SunfireTM C18 column, 5 μm (4.6 × 150 mm) (Waters^®) attached to a guard column (SunfireTM C18, 5 μm, 4.6 × 20 mm, Waters^®). The mobile phase consisted of a gradient of H₂O (A):CH₃CN (B) with a flux rate of 2 mL/min for 60 min (5%–100% B) and a UV detection wavelength of 210–600 nm.

A 900 µL aliquot from the elicited culture medium was extracted with an equal amount of ethyl acetate in a 2 mL microcentrifuge tube by vortexing for 30 s. Then, 500 µL of the upper organic phase was removed, transferred to an amber HPLC vial, and dried under a nitrogen stream using a Reacti-Vap III apparatus (Thermo Fisher Scientific, Waltham, MA, USA). HPLC analyses were performed in an Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA). The extract was resuspended in 500 µL of MeOH and analyzed by reversed-phase HPLC. Briefly, the chromatography was performed in a SunFire^TM C18 column (5 µm, 4.6 × 250 mm, UV detection at 340 nm) (Waters, Milford, MA, USA) at 40 °C and a flow rate of 1.0 mL/min. The mobile phase was composed of MeOH (A) and 0.5% HCO₂H (v/v) (B). The column was initially calibrated with B for 1 min. Then, a linear gradient was performed using the following program: 60% A to 65% A for 1–20 min, 65% A and 35% B to 100% B for 20–25 min, and 100% B for 25–30 min. Calibration curves for reference compounds were established previously [36 (link)]. Similarly, re-elicited culture medium was extracted and analyzed as described above.

Fresh-frozen tissues were washed once with PBS, and homogenized in 10% Trichloroacetic Acid (T6399, Sigma-Aldrich, St. Louis, MO, USA). After incubating on ice for 10 min, the mixtures were centrifuged at 12,000 g for 15 min at 4 °C, and the nucleotide containing supernatant was washed 4 times with water-saturated diethyl ether. Then the supernatant was froze at −80 °C, and freeze-dried at −20 °C overnight use FreeZone Freeze Dry Systems (LABCONCO, Inc., MO, USA). The extracted nucleotides were dissolved in 100 μL ultrapure water and centrifuged at 8,000 rpm for 5 min. Supernatant was filtered with 0.45 μm filter and then analyzed by HPLC on a Agilent 1260 (Agilent Technologies, Inc., CA, USA) equipped with a Waters SunFireTM C18 column (250 mm × 4.6 mm i.d., 5 μm, Waters, Milford, MA, USA) and detected by UV-VIS absorbance at 260 nm. The nucleotides were eluted with isocratic elution (flow rate: 0.75 ml/min, Column temperature: 25 °C) using a mixture of 10% acetonitrile and 90% water (0. 01 mol/L Tetrabutylammonium bromide, 0.05 mol/L Na₂HPO₄-NaH₂PO₄, pH6.4). The amount of each nucleotide was determined by external standard method. The following standards were used: ATP (A2383), ADP (A2754), AMP (01930), IMP (57510), GMP (G8377), GDP(G7127), GTP(G8877) (Sigma-Aldrich, St. Louis, MO, USA).

Analyses were conducted with Separations Module 2695 (Waters, USA), using the UV/Visible 2489 detector (Waters). Determination of prunasin was performed isocratically using the SunFireTM C18 column (4.6 × 250 mm, 5 μm) together with the pre-column Waters (4.6 × 20 mm). The temperature of the column was stabilized at 40 °C. The methanol: water (15: 85 v/v) solution was used as eluent at 1.5 ml/min flow rate, and UV detection was set at 218 nm.
The concentration of prunasin in extracts was calculated from the calibration curve obtained using commercially available chromatographically purified prunasin (ABCR GmbH & Co. KG, Karlsruhe, Germany) as a standard. Prunasin concentration was first expressed as mg g⁻¹ root fresh mass, and it was recalculated using fresh weight to dry weight root ratio and expressed as mg g⁻¹ DM.

Compounds 1d–3d and 1e–3e were used as intermediates for the synthesis of hydrozinecarboximidamide-HNK derivatives. In brief, the compounds 1d–3d (0.05–0.28 mmol) and acetic acid/aminoguanidine carbonate (0.18–0.39 mmol) in ethanol were stirred for 3–4 h at 65 °C, and then detected by TLC analysis, respectively. The reaction was quenched with water and extracted twice with ethyl acetate. The combined extract was dried over Na₂SO₄ and concentrated under reduced pressure. Finally, the residue was purified by a 2535Q semi-prep HPLC system (Waters, USA) using a SunFire^TM C18 column (250 × 10 mm) with 35–55%% acetonitrile to afford 1f–3f and 1g–3g.

The CE was prepared by mixing it with water (Burdick & Jackson, USA) and methanol (Burdick & Jackson) at a ratio of 30:70 (v/v) to 500 mg/L, followed by stepwise dilution to 1 mg/L. Anthocyanin was also diluted to 500 mg/L with the same solvent and used as a standard stock solution. For quantitative analysis of the anthocyanin content in CE, a standard curve was generated by diluting the anthocyanin to 0.1, 0.2, 0.5, 1, and 2 mg/L, and the analysis was repeated three times. All substances were analyzed by an injection of 10 µL of the sample into Shimadzu HPLC i-Series LC-2030 LT (SHIMADZU, Japan) and separation at a flow rate of 1.0 mL/min through a SunFireTM C-18 column (4.6 × 250 mm, 5 μm, Waters, Germany). In addition, mobile phase A consisted of 0.1% trifluoroacetic acid (Sigma-Aldrich, St. Louis, MI, USA) added to water, and mobile phase B consisted of 0.1% trifluoroacetic acid added to acetonitrile (Burdick & Jackson) (v/v). The change in the ratio of the mobile phases is shown in Table 1, and the absorbance at 520 nm was detected using deuterium (D2) lamps (SHIMADZU) over a 35-min period.