Ezchrom elite

Chromatographic analysis of ELH was performed with a Hitachi HPLC system, and data were processed using EZchrom Elite software (Lachrom Elite; Hitachi High-Technologies Co., Tokyo, Japan). Separation was performed with a Phenomenex Luna C₁₈ column (Part No. 00G-4252-E0, 5 μm particle size, 100 Å, 4.6 × 250 mm, Phenomenex Co., Torrance, CA, USA) at 25 °C, and the injection volume was 3 μL. Gradient elution was performed using solvent A (1% aqueous acetic acid, v/v) and solvent B (100% acetonitrite); the gradient flow was as follows: 0–15 min with 14.5% B, 15–35 min with 14.6% B, 35–45 min with 100% B, and 45–60 min with 14.5% B. The flow rate was 1 mL/min, and HPLC chromatograms were obtained using a UV detector at 190–400 nm. Standard samples including isoorientin and p-coumaric acid (Sigma) were dissolved in methanol at 100 ppm, and the ELH sample was dissolved in methanol at 20 mg/mL.

Standard amino acid mixtures, including Type B and Type AN‐II, were used to quantify the free amino acid contents. Five grams of sample was weighed and homogenized with 15 ml of 5% trichloroacetic acid, followed by centrifugation at 10,000 g at 4 ℃ for 15 min in a HITACHI CR21GⅢ centrifuge. Five milliliters of the supernatant and 5 ml of hexane were mixed and vortexed. Then, 1 ml of the lower layer was taken and filtered using a 0.2‐μm filter. Three replicates were analyzed for each sample.
The analytical equipment used was the Amino Acid Automatic Analyzer L‐8900 (Hitachi). The guard column was AN0‐9256, the main column was a PF column (488511), and the ammonia filter column was 19664. The column temperature ranged from 30 to 70°C (increase 1°C/step). The reactor heater temperature was 135°C. The visible detector wavelengths were 570 nm and 440 nm (for proline). The column flow rate was 0.35 ml/min. The total analysis time was 157.3 min.
The amount of each amino acid in the samples was calculated with reference to the standard sample using the EZChrom Elite (Hitachi High‐Technologies Corporation, 2004) software, and the content of each amino acid was expressed as a percentage of the total sample weight. Determinations were made with three replications for each sample.

The HPLC-DAD system (Hitachi, Tokyo, Japan) consisted of a pump (L-2130), autosampler (L-2200), column oven (L-2300) and UV/VIS diode array detector (L-2455). The output signal of the detector was recorded using EZChrom Elite software for Hitachi. For sample analysis, an OptimaPak C₁₈ column (4.6 × 250 mm, 5 μm; RS Tech Co., Daejeon, Korea) was used, and the column oven temperature was kept at 35°C. The injection volume was 20 μL, and the flow rate of the mobile phase was 1.0 mL/min. The wavelength of the UV detector was set at 220 nm. The mobile phase was water containing 0.1% TFA and acetonitrile, with gradient elution at a flow rate of 1.0 mL/min (Table 3).