Shim pack gis ods column

The CSL extracts (CSL1-CSL4) were quantitatively analyzed using HPLC-DAD (SPD-20A, Shimadzu Co., Japan). Seven standards (gallic acid, epicatechin, EGCG, rutin, tannic acid, naringin, and quercetin) were selected for experiments, and HPLC analysis conditions were as follows: Shim-pack GIS-ODS column (C18, 4.6 × 250 mm, 5.0 μm, Shimadzu Co.), flow rate of 0.7 mL/min, temperature 30 °C, injection volume 20 μL, and a UV detector wavelength of 280 nm. For the mobile phase, 0.1% acetic acid in water (solvent A) and 0.1% acetic acid in methanol (solvent B) were used. The gradient conditions of the mobile phase were 0 min: B (10%), 0–5 min: B (10%), 5–15 min: B (40%), 15–45 min: B (60%), 45–55 min: B (80%), 55–60 min: B (100%), 60–65 min: B (10%), 65–70 min: B (10%). The injection volume was 20 μL. All samples used for analysis were filtered with a 0.45 µm filter.

The BCL was analyzed quantitatively using HPLC-DAD (SPD-20A, Shimadzu Co., Japan). Thirteen standards ((1) gallic acid, (2) catechin, (3) epigallocatechin gallate, (4) epicatechin, (5) caffeic acid, (6) chlorogenic acid, (7) ethyl gallate, (8) p-coumaric acid, (9) ferulic acid, (10) benzoic acid, (11) rutin, (12) quercetin, and (13) luteolin) were selected for experiments, and HPLC analysis conditions were as follows: Shim-pack GIS-ODS column (C18, 4.6 × 250 mm, 5.0 μm, Shimadzu Co.), flow rate of 0.7 mL/min, temperature 30 °C, injection volume 20 μL, and a UV detector wavelength of 280 nm. For the mobile phase, 0.1% acetic acid in water (solvent A) and 0.1% acetic acid in methanol (solvent B) were used. The gradient conditions of the mobile phase were 0 min: B (10%), 0–5 min: B (10%), 5–15 min: B (40%), 15–45 min: B (60%), 45–55 min: B (80%), 55–60 min: B (100%), 60–65 min: B (10%), 65–70 min: B (10%). The injection volume was 20 μL. All samples used for analysis were filtered with a 0.45 µm filter. These data were obtained through the company SUMSUMBIO. Co., Ltd.

Powders (20 g) of leaves, stems, roots, early seeds and late seeds that were the same as those used for transcriptome analysis were prepared using an electric blender and freeze-drier and extracted with 200 ml of 70% methanol for 2 days using an electromagnetic stirrer. After filtration, extracts were concentrated on a rotatory evaporator under vacuum pump and chromatographed on an Agilent 1100 series HPLC system with Shimadzu shim-pack GIS-ODS column (4.6 mm × 150 mm, 5 um). The mobile phase consisted of acetonitrile (A) and 5% aqueous formic acid solution (B). The flow rate was maintained at 1 ml/min and samples were eluted with the following gradient: 5–15% B (0–10 min), 15–25% B (15–40 min), 25–50% B (15–40 min), 50–80% B (40–50 min) and 80–5% B (50–58 min). Chromatograms were recorded at 280 nm. A coixol standard was purchased from ChemFaces Biochemical Co. Ltd. (Wuhan, China) and used to generate a calibration curve for quantification of coixol. HPLC analyses were performed with at least two independent biological samples.