Scopolin

Ultra-pure water (18 mΩ) was obtained from a Milli-Q water purification system (Millipore Co., Ltd., Milford, MA, USA). High-performance liquid chromatography (HPLC)-grade methanol, acetic acid, and 56 analytical standards, including catechinic, scopolin, chlorogenic acid, epicatechinic, vanillic acid, caffeic acid, purerarin, syringic acid, daidzin, glycitin, scopoletin, eriocitrin, umbelliferone, p-coumaric acid, dihydroquercetin, sinapic acid, genistin, liquiritin, ferulic acid, salicylic acid, rutin, isoferulic acid, m-coumaric acid, naringin, hesperidin, resveratrol, xanthotoxol, silydianin, sinapyl alcohol, o-coumaric acid, liquiritigenin, kaempferol, 2’-hydroxygenistein, eriodictyol, daidzein, psoralen, glycitein, quercetin, didymin, bergaptol, naringenin, luteolin, cinnamic_acid, hesperetin, genistein, bergapten, diosmetin, isoliquiritigenin, coumestrol, sinensetin, formononetin, medicarpin, imperatorin, biochanin A, tangeretin, and rotenone (displayed in Table S2), were purchased from Sigma-Aldrich Co., Ltd. The stock solutions of these authentic standards were 10.0 mg of each standard dissolved in 10 mL methanol. Then, the stock solutions were diluted to various concentrations before analysis. All stock solutions were sealed with Parafilm^® and stored in a −20 °C freezer.

All buffer constituents and solvents were of reagent or HPLC grade and obtained from either Roth (Karlsruhe, Germany), Sigma-Aldrich (St Louis, MO, USA) or J. T. Baker (Deventer, The Netherlands). Coumarin standards were purchased from PhytoLab (Vestenbergsgreuth, Germany; scopolin, isofraxidin, fraxin), and Sigma-Aldrich (esculin, esculetin, fraxetin, 4-methyl-umbelliferon, scopoletin). [2,2,4,4-²H]citric acid and [2,3,3-²H]malic acid were from CDN isotopes (Point Claire, QC, Canada), [2,2,3,3-²H]succinic acid and methoxyamine HCl from Sigma-Aldrich, and Silyl-991 from Macherey-Nagel (Düren, Germany).

The HPLC analysis for comparison of the calluses and leaves of WS in vitro was performed using an Agilent 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA), equipped with a diode array detector, binary gradient pump, autosampler and vacuum degasser. The indicated compounds were eluted using a Capcell Pak MGII ODS column (C18, 4.6 mm I.D. × 150 mm, 3 μm particle size) at 35 °C with a flow rate of 0.35 mL/min. The injection volume was 15 µL with needle wash. The mobile phase comprised a mixture of aqueous formic acid (0.1%, v/v) and 0.5% acetonitrile. Scopolin and scopoletin (Sigma–Aldrich) as markers, were eluted under gradient conditions. Data were collected and integrated using Agilent Chemstation B.04.01 software. The standard solutions of Scopolin and scopoletin were prepared using 70% aqueous methanol (v/v). The in vitro callus and leaves of WS were extracted with the same solvent used in the standard solution and compared using HPLC analysis.