Thermo finnigan surveyor

Phyllodulcin was extracted from hydrangea leaves by following the method reported previously [28 (link)]. Briefly, leaves were harvested in October 2013 (Sugukmiso Co., Daegu, Korea) and freeze-dried until phyllodulcin extraction. Dried hydrangea leaves were drenched in distilled water for 12 h. Phyllodulcin was extracted by using 75% (v/v) ethanol. The extract of hydrangea leaves was passed through a mixed-bed ion exchanger column. Phyllodulcin was isolated by using a preparative high-performance liquid chromatography system that was equipped with an autosampler and photodiode array (PDA)-UV detector (Thermo-Finnigan Surveyor, Thermo Scientific, Sunnyvale, CA, USA) for purifying phyllodulcin. Final purity and yield of phyllodulcin were 97% and 2.12% (dry basis), respectively [28 (link)]. Purified phyllodulcin was kept in an auto-desiccator (Sanpla Dry Keeper, Sanplatec Corp, Osaka, Japan) until it was used for experiments.

Phenolic compounds were analysed on an HPLC system (Thermo Finnigan Surveyor, Thermo Scientific, San Jose, CA, USA) equipped with a DAD detector set (280, 350, and 530 nm) following the procedure of Mikulic-Petkovsek et al. [61 (link)]. A Gemini C18 (Phenomenex, Torrance, CA, USA) column (150 × 4.6 mm i.d., 3 μm) was used at 25 °C. The samples were eluted with aqueous 0.1% formic acid and 3% acetonitrile in double-distilled water (A) and 0.1% formic acid/3% double-distilled water in acetonitrile (B) according to linear gradients reported by Wang et al. [63 (link)]. The Excalibur software was used for spectral data analyses (Thermo Fisher Scientific, Waltham, MA, USA). All phenolic compounds were identified using a mass spectrometer Thermo Finnigan LCQ Deca XP Mass (Thermo Fisher Scientific, Waltham, MA, USA) with an electrospray interface (ESI) operating in negative and positive ion modes, performing analyses under the same conditions as reported by Mikulic-Petkovsek et al. [64 (link)]. Identification of the phenolic compounds was established based on their retention times and their PDA spectra in comparison with standard phenolics and based on fragmentation patterns in different MSn modes compared with literature data. Contents of phenolics were expressed in mg/g dry weight (DW) of plant material. All analyses were performed in triplicate.