2 2 azobis 2 methylpropionamidine dihydrochloride aaph

Water was purified with a milli-Q system. Methanol was provided from Honeywell (Seelze, Germany). The 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylique acid (Trolox^®), formic and chlorogenic (5-O-caffeoylquinic acid) (99%) acids were provided by Acros (Morris Plains, NJ, USA). The p-coumaric (98%) and rosmarinic (98%) acids, hesperidin (hesperetin-7-O-rutinoside) (80%), quercetin (98%) and rutin-hydrate (quercetin-3-O-rutinoside) (94%) were provided by Sigma (Steinheim, Germany). Hyperoside (quercetin-3-O-galactoside) (98%) was provided by Extrasynthese (Genay, France). The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Aldrich (L’Isle d’Abeau Chesnes, France). The 2,2′-Azobis (2-methylpropionamidine) dihydrochloride (AAPH) and fluorescein (FL) were obtained from Acros Organics (Noisy-Le-Grand, France). The following natural products were present in the chemical library of the SONAS laboratory: neo-chlorogenic acid (3-O-caffeoylquinic acid) (83%) isolated from the petals of Hydrangea, centaureidin (50%) from Alnus glutinosa young shoots, linarin/acaciin (acacetin-7-O-rutinoside) (96.4%), trans-tiliroside [kaempferol-3-O-(coumaroyl)-glucoside] (76.1%) and thymidine (62.4%) from Tilia tomentosa buds.

The assay was performed on a LS55 spectrofluorimeter (Perkin-Elmer, Leatherhead, UK) using 96-well white polystyrene microtiter plates (Porvair Sciences, Leatherhead, UK) according to a method described by Fredotovic et al. [73 (link)] and Nazlić et al. [74 (link)]. Each reaction contained 180 µL of fluorescein (1 µM), 70 µL 2,2′-Azobis(2-methyl-propionamidine) dihydrochloride (AAPH, Acros Organics, Fair Lawn, NJ, USA) (300 mM), and 30 µL of plant extract or reference standard Trolox (6.25–50 µM) (Sigma–Aldrich, St. Louis, MO, USA). All experimental solutions were prepared in a phosphate buffer (0.075 mM, pH 7.0). The extract of essential oil was prepared in acetone (20 mg/mL). This solution was further diluted 400× with the phosphate buffer for the experiments. For the hydrosol analyses we used total hydrosol diluted 10x. Measurements were performed in triplicate. ORAC values for essential oil were expressed as µmol of Trolox equivalents (TE) per g of essential oil and for hydrosol as µmol of Trolox equivalents (TE) per L of hydrosol.

Hesperidin, hesperetin, narirutin, naringenin, DPPH, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), ferrous sulfate heptahydrate, fluorescein sodium salt, N-hippuryl-his-leu hydrate (HHL), lung acetone powder from rabbit, xanthine oxidase from bovine milk, allopurinol, ρ-nitrophenyl-β-D-glucopyranoside (ρ-NPG), acarbose, α-glucosidase from Saccharomyces cerevisiae, ρ-nitrophenyl butyrate (ρ-NPB), lipase from porcine pancreas, orlistat, and 3-(N-Morpholino)propane sulfonic acid (MOPS) were purchased from Sigma Chemical Co. (St. Louis, MO). 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) was purchased from Acros organics (Geel, Belgium) and captopril was purchased from Tokyo Chemical Industry Co., Ltd. (Kita-ku, Tokyo, Japan). Sinensetin, nobiletin, and tangeretin were purchased from Avention (Yeonsu-gu, Incheon, Korea). hesperetin-7-O-glucoside and prunin were purchased from Extrasynthese (Genay, France). Acetic acid was purchased from Junsei Chemical Co., Ltd (Chuo-ku, Tokyo, Japan) and HPLC grade acetonitrile and methyl alcohol were purchased from Daejung Chemicals & Metals Co., Ltd (Shiheung, Gyeonggi, Korea).