Mill ω purification system

Methanol and formic acid HPLC grade were purchased from VWR (Milano, Italy). HPLC grade water (18 mΩ) was prepared by a Mill-Ω purification system (Millipore Co., Bedford, MA, USA). n-Hexane and n-butanol were achieved from VWR. Chloroform, glacial acetic acid and Methanol were purchased from Carlo Erba (Milano, Italy). The reagents as sodium acetate trihydrate, 2,4,6-tripyridyl-s-triazine (TPTZ), iron (III) chloride (FeCl₃∙6H₂O), Folin-Ciocalteu reagent, carbonate sodium, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical, β-carotene, linoleic acid, Tween 20, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), gallic acid, butylated hydroxytoluene (BHT), β-nicotinamide adenine dinucleotide reduced form (NADH), phenazine methosulfate (PMS), nitrotetrazolium blue chloride (NBT), l-ascorbic acid, potassium persulfate, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), α-glucosidase, acarbose, potassium phosphate buffer solution, 4-nitrophenyl α-d-glucopyranoside were obtained from Sigma-Aldrich S.p.a. (Milano, Italy). Phenols reference standards were obtained in our laboratories by previous isolation from plant materials and characterization by 1D and 2D NMR experiments.

Analytical grade methanol was obtained from Merck (Darmstadt, Germany and Mollet del Vallés, Spain); HPLC grade water (18 mΩ) was prepared by a Mill-Ω purification system (Millipore Corp., Bedford, MA, USA). 2,2-diphenyl-1-picryl hydrazyl (DPPH) in free radical form, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic), and the standards of phenolic compounds were purchased from Sigma (St. Louis, MO, USA and Steinheim, Germany).

Bovine serum albumin (BSA), d,l-dithiothreitol (DTT), d,l-glyceraldehyde (GAL), EDTA and DSC18 hydrophobic interaction cartridges Supelco Discovery were purchased from Sigma-Aldrich (Saint Louis, MO, USA). NADPH, l-idose, soyasaponin Ba (SSBa) and soyasaponin Bb (SSBb) were supplied by Carbosynth (Compton, England); YM10 ultrafiltration membranes were obtained from Merck-Millipore (Darmstad, Germany); soyasaponin Bd (SSBd) was obtained from ALB Technology Limited (Mongkok Kowloon, Hong Kong); PTFE filtration membranes 0.45 µm pore size were from Phenomenex Italy (Bologna, Italy); HPLC grade methanol, formic acid, and acetic acid were purchased from VWR (Milano, Italy). HPLC grade water (18 MΩ) was prepared by a Mill-Ω purification system (Millipore Corp., Bedford, MA). All other chemicals were of reagent grade.
Dry seeds of yellow Zolfino landrace were obtained from the farm Agostinelli Mario in Leccio-Reggello (Florence, Italy; 43° 42' N, 11° 27' E) and authenticity was confirmed by comparing their features with those registered in the “Regione Toscana” germplasm data bank (access VE_027): http://germoplasma.arsia.toscana.it/.