Luteolin

The following chemicals of analytical and gradient grade purity were used: 2-aminoethyl-diphenylborate, 5,5 dimethyl-1-pyrroline-N-oxide (DMPO), 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulphonic acid) salt/cation radical (ABTS/ABTS^•+), acetonitrile, dimethyl sulfoxide (DMSO), (±)-catechin, chlorogenic acid, ethanol, ferulic acid, Folin–Ciocalteu phenol reagent, gallic acid, hesperidin, myricetin, p-coumaric acid, sinapic acid, quercetin, and rutin hydrate (Sigma-Aldrich, St. Louis, MO, USA); potassium persulfate, potassium dihydrogen phosphate, sodium hydrogen phosphate (Merck, Darmstadt, Germany); caffeic acid and luteolin (Alfa Aesar, Ward Hill, MA, USA); formic acid, sodium carbonate, sodium hydroxide (Lachema, Brno, Czech Republic), standard solutions of elements concentration 1 g/L (Analytika, Prague, Czech Republic) and deionized water purified by a Milli-Q A10 Gradient (Millipore Corp., Burlington, MA, USA).

Standard solutions were prepared for the fifteen investigated phenolic compounds and flavonoids, namely: kaempferol, luteolin, phenylacetic acid (all Alfa Aesar, Ward Hill, MA, USA), apigenin, chrysin, quercetin, p-coumaric acid, naringenin (all Sigma-Aldrich, Merck, Darmstadt, Germany), ferulic, syringic, vanillic, caffeic, ellagic, gallic and benzoic acids (Acros Organics, Geel, Belgium). The solutions were prepared by dissolving the standards in HPLC grade methanol (ultragradient grade; Carlo Erba, Milan, Italy) to produce stock solutions of 100 mg/L, which were then used to prepare 50, 40, 30, 20 and 10 mg/L solutions for the standard plots. In addition, a mixture containing 30 mg/L of each phenolic compound in methanol was also prepared. Two mixtures of p-coumaric and ferulic acid were prepared, one with equal concentrations of 50 mg/L each and the other with 30 mg/L p-coumaric and 70 mg/L ferulic acid. Another similar set of mixtures was also prepared using vanillic and caffeic acids. We measured the absorbance of the samples of 100 mg/L of each phenolic compound and flavonoid with a UV-Vis spectrophotometer (UV-2600; Shimadzu, Tokyo, Japan) at wavelengths between 180 and 480 nm to find the optimum wavelength for the HPLC-DAD measurements.

The specific compounds, were: Caffeic acid, CAPE, chrysin, luteolin, daidzein, suberic acid, apigenin, (Alfa Aesar), pinocembrin, isorhamnetin, isosakuranetin, vitexin, orientin, rosmarinic acid, myricetin, vanillin, ursolic acid, hydroxytyrosol, tangeretin, chrysoeriol, betulinic acid, eriodictyol, sakuranetin, naringenin, t-cinnamic acid, genistein, diosmetin, resveratrol, galangin, pinocembrin 7-methyl ether, techtochrysin, (Extrasynthese) rutin, isoferulic acid, ellagic acid, kaempferol, quercetin, corosolic acid, acacetin, diosmin, protocatechuic acid ethyl ester, hesperetin, phloridzin, chlorogenic acid, p-coumaric acid, (±)catechin, maslinic acid, (Sigma Aldrich), rhamnetin, syringic acid, protocatechuic acid, ferulic acid, kaempferide, adipic acid, pinostrobin, gallic acid, pinobanksin (Fluka), naringin, hesperidin, (Acros Organics) pinobanksin-3O-acetate (Interchim Inc), cinnamylidenacetic acid, artepillin C (Wako Chemicals). o-Orselllinaldehyde was purchased from Santa Cruz Biotechnology (USA).
Water, acetonitrile, mEthanol and formic acid were purchased from Fisher Scientific, UK and they were of LC-MS grade. Ethanol was purchased from Merck, Germany. PTFE filters (0.45 μm) were obtained from Macherey-Nagel, Germany.