Chlorogenic acid

From Sigma-Aldrich (St. Louis, MO, USA): Amberlite XAD7^®, α-amylase from porcine pancreas (A3176; EC 3.2.1.1), α-glucosidase from Saccharomyces cerevisiae (G5003; EC 3.2.1.20), DPPH (2,2-diphenyl-1-picrylhydrazyl radical), 3,5-dinitrosalicylic acid, catechin, CuCl₂, lipase from porcine pancreas type II (L-3126; EC 3.1.1.3), 4-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl palmitate, quercetin, sodium acetate, starch, TPTZ (2,4,6-tri(2-pyridyl)1,3,5-triazine), and triton X-100. From Merck (Darmstadt, Germany): ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), FeCl₃ × 6H₂O, neocuproin, potassium sodium tartrate, and HPLC-grade methanol. Ammonium acetate was from JT Baker (Xalostoc, Mexico). The following standards were from PhytoLab (Vestenbergsgreuth, Germany): malvidin-3-glucoside chloride (89728, 99.3% purity), quercetin-3-glucoside (89230, 99.1% purity), and chlorogenic acid (89175, 98.9% purity). Orlistat was from Laboratorio Chile (Santiago, Chile).

Extraction and chromatography solvents, ethanol (EtOH), methanol (MeOH), and acetic acid (AcOH) were HPLC certified. The polyphenol standards used in this study, such as chlorogenic acid, 3-O-caffeoylquinic acid, 1,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid, were purchased from PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany). N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), lipopolysaccharide (LPS), α-tochopherol and hydrogen peroxide solution (H₂O₂) were purchased from Sigma Aldrich Co. (Milano, Italy). Human TGF-β recombinant protein, 4,4-difluoro-3a,4a-diaza-s-indacene (BODIPY), dextran fluorescein 3000 MW and the Griess reagent [N-(1-naphthyl)ethylenediamine and sulfanilic acid] were purchased from Invitrogen-Life Technologies (Carlsbad, CA, USA).

Formic acid (>98% purity), acetonitrile (LC-MS grade), methanol, rutin, quercetin, apigenin and kaempferol were purchased from Merck (Darmstadt, Germany). apigenin 7-O-glucoside, quercetin 3-O-glucoside, kaempferol 3-O-rutinoside and luteolin were purchased from Extrasynthese (Genay Cedex, France). Cynarin, chlorogenic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, luteolin 7-O-glucuronide, apigenin, 7-O-glucoside, luteolin 7-O-glucoside and diosmetin were purchased from Phytolab (Vestenbergsgreuth, Germany). Bond Elut Jr 500 mg SPE-C18 cartridge was from Agilent (Santa Clara, California, USA). A Milli-Q purification system (Millipore, Bedford, MA, USA) was used to obtain the deionised water (18.2 MΩ cm). All chemicals used for biological assays were obtained as pure commercial products from Sigma Chemical Co (St. Louis, USA) and used without further purification. Spectrophotometric determinations were obtained with an Ultrospec 2100 spectrophotometer (Biochrom Ltd, Cambridge, England) using a 1 cm length path cell.

Fennel (Anethum foeniculum L.) seeds were obtained from Baker Creek Heirloom Seed Company. All the fennel plants including the green variety (Florence) and purple variety (Bronze) were grown in a greenhouse with a 16-h photoperiod at 25°C. Besides, tomato (Solanum lycopersicon Mill.) and tobacco plants (Nicotiana benthamiana) were also grown in the same conditions. The edible tender leaves were carefully sampled at 60 days after sowing seeds. Anthocyanin standards including pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside and other phenolic compounds (quercetin-3-O-rutinoside, kaempferol-3-O-glucoside and chlorogenic acid) were purchased from PhytoLab (Germany). The other solvents and reagents were purchased from MERCK (Germany) and Sigma-Aldrich (St. Louis, MO).

Formic acid (LC-MS grade) was obtained from Merck KGaA (Darmstadt, Germany). Acetonitrile (LC-MS grade) and methanol (LC-MS grade) were purchased from VWR International Srl (Milan, Italy). Liquid nitrogen was purchased from Rivoira (Milan, Italy). Sodium fluoride, phosphoric acid, glacial acetic acid, TPTZ, ABTS, iron (III) chloride hexahydrate, Trolox, potassium chloride, (+)-catechin, sodium carbonate, F-C reagent, quercetin-3-xyloside, 3,4-dihydroxybenzoic acid, naringenin-7-glucoside, and phloridzin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Quercetin-3-rutinoside and potassium peroxydisulfate were purchased from Roth (Karlsruhe, Germany). Quercetin-3-glucoside, neochlorogenic acid, cryptochlorogenic acid, cyanidin-3-galactoside chloride, and malvidin-3-glucoside chloride were purchased from Extrasynthese (Genay, France).
(−)-Epicatechin, procyanidin B1, procyanidin B2, procyanidin C1, chlorogenic acid, cyanidin-3-glucoside chloride, cyanidin-3-arabinoside chloride, phloretin, quercetin-3-arabinoside, quercetin-3-galactoside, quercetin-4’-glucoside, and quercetin-3-rhamnoside were purchased from Phytolab (Vestenbergsgreuth, Germany). Sodium acetate anhydrous was purchased from CHEMSOLUTE^® (Renningen, Germany). Hydrochloric acid from Fisher Chemical (Pittsburgh, UK). Deionized water was from MilliQ apparatus (Millipore Corp., Bedford, MA, USA).

Chromatography was performed on silica gel F254 HPTLC pre-coated plates using as mobile phase ethyl acetate–formic acid–glacial acetic acid–water (100:1:11:26) for identification of polyphenols [10 ] and ethyl acetate–methanol–water (81:11:8) for ecdysteroids [10 ]. Samples were applied on the plates as band of 7 mm width using a Camag Linomat V sample applicator at the distance of 14 mm from the edge of the plates. The fingerprints were evaluated in visible mode with a WinCats software after derivatization with NP-PEG for polyphenols or after drying the plate at 110 °C for 10 min, for ecdysone visualization. Reference compounds for HPTLC analysis were purchased from Sigma-Aldrich, Germany (rutin, chlorogenic acid, hyperoside) and from PhytoLab, Baden-Wurttemberg, Germany (β-ecdysone).