Umbelliferone 7 hydroxycoumarin

Acetonitrile and water were LC-MS grade and obtained from Burdick and Jackson from Honeywell (Muskegon, MI, USA). Methanol was purchased from J. T. Baker (Phillipsburg, NJ, USA). Formic acid was LC/MS grade and obtained from Fisher Scientific (Hampton, NH, USA). Umbelliferone (7-hydroxycoumarin) and raffinose were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Biological Samples: Pecan leaves were collected in triplicate from 3 individual trees per variety from the Samuel Roberts Noble Foundation McMillan pecan orchard (Ardmore, OK, USA) in June 2014 and August 2015. Fresh leaf samples were transported from the field over dry ice and then frozen immediately in liquid nitrogen upon arrival in the laboratory and stored at −80 °C. Additional samples were collected in triplicate or quadruplicate (three or four individual trees) from the University of Missouri Horticulture and Agroforestry Research Center (New Franklin, MO, USA) in September 2016, the University of Missouri Southwest Research Center (Mt. Vernon, MO, USA) in May 2017, and Kansas State University Pecan Experiment Field (Chetopa, KS, USA) in May 2017. Samples were handled in the same manner and stored at −80 °C until use.

The following chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO): protease inhibitor cocktail tablets, α-naphthyl acetate, fast blue B salt, 1-chloro-2,4-dinitrobenzene (CDNB), L-glutathione reduced (GSH), 4-nitrophenyl-α-D-glucopyranoside (pNPG), dopamine hydrochloride, acetylthiocholine iodide (ATC), 5,5`-dithio-bis(2-nitrobenzoic acid) (DTNB), ρ-hydroxybenzhydrazide (PAHBAH), umbelliferone (7-hydroxycoumarin), 7-ethoxycoumarin (7-EC), oxidized glutathione (GSSG), glutathione reductase, β-nicotinamide adenine dinucleotide phosphate (reduced β-NADPH).

Acetylcholine chloride (ACh), papaverine hydrochloride, hexamethonium chloride, N_ω-nitro-L-arginine methyl ester hydrochloride (L-NAME), histamine dihydrochloride, serotonin hydrochloride (5-hydroxytryptamine or 5-HT), pyrilamine maleate, ketanserin (+) tartrate, verapamil hydrochloride, umbelliferone (7-hydroxycoumarin), herniarin (7-methoxycoumarin), and loperamide hydrochloride were obtained from Sigma (ST. Louis, Mo, USA). Calcium chloride (CaCl₂) and potassium chloride (KCl) were bought from J.T. Baker. All chemicals were dissolved or suspended in saline solution (0.9%) and prepared on the day of the experiments. Saline solution was used as vehicle (VEH).