Hip his leu

ACE activity was measured in plasma, heart, aorta, brain, kidney and lung using a
fluorometric method adapted from Friedland and Silverstein.^{29 (link)} Briefly, triplicate tissue and
plasma samples (3 µL) were incubated for 15-90 minutes at 37ºC with 40
µL of assay buffer containing the ACE substrate 5 mM Hip-His-Leu (Sigma).
The reaction was stopped by the addition of 190 µL of 0.35 M HCl. The
generated product, His-Leu, was measured fluorometrically following 10 min of
incubation with 100 µL of 2% o-Phthalaldehyde in methanol. Fluorescence
measurements were taken at 37ºC in a FLUOstar Optima plate reader (BMG Labtech,
Offenburg, Germany) with 350 nm excitation and 520 nm emission filters. The
fluorescence plate reader was controlled using the FLUOstar Optima Software.
Black 96-Well polystyrene microplates (Biogen Cientifica, Madrid, Spain) were
used. A calibration curve with ACE from the rabbit lung (Sigma) was included in
each plate.

Ascorbic acid, xanthine, XO grade I from buttermilk (EC 1.1.3.22), nitro blue tetrazolium (NBT), hydrogen peroxide (H₂O₂), 2-deoxyribose, ferrous sulfate, ACE, and Hip-His-Leu (HHL) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Water, dichloromethane, and methanol were purchased from J. T. Baker (Phillipsburg, NJ, USA). All other reagents were of analytical grade. Garlic was purchased from the Chungbuk Agriculture and Marine Products Market in Korea in June 2007 and was stored at −20°C. Heat treatment was performed using a temperature- and pressure-controlling apparatus (Jisico, Seoul, Korea). The samples were heated at temperatures of 130°C for 2 h. Thiacremonone was isolated according to the Hwang et al. method (13 ). Heated garlic juice was partitioned consecutively in a separating funnel using ethyl acetate. Isolation of thiacremonone from the ethyl acetate layer of heated garlic juice was subjected to column chromatography on silica gel. The fractions included thiacremonone were purified by preparative reverse phase-HPLC (Discovery^® C18 column; 250×10 mm, i.d., 5 μm; Supelco, Bellefonte, PA, USA) on a SP930D solvent delivery pump (Younglin Instrument, Anyang, Korea) equipped with a UV detector, operating at 365 nm, at room temperature, and a flow rate of 3.5 mL/min. The pure thiacremonone was obtained after evaporating the solvents using a rotary evaporator.