DPPH• Free-Radical Scavenging Assay. The antioxidant
activity of Psidium was measured in terms of radical scavenging
ability, according to the DPPH• method (16). The parameter
EC50, which reflects 50% depletion of DPPH• free-radical,
was expressed in terms of grams of Psidium equivalent per
gram of DPPH• in the reaction medium. The time taken to
reach the steady state at EC50 (TEC50) was calculated. The antiradical
efficiency (AE), a parameter defined by Sa´nchezMoreno
et al. (16) which combines both factors, was also
calculated in order to recognize both effects of the abovementioned
parameters: AE ) 1/EC50TEC50.
FRAP Assay. The antioxidant capacity of each sample was
estimated according to the procedure described by Benzie et
al. (17) with some modifications introduced in our laboratory.
The readings at 30 min were selected for calculation of FRAP
values (18). Methanolic solutions of known Trolox concentrations
were used for calibration.
In vitro Copper-Induced Oxidation of Human Low-Density
Lipoprotein Assay. Low-density lipoprotein (LDL) was obtained
from Ramo´n y Cajal Hospital, Madrid, Spain. The plasma was
collected from a patient with homozygous familial hypercholesterolemia.
LDL was isolated following the procedure of Tani
(19) as described in detail elsewhere (20).
In vitro LDL oxidation was performed by the procedure of
Esterbauer et al. (21), as modified by Sa´nchez-Moreno et al.
(20) to avoid the influence of LDL status. In this system, LDL
hydroperoxidation was estimated on the basis of formation of
conjugated dienes by measuring the change in absorption at
234 nm. The parameter CLT50 was calculated to measure the
concentration of antioxidant that increases the lag time to 50%
greater than that of the control.
Statistical Analysis. Results are expressed as mean values
( standard deviation. Comparison of means of three measurements,
using a significance level of p < 0.05, was performed
by one-way analysis of variance (ANOVA) using the Statgraphics
Computer System, version 5.1.