TAA was quantified according to Arnao, Cano, and Acosta
(2001), which enables the determination of TAA due to both hydrophilic
and lipophilic compounds in the same extraction. Briefly, for
each sub-sample, 5 g of tissue were homogenised in 5 ml of 50 mM
phosphate buffer pH = 7.8 and 3 ml of ethyl acetate, and then centrifuged
at 10,000 g for 15 min at 4 C. The upper fraction was used
for total antioxidant activity due to lipophilic compounds (L-TAA)
and the lower for total antioxidant activity due to hydrophilic compounds
(H-TAA). In both cases, TAA was determined using the
enzymatic system composed of the chromophore 2,2´ -azino-bis-
(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS),
the horse radish peroxidase enzyme (HRP) and its oxidant substrate
(hydrogen peroxide), in which ABTS+ radicals are generated
and monitored at 730 nm. The decrease in absorbance after adding
the pomegranate extract was proportional to the TAA of the sample.
A calibration curve was performed with Trolox ((R)-(+)-6-hydroxy-
2,5,7,8-tetramethyl-croman-2-carboxylic acid) (0–20 nmol)
from Sigma (Madrid, Spain), and results are expressed as mg of
Trolox equivalent 100 g1. All reagents were purchased from Sigma,
Sigma–Aldrich, Madrid, Spain. A relative calibration procedure
was performed using Trolox at 1–5 lg in the reaction medium,
which showed linearity with the absorbance at 730 nm (y =
0.138 +0.033; R2 = 0.999; for H-TAA, and y = 0.093 +0.068;
R2 = 0.998; for L-TAA).
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