Agriculture Reference
In-Depth Information
with 40 mL of extraction solution consisting of methanol and distilled water (80% v/v) for 2 h
in the dark, at room temperature. The mixture was centrifuged in two sequential times for 15
min at 3500 rpm, and supernatant was filtered through a 0.45 µm Minisart filter prior to
analysis.
Determination of Individual Phenolic Compounds
Samples were prepared according to the method of Hertog et al. (1992) and analyzed
using an Agilent 1260 series HPLC (Agilent Technologies, Santa Clara, CA, USA) linked to
a ChemStation data handling system, using a ZORBAX Eclipse Plus C18 column (4.6 х 150
mm, 3.5 µm particles).
The injection volume was 5 µL and the temperature was set at 30 ºC. Solvent A was 1%
formic acid and solvent B was acetonitrile. The gradient used was as follows: 0─10 min, 10%
of B in A; 10─25 min, 15─50% of B in A; 25─30 min, 50-80% of B in A; 30─32 min, 10%
of B in A. By using this gradient (flow rate 0.5 ml/min), a good level of purity and separation
was achieved in the fruit samples. The HPLC equipment was used with a diode array detector
(DAD).
Phenolic compounds were detected at 260 nm (protocatechuic acid, ellagic acid), 280 nm
(gallic acid,
p
-coumaric acid), 360 nm (kaempferol), and 520 nm (cyanidin-3-glucoside,
pelargonidin-3-glucoside). Phenolic compounds were identified according to the peak
retention time (RT) and UV/Vis spectra by comparing them with those of the standards. The
quantities of the different phenolic compounds were based on peak areas, and expressed as
mg/100 g FW.
Determination of Total Phenolic Content (TPC)
The total phenolic content was determined using a modified Folin-Ciocalteu colorimetric
method (Liu et al., 2002), with results expressed as milligrams of gallic acid equivalents
(GAE) /100 g fresh weight. Shortly, 40 μL of fruit extracts or gallic acid standard solution
was mixed with 3.16 mL of distilled water. In the next phase, 200 μL of Folin-Ciocalteu
reagent was added and allowed to stand for 8 minutes before adding 600 μL of 20% Na
2
CO
3
solution. The solution was well mixed and absorbance at 765 nm against an appropriate blank
was determined after 2 hours.
Determination of the Total Antioxidant Capacity (TAC)
Antioxidant capacity was determined using the DPPH method reported by Brand-
Williams et al. (1995) with modifications (Sanchez-Moreno et al., 1998). An aliquot of 0.1
mL of the fruit phenolic extraction was added to 3.9 mL of DPPH solution in methanol (0.060
mM) and vortexed.
A control sample, containing the same volume of solvent in the place of the extraction,
was used to measure the maximum DPPH absorbance. After the reaction was allowed to take
place in the dark for 30 minutes, the absorbance at 515 nm was recorded to determine the
Search WWH ::
Custom Search