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Comm. Appl. Biol. Sci, Ghent University, 75/4, 2010
1
THE ANTIOXIDANT CAPACITY OF LEEK (
ALLIUM AMPE-
LOPRASUM
VAR.
PORRUM
)
N. BERNAERT1,2, B. VAN DROOGENBROECK2, C. BOUTEN3, D. DE PAEPE2, E.
VAN BOCKSTAELE1,2, H. DE CLERCQ2, D. STEWART4, M. DE LOOSE2,5
1Department of Plant Production, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
2Research group Product quality and innovation, Unit Technology and Food (T&V), Institute for
Agricultural and Fisheries Research (ILVO), Burg. Van Gansberghelaan 115, 9820 Merelbeke,
Belgium
3 Department of Biosciences and Landscape Architecture, Ghent University College, Voskenslaan
270, 9000 Ghent, Belgium
4Plant Products and Food Quality Programme, Scottish Crop Research Institute (SCRI), Inver-
gowrie, Dundee, Scotland
5Department of Plant Biotechnology and Genetics, Ghent University, 9000 Ghent, Belgium
INTRODUCTION
Leek (Allium ampeloprasum var. porrum) is a worldwide grown vegetable,
although its importance is most significant in the temperate zone of Europe
(De Clercq et al., 2003). The largest areas of leek cultivation can be found in
West European countries where it is cultivated on about 30,000 ha (Declercq
et al., 2010). Plants of the genus Allium have been recognized as rich sources
of secondary metabolites. They contain high concentrations of organic sul-
phur compounds, which are considered to be an important factor responsi-
ble for the health promoting and related biological effects of alliums but also
for their characteristic pungent aroma and taste (Lanzotti, 2006). Also phe-
nolic compounds, such as flavonoid glycosides, which are associated with
health benefits, are found in leek (Fattorusso et al., 2001). Next to these
compounds, also lutein, β-carotene and vitamin C are quantified in this veg-
etable (Hart & Scott, 1995; Proteggente et al., 2002).
The measurement of the antioxidant capacity of food products is a matter of
growing interest because it may provide a variety of information, such as
resistance to oxidation, quantitative contribution of antioxidant substances,
or the antioxidant activity that they may present inside the organism when
ingested (Zulueta et al., 2009). In this study, the antioxidant capacity is de-
termined, using the Oxygen Radical Absorbance Capacity (ORAC) assay,
developed initially by Cao et al. (1993) and the Ferric Reducing Antioxidant
Power (FRAP) assay.
MATERIAL AND METHODS
Plant material
Thirty one leek cultivars were included in the first growing season (hybrids,
open pollinated cultivars, breeder selections and old cultivars), which were
sown in triplicate in April 2009 in a greenhouse. In June 2009, three repeti-
tions of 15 plants of each cultivar were planted in a randomized block design
2
in the field. In the optimal harvest period (from September until March), they
were harvest manually according to their type. After harvest, they were
transported in water to the lab and immediately cut into small pieces, green
and white part separately. They were stored temporary in a freezer of -80°C
before the freeze-drying process. The freeze-dried samples were milled into a
powder and stored in tubes at 4°C until the time of analysis. The samples for
FRAP analyses were send to the Scottish Crop Research Institute.
ORAC assay
The ORAC assay is based on the oxidation of fluorescein by peroxyl radicals
(ROO°). Free radicals are generated by the water soluble compound 2,2’-
azobis-2-methyl-propanimidamide (AAPH). The loss of fluorescence of fluo-
rescein is an indication of the extent of damage from its reaction with the
peroxyl radical. In the presence of antioxidants, ROO° abstracts a hydrogen
atom from the antioxidant to form hydroperoxide (ROOH) and a stable anti-
oxidant radical (ArO°); as a result, the damage to fluorescein induced by
peroxyl radical is inhibited. The protective effect of an antioxidant is meas-
ured by assessing the area under the fluorescence decay curve (AUC) of the
sample compared to a standard curve obtained using various concentrations
of the water soluble vitamin E analog Trolox. The ORAC procedure estab-
lished by Prior et al. (2003) and the application note of Ganske & Dell
(BMGlabtech) was followed. In every working well 25 μL Trolox dilution,
sample dilution or phosphate buffer was pipetted in triplicate for the calibra-
tion curve, sample or blank respectively. 150 µL fluorescein solution was
added to each well. The microplate was sealed followed by an incubation for
30 min at 37°C in the FLUOstar OPTIMA. After incubation, fluorescence
measurements (Ex. 485 nm, Em. 520 nm) were taken every 90 sec to deter-
mine the background signal. After 3 cycles, 25 μl of AAPH was injected
manually. The test was resumed and fluorescent measurements were taken
up to 90 minutes. Calculations were based on the area under the fluores-
cence decay curve. ORAC values were expressed in µmol Trolox equivalents
per gram of dry weight (µmol TE/g DW).
FRAP assay
The FRAP assay is based on the oxidation of antioxidants by oxidants. As a
result, a single electron is transferred from the antioxidant molecule to the
oxidant. The change of absorbance is measured by an ultraviolet-visible
spectrometer and the absorbance value is used as the quantitation for the
reducing capability of the antioxidant. The FRAP analysis was based on
Deighton et al. (2000). FRAP reagent was freshly prepared to comprise 1 mM
2,4,6-tri(2-pyridyl)-1,3,5- triazine and 2 mM ferric chloride in 0.25 M sodium
acetate, pH 3,6. A 50 µl aliquot of the leek extract and 50 µl water was added
to 900 µl of FRAP reagent and mixed. After incubation at ambient tempera-
ture for 4 min, absorbance at 593 nm was determined against a water blank.
Calibration was carried out using a 100-1000 µM ferrous ion standard curve
produced by addition of freshly prepared ammonium ferrous sulphate. FRAP
Comm. Appl. Biol. Sci, Ghent University, 75/4, 2010
3
0
10000
20000
30000
40000
50000
60000
70000
01000 2000 3000 4000 5000 6000
time (s)
Units
values obtained from leek are presented as micromolar ferrous ion (ferric
reducing power) of a 100% extract.
RESULTS AND DISCUSSION
The green part of leek showed a higher
AUC in the ORAC assay, which results
in a higher antioxidant activity in com-
parison with the white part (Figure 1).
The antioxidant capacities of the white
stem of the leek cultivars to the peroxyl
radicals ranged from 26.91 to
88.07 µmol TE g-1 DW (ORAC assay).
The antioxidant reducing potential on
ferric ion of the white stem of the leek
cultivars on the other hand ranged from
154.14 to 898.04 µM ferric reducing
antioxidant power (FRAP). Figure 2
shows the ORAC and FRAP results of
the green leaves of the 31 cultivars.
Figure 2. The antioxidant activity of 31 leek cultivars expressed as µmol TE.g-1 DW (■)
and µM ferric reducing antioxidant power (□)
Figure 1. The fluorescent decay in the ORAC assay
for the white (■) and green part (о) of leek
4
The green part of leek shows a significant higher antioxidant capacity in
comparison with the white part. Phenolic compounds will be partly respon-
sible for this difference, because their biosynthesis requires the presence of
light.
The data collected with the ORAC and FRAP assay for the white part of leek,
shows a good correlation. This is however not the case for the green leaves
(Figure 3).
(a) (b)
Figure 3. The correlation between the ORAC and FRAP assay for the green leaves (a), R²= 0.01 and
the white shaft (b), R²=0.67
CONCLUSION
The determination of the antioxidant activity can give a general overview of
the strength and amount of antioxidants present. In a later phase its more
interesting to analyze specific antioxidants which are responsible for the
antioxidant capacity in the different plant parts sampled.
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