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ABSTRACT
The influence of extraction medium and sample preparation on the total antioxidant capacity (TAC) of pea plants leaves is
investigated. Sodium acetate buffer (pH 5), potassium phosphate buffer (pH 7.5), methanol or 0.1% w/v trichloroacetic acid
(TCA) were used for homogenization. TAC was measured in the crude supernatants (one-step procedure). The pellets were
extracted with acetone and their TAC values were added to those from the previous assay (two-step procedure). The values
for TAC of the acetone extract, the low-molecular and protein fractions derived from a partial fractionation of the probes are
used in the proposed by us new three-step method. The results indicate that TAC of the probes processed with the two or threestep procedure is significantly higher than that of the crude supernatants (one-step procedure). On the basis of this study we
recommend two-step protocol as a minimum and buffer with slightly acid pH or TCA for extraction when plant material is
investigated. The highest results were obtained when the new three-step procedure for sample preparation and sodium acetate
buffer as an extracting agent were applied. Moreover, the separation of the cellular antioxidants into three different fractions is
especially useful for studying the effects of aging, processing and storage on the antioxidant capacity.
Keywords: antioxidant capacity, pea plants, TEAC, FRAP,
sample preparation
Introduction
The antioxidant system in plants is very complex, with
antioxidants having different targets, sizes and interactions
with each other. Halliwell (8) defined biological antioxidants
as molecules which, when present in small concentrations
compared to the biomolecules they are supposed to protect,
can prevent or reduce the extent of oxidative destruction of
biomolecules. They can be divided into enzymatic (e.g
superoxide dismutase, catalase, glutathione peroxidase) and
nonenzymatic (e.g. glutathione, vitamin E, ascorbic acid).
Total Antioxidant Capacity (TAC) is a parameter that takes into
account all the synergistic and cumulative interactions between
the known and unknown antioxidants present in the sample.
Several methods have been developed to measure the TAC of
biological samples including plants. Some of the most used
are TRAP (Total Radical-Trapping Antioxidant Parameter),
TEAC (Trolox Equivalent Antioxidant Capacity), ORAC
(Oxygen-Radical Absorbance Capacity), FRAP (Ferric/
Reducing Antioxidant Power) (24). Sample preparation and
extraction solvents are vital (3) but the information on how it
affects the subsequent measurements is insufficient. Different
authors used various protocols for extraction of antioxidants.
Usually, water-soluble and water-insoluble fractions were
assessed separately (two-step procedure). For extraction of
water-soluble antioxidants water or different buffers were used.
Water-insoluble antioxidants were extracted with acetone or
chloroform. Numerous authors used solely methanol (one-step
procedure) as an extracting agent.
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TAC of
crude extract (1)
Supernatant
Pellet
Saturation with TCA
Centrifugation, 12 000g
for 15 min.
Supernatant
Centrifugation, 15 000g
for 15 min.
TAC of water-insoluble
lypophilic fraction (2)
Pellet
Calculation of TAC:
TAC of
low-molecular fraction (3)
TAC of
protein fraction (4)
Fig. 1. Outline of procedure for sample preparation for detailed protocols see
Materials and Methods)
Acetone extract
%
8.060.78
43.1
8.060.78
32.5
7.580.485
32.4
7.580.485
32.4
6.940.50
30.5
mol Trol.
Low-molecular fraction
%
6.280.51
25.3
6.980.53
29.8
mol Trol.
Protein fraction
%
10.470.08
42.2
8.840.38
37.8
mol Trol.
Total
mol Trol.
10.66
18.72
24.81
n.d.
7.58
23.38
15.81
22.75
10.41
557
the exception of the extract with phosphate buffer, data for TAC
according to the FRAP assay were very similar.
The two-step procedure for sample preparation allows the
antioxidant capacity of both water-soluble and water-insoluble
lipophilic antioxidants to be measured. Normally, the probe is
initially homogenized with water or buffer and afterwards the
insoluble pellet is extracted with an organic solvent. Acetone
and chloroform are usually used in this step. Numerous studies
of the TAC in foods and beverages are carried out with the twostep procedure (16, 18, 21). In our work the data from the twostep protocol were 1.5 to 3-fold higher than those of the crude
extract for both TEAC and FRAP assay (Tables 1, 2). The
TEAC values obtained with TCA as an extracting agent were
the highest (Table 1). In the case of the FRAP assay the highest
results were observed with sodium acetate buffer (Table 2).
The low-molecular antioxidants, the water-insoluble
lypophilic antioxidants, and the scavenging activity of the
proteins against free radicals can be assessed with the three
step procedure. The highest values for TAC were obtained by
both analytical methods when the three-step procedure after
homogenization with sodium acetate buffer was applied.
Moreover, the contribution of the antioxidant compounds
present in the three fractions to the total antioxidant capacity
can be outlined. The principal antioxidants related to the lowmolecular fraction included glutathione, ascorbate, free proline
and various phenolic compounds. The tripeptide glutathione
is the main non-protein, acid-soluble thiol compound in most
organisms (1). Analogically, vitamin C is a water-soluble
antioxidant that has a high reducing potential and is a powerful
scavenger of oxygen radicals and quencher of singlet O2.
Ascorbate co-operates with glutathione in glutathione-ascorbate
shuttle the main mechanism of detoxifying the AOS in plants
(17, 29). Carotenoids and Vitamin E are easily extracted from
the pellet with acetone. Vitamin E is a generic term for a series
of natural tocopherols and tocotrienols (, , and d) also
called tocols, and it is regarded as the most important lipidsoluble antioxidant (9). The major role of carotenoids in plants
is light harvesting as auxiliary components and quenching of
excited states that might be formed during photosynthesis (12).
TABLE 2
Acetone extract
mol Fe2+
%
13.870.61
43.6
13.870.61
44.8
12.860.04
70.2
12.860.04
63.1
8.580.47
32.8
-
Low-molecular fraction
mol Fe2+
%
15.660.32
50.4
5.070.04
24.9
-
Protein fraction
mol Fe2+
%
1.500.01
4.8
2.460.03
12.0
-
Total
mol Fe2+
17.95
31.87
31.03
5.45
18.31
20.39
18.43
27.01
19.58
Measurements are made after homogenization with (1) sodium acetate buffer with pH 5, (2) potassium phosphate buffer with pH 7.5, (3) 0.1% TCA and (4)
methanol. As Total is represented the capacity of the sample calculated as a sum of the different fractions according to: a one-step procedure, b two-step
procedure, c three-step procedure. Data are presented as mol Fe2+ g-1 FW SE (n= 6), and respective % from total TAC (at the right side of each value). For
details see materials and methods.
558
Conclusions
Generally, based on our results we recommend the newest
three-step procedure and homogenization of the samples with
moderate acid pH buffer, e.g. acetate buffer pH 5, as the most
reliable way of evaluating the TAC of plant material. The twostep procedure can be used as an alternative. Most appropriate
solvents for the initial extraction are TCA or acetate buffer. The
water-insoluble lypophilic antioxidants are easily extracted
with acetone. The two-step procedure is simple to perform and
is suitable for large-scale investigations. Finally, the one-step
procedures for sample preparation regardless of the extraction
solvent are unsatisfactory.
Acknowledgements
This study is partly supported by projects CC1413 and MU-B1505 (Ministry of Education and Science of Bulgaria).
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BIOTECHNOL. & BIOTECHNOL. EQ. 22/2008/1