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P. Kerchev1 and S. Ivanov2
Bulgarian Academy of Sciences, Institute of Plant Physiology, Sofia, Bulgaria1
Centre of Food Biology, Sofia, Bulgaria2
Correspondence to: Sergei Ivanov
E-mail: sivanov714@abv.bg

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

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.

The purpose of this work is to determine how different

extraction mediums and sample preparation affect the TAC of
plant material. In addition to the one and two steps procedures
used by other authors, we developed a three-step method
for sample preparation. The new approach shows highest
values for TAC in comparison to the standard procedures.
Moreover, our method discriminates between water-soluble,
water-insoluble lipophilic and protein antioxidants within one
sample. For TAC evaluation two widespread assays, TEAC
and FRAP were used.

Materials and Methods

In the experiments, pea plants (Pisim sativum, L.) cv. Pleven
4 were used. Seeds were soaked in tap water for 6 hours,
and germinated for 3 days on a moisturized filter paper in a
germination chamber at 25C. Plants were grown hydroponically
on a half-strength Hoaglands solution with trace microelements
in a growth chamber (photoperiod 12/12h, photon flux density
70-90 mol.s-1.m-2, temperature 25C, relative air humidity 6070%). All samples were collected from second leaf stage.
A scheme describing the procedure for sample preparation
is presented in Fig. 1. The plant material for homogenizations
was gathered at the same time from a random sample. The
0.5 g leaves were ground with a pestle and mortar in sodium
acetate buffer (0.1 M, pH 5), potassium phosphate buffer
(0.1 M, pH 7.5), methanol or 0.1% (w/v) trichloroacetic acid,
respectively. The homogenate were centrifuged at 15 000 g for
30 min and the resulting supernatants (crude fraction) were
analyzed for TAC, as reported below (one-step procedure).
In the two-step procedure all pellets, except that from the
methanol slurry, were extracted with 3 mL of acetone under

agitation for 30 min at room temperature, centrifuged at

15 000 g for 15 min and the supernatants collected for analysis
(acetone fraction). For the three-step procedure the initial
supernatants from phosphate and acetate buffers were further
processed by adding 60% TCA to a final concentration of 1%.
After centrifugation at 12 000 g for 15 min in the supernatants
TAC (low-molecular fraction) was evaluated. The protein
pellets were resuspended in a detergent solution (6 M Urea,
0.1 M acetate buffer, pH 5) and used in the analyses (protein
fraction). The presented results are means + SE (standard error)
from three independent experiments.
Plant material ~ 0.5 g
Centrifugation, 15 000g for 30 min.

TAC of
crude extract (1)


Saturation with TCA
Centrifugation, 12 000g
for 15 min.


Extraction with acetone

for 30 min at room temp.

Results and Discussion

Centrifugation, 15 000g
for 15 min.
TAC of water-insoluble
lypophilic fraction (2)


Calculation of TAC:

TAC of
low-molecular fraction (3)

TAC of
protein fraction (4)

of the results. The results were calculated by standard curves

prepared with known concentrations of Trolox, and were
expressed as mol Trolox/g FW.
The FRAP assay is based on the reduction of the Fe3+
TPTZ complex to the ferrous form at low pH (4). The working
solution is prepared daily according to a procedure described
by the authors of the method. Freshly prepared FRAP reagent
(0.2 mL) was added to 20 L of the diluted sample on a 96-well
micro plate and the absorbance at 593 nm was recorded after a
30 min incubation at 37C. A blank probe without plant extract
is used for correction of the results. The results were calculated
by standard curves prepared with known concentrations of
Fe2+, and were expressed as mol Fe2+/g FW.
All spectrophotometrical measurements were made with
microplate instrument (Multiscan Spectrum, Thermo Electron

One-step TAC= (1)

Two-step TAC= (1) + (2)
Three-step TAC= (2) + (3) + (4)

Fig. 1. Outline of procedure for sample preparation for detailed protocols see
Materials and Methods)

The TEAC assay is based on the ability of antioxidant

molecules to quench the long-lived ABTS+ radical, a bluegreen chromophore with characteristic absorption at 734 nm,
compared with that of Trolox, a water-soluble vitamin E analog.
A stable stock solution of ABTS+ was produced by reacting
a 7 mM aqueous solution of ABTS with 2.45 mM potassium
persulfate (final concentration) and allowing the mixture to
stand at dark at room temperature for 12-16 h before use (22).
The plant sample (20 l) is added to 200 l of the reagent in a
96-well micro plate. The absorption is read after 30 min at 734
nm. A blank sample without plant extract is used for correction

Both TEAC and FRAP methods were used for determination of

TAC in the crude extracts according to the one-step procedure.
The values obtained by the TEAC assay varied from 10.41
mol Trolox equivalents/g FW for the probe homogenized
with methanol to 15.81 for the probe homogenized with 0.1%
TCA (Table 1). The result concerning the usage of phosphate
buffer was not taken into consideration. Analogically, when the
FRAP method was used the TAC of the sample homogenized
with phosphate buffer was very low 5.45 mol Fe2+ equivale
nts/g FW (Table 2). The antioxidant activities of the remaining
samples were in the same range (acetate buffer 17.95; 0.1%
TCA 18.43; methanol 19.58 mol Fe2+/g FW).
Water-insoluble lipophilic antioxidants present in the
acetone extract from the initial pellet, which consisted of
cell walls, broken nucleus, chloroplasts, and mitochondrion
possess less activity than the crude extract. The values ranged
from 6.94 (0.1% TCA) to 8.06 mol Trolox/g FW (acetate
buffer) (Table 1). The data obtained by the FRAP assay
were similar. The highest result was obtained for the probes
initially homogenized with acetate buffer 13.87 mol Fe2+/

TAC according to the TEAC assay

Crude extract
mol Trol.
a 10.660.42
1 b 10.660.42
a n.d.
2 b 29.8
a 15.810.22
b 15.810.22
4 a 10.410.05

Acetone extract
mol Trol.

Low-molecular fraction
mol Trol.

Protein fraction
mol Trol.

mol Trol.


n.d. not detected

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 Trolox g-1 FW SE (n= 6), and respective % from total TAC (at the right side of each value).
For details see materials and methods.



g FW (Table 2). The share of the water-insoluble lipophilic

antioxidants, calculated as a percent of the total antioxidant
activity was estimated and is also present in the tables. When
the two-step procedure was used their part was about 44%
for the acetate buffer, 30% for TCA and up to 70% after
homogenization of the leaf material with phosphate buffer and
consequential evaluation with the FRAP assay (Table 2). If
three-step procedure is used the percentages of water-insoluble
lipophilic antioxidants are 44.7% and 32.5% (FRAP, TEAC
acetate buffer, Tables 1, 2), and 63.1% and 32.4% (FRAP
phosphate buffer, Table 2).
After the addition of TCA to the crude extracts obtained
by homogenization with phosphate and acetate buffers the
proteins present in the sample precipitate. This step allows
each probe to be divided into three fractions - low-molecular
fraction, acetone extract from the initial pellet and protein
fraction (three-step procedure). The TAC of the low-molecular
fraction according to the TEAC assay is similar regardless
of the initial homogenization technique (Table 1). When
evaluated with the FRAP assay the probes homogenized with
sodium acetate buffer had substantially higher values (Table
2). The protein fraction showed different antioxidant capacity
depending on the method for TAC estimation. According to the
TEAC (Table 1) and FRAP (Table 2) assays their contribution
to the TAC of the sample is 42.2% (TEAC acetate buffer),
37.8% (TEAC phosphate buffer), 4.8% (FRAP acetate
buffer), and 12% (FRAP phosphate buffer), respectively.
The low values for the FRAP method are probably due to the
precipitation of proteins at low pH (5).
The one-step procedure for sample preparation due to its
simplicity is used by many authors. Usually, the material is
homogenized with water (2, 13, 14, 27), water/acetone (19, 30),
pure methanol (20), water/ethanol (or ethyl acetate, diethyl ether,
chloroform) in different ratios (7, 10, 25, 26), acid (16), neutral or
alkaline buffers (11, 28). With the exception of TCA, the solvents
applied in our study are widely used for evaluation of TAC. The
results indicate that if one-step procedure is used the highest
result is obtained for the probes homogenized with TCA (TEAC
Table 1) and with methanol (FRAP Table 2). However, with

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).

TAC according to the FRAP assay

Crude extract
mol Fe2+
a 17.950.24
1 b 17.950.24
a 5.450.10
2 b 5.450.10
a 18.430.34
b 18.430.34
4 a 19.580.40

Acetone extract
mol Fe2+

Low-molecular fraction
mol Fe2+

Protein fraction
mol Fe2+

mol Fe2+

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.



The protein fraction included proteins in pellet, protein-bound

phenolic compounds, and various high-molecular biopolymers.
The primary antioxidants in the protein fraction are the reactive
thiol groups of the cysteine residues and some membranebound molecules. Interestingly, when evaluated according to
the TEAC assay, the protein fraction contributed 42.2% (for
acetate buffer) to the total antioxidant capacity (Table 1),
whereas this percent was significantly lower (4.8% for same
buffer) when the fraction was assessed for its reducing (FRAP)
capacity (Table 2). This is due to the fact that the FRAP assay
does not measure thiol antioxidants (23).
As a whole, the changes in the total antioxidant capacity
of plant products could be evaluated more accurately when
the probes are fractionated into 3 fractions. This approach is
especially useful for studying the effects of aging, processing
and storage on the antioxidant capacity. For example, during
storage of potato tubers (6) were observed a decrease in the
levels of the water-soluble ascorbate, whereas the contents of
the lypophilic tocopherol remained virtually unchanged (15).
The degree of degradation of the water-soluble and waterinsoluble lypophilic antioxidants in plant products is variable
and most probably depends substantially on the severity and
duration of processing, packaging material, storage conditions,
etc. Moreover, the initial levels of antioxidant compounds
vary significantly among species. The effects of geographical
locations, cultivar, and ripening should also be kept in mind
(10, 18). All this sets the necessity for a complex approach in
sample preparation prior to measuring the antioxidant capacity
of plant material.

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.

This study is partly supported by projects CC1413 and MU-B1505 (Ministry of Education and Science of Bulgaria).

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