ISSN 1021-4437, Russian Journal of Plant Physiology, 2007, Vol. 54, No. 4, pp. 553–558. © Pleiades Publishing, Ltd., 2007.
Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 623–628.
METHODS
Analysis of Salicylic Acid in Willow Barks and Branches
by an Electrochemical Method*
J. Petreka, L. Havela, J. Petrlovab, V. Adamb, c, D. Potesilb, c, P. Babulad, and R. Kizekb
a Department
of Plant Biology, Faculty of Agronomy, Mendel University of Agriculture and Forestry,
Zemedelska 1, CZ-613 00 Brno, Czech Republic
b Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University of Agriculture and Forestry,
Zemedelska 1, CZ-613 00 Brno, Czech Republic;
fax: +420-5-4521-1128; e-mail: [email protected]
c Department of Analytical Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
d Department of Natural Drugs, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic
Received February 20, 2006
Abstract—An electrochemical method for measuring free salicylic acid (SA) was optimized and used to detect
its content in barks and branches of thirteen Salix species. We utilized square wave voltammetry method in combination with pencil lead, the detection limit of which was 1.7 ng/ml of salicylic acid. The highest contents of
free SA were observed in the bark of S. laponum (3.0 mg/g fr wt) and in the branches of S. purpurea, cv. Nana
(2.1 mg/g fr wt) and S. planifolia (2.2 mg/g fr wt). The technique utilized for determination of SA in willow
tissues has a much broader dynamic range and lower limit of detection in comparison to both linear sweep and
cyclic voltammetry because of its efficient discrimination of capacitance current.
DOI: 10.1134/S1021443707040188
Key words: Salix - salicylic acid - electrochemical method - square wave and cyclic voltammetry
INTRODUCTION
Salicylic or 2-dihydroxybenzoic acid (SA) is a compound, which has been shown to play an important signaling role in the activation of various plant defense
responses following pathogen attack. These responses
include the induction of local and systemic disease
resistance, the potentiation of host cell death, and the
limitation of pathogen spread [1, 2]. The changes in SA
content in plants exposed to stresses were published
[3, 4]. SA is also an endogenous growth regulator fulfilling numerous functions in plants under normal
conditions [5].
A number of analytical approaches such as UV
spectrometry and spectrofluorimetry, flow injection
analysis, both for nonplant materials, as well as HPLC
for plants [6–9] coupled with different detectors were
suggested for determination of SA and its derivates.
Some papers were based on electrochemical properties
of salicylates and other phenolic compounds for their
determination [10–15]. Among the broad range of electrochemical methods, only differential pulse voltammetry has been used for determination of salicylic acid
[12, 15]. We followed the results obtained by Torriero
* The text was submitted by the authors in English.
Abbreviations: CV—cyclic voltammetry; SA—salicylic acid;
SWV—square wave voltammetry.
[12], but employed another electrochemical method
(square wave voltammetry) for the detection of salicylic acid. Primarily, we optimized the method and then
used it for the determination of free salicylic acid content in barks and branches of thirteen widely spread
Salix species.
MATERIALS AND METHODS
Chemicals. All chemicals were purchased from
Sigma-Aldrich (United States). The stock standard
solutions of salicylic acid at 50 mg/ml were prepared in
methanol and stored in the dark at –20°C. Standard
solutions were prepared daily by dilution of the stock
solution. The pH value was measured by using
pH-meter (MultiLab Pilot; Germany), controlled by
personal computer program (MultiLab Pilot).
Preparation of plant samples. Samples of barks and
two-years-old branches of willows (S. laponum,
S. planifolia, S. foetida, S. foetida × S. hastata,
S. “Aegma Brno” (S. aegyptica × S. magnifica),
S. purpurea cv. Nana, S. reinii, S. repens, S. erythrotoflexuosa, S. caterii, S. fragilis, S. triandra, and S. viminalis) were collected in July 2005 in the Botanical garden of Mendel University of Agriculture and Forestry
in Brno. The age of the trees varied from 5 to 40 years.
The samples (0.5–2.0 g) were placed into polyethylene
vials, where we added 15 ml of 99.99% methanol. After
553
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PETREK et al.
Table 1. Basic voltammetric characteristics of salicylic acid
and its derivates (n = 5) measured by cyclic voltammetry
Salicylates
Salicylic acid
Thiosalicylic acid
Acetylsalicylic acid
5-sulfosalicylic acid
3,5-dinitrosalicylic acid
Peak potential
Peak height
for concentration at 100 µg/ml,
100 µg/ml, V
µA ± S.D.
1.09
0.70
–
1.15
–
19.8 ± 1.6
0.030 ± 0.002
–
2.00 ± 0.08
–
Notes:A dash—not detected; S.D.—standard deviation.
that, the samples were homogenized by shaking on Vortex-2 Genie (400 rpm, Scientific Industries, United
States) at 4°ë for 72 h [9]. The optimization of the sample volume for SA determination is described in
“Results”.
Electrochemical measurement. Electrochemical
analyses of pure SA and willow tissues were performed
with an AUTOLAB analyzer (EcoChemie, The Netherlands). All experiments were carried out at room temperature. The raw data were treated using the Savitzky
and Golay filter (level 2) and the moving average baseline correction (peak width 0.03) of the GPES software.
Britton–Robinson buffer (pH 1.81) consisting of 0.4 M
H3PO4, 0.4 M CH3COOH, and 0.4 M H3BO3 was used
as supporting electrolyte. Its pH was adjusted by 0.2 M
NaOH.
Cyclic voltammetry. The three-electrode system
included the carbon paste working electrode, an
Ag/AgCl/3 M KCl reference electrode, and a carbon
auxiliary electrode. The cyclic voltammetric parameters were as follows: initial potential of 0 V, vertex
potential 1.2 V, end potential 0 V, step potential 5 mV.
Square wave voltammetry. The three-electrode system consisted of the graphite pencil working electrode,
an Ag/AgCl/3 M KCl reference electrode and a carbon
auxiliary electrode. The square wave voltammetric
parameters were as follows: initial potential of 0.7 V,
end potential 1.5 V, frequency 260 Hz, amplitude
50 mV, step potential 10 mV.
Preparation of carbon paste electrode. The carbon
paste (about 0.5 g) was made of 70% graphite powder
(Sigma–Aldrich) and 30% mineral oil (Sigma–Aldrich;
free of DNase, RNase, and protease) according to [16,
17]. This paste was housed in a Teflon body having a
2.5-mm-diameter disk surface. Prior to measurements,
an electrode surface was renewed by polishing with a
soft filter paper. Then, the surface was ready for measurement of a sample volume of 5 µl.
Preparation of graphite pencil electrode. The pencil
leads with a lead diameter of 500 µm and total length of
60 mm were purchased from Kohinor (Czech Republic). The immersing of 3 mm of the pencil lead tip into
a solution resulted in an active electrode area of
4.91 mm2. The electrodes were used without any pretreatment. They were polished mechanically with
0.1 µm alumina (ESA, United States) [18]. Utilization
of graphite pencil electrode as a working electrode has
been also published [19, 20].
Accuracy, precision, and recovery. Accuracy, precision, and recovery of SA were evaluated in homogenates from barks and branches of S. purpurea, cv. Nana,
spiked with standard. Before extraction, 100 µl SA
standard and 100 µl water were added to willow tissue
sample. Detection limit was measured as a three-fold
excess over noise level, but quantification was reliable
at tenfold excess. Precision (coefficient of variation) of
intra-day assaying was performed in 6 homogenates.
Inter-day variation was determined by analysing six
homogenates over a 5-day period, in the intervals the
samples were kept in the dark at –20°ë prior to analysis. Homogenates were assayed blindly and SA concentrations were derived from the calibration curves.
Accuracy was evaluated by comparing the estimated
concentration with the known concentrations of SA.
We estimated accuracy, precision, and recovery according to [21].
RESULTS
Primarily, we studied the basic electrochemical
characteristics of the SA and its derivatives, which
could interfere during analysis. Thus, we utilized cyclic
voltammetry (CV), because CV belongs to fundamental electrochemical techniques that can be used to study
of basic electrochemical properties and behavior of
compounds of interest [22, 23]. CV voltammograms of
salicylic acid measured at various scan rates (25, 50, 75,
100, and 125 mV/s) are shown in Fig. 1a. It clearly follows from the results obtained that SA gives the signal
at a potential of 1.09, which increased with concentration elevation and scan rate (Fig. 1b).
As SA has many derivatives, we were interested if
they could interfere with the electrochemical determination of SA. We analyzed four derivatives of salicylic
acid, namely thiosalicylic acid, acetylsalicylic acid,
5-sulphosalicylic acid, and 3,5-dinitrosalicylic acid.
Acetylsalicylic acid and 3,5-dinitrosalicylic acid did not
produce any electrochemical signal (Table 1). On the
other hand, thiosalicylic and 5-sulphosalicylic acids
were electro-active and gave their signals at the potentials of 0.70 and 1.15 V, respectively. It clearly follows
from the results obtained that electroactivity of salicylates strongly depends on the group bound to the SA.
Only sulfur-substituted salicylic acid exhibited the
electrochemical response, but the peak heights were
much lower in comparison with the nonsubstituted one.
As for the potentials producing the signals, they were
different enough to distinguish SA from its derivates.
At the same time, cyclic voltammetry is not enough
sensitive to determine SA in biological samples. For
this reason, we attempted to determine SA by square
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ANALYSIS OF SALICYLIC ACID IN WILLOW BARKS AND BRANCHES
555
Cyclic voltammetry
5
(‡)
60
(b)
50
4
3
40
3
40
Peak height, µÄ
Peak height, µÄ
50
30
30
2
10 µÄ
20
1
20
2
1
scan
10
10
0.4
0.6
0.8
Potential, V
1.0
1.2
0
100 200 300 400
Scan rate, mV/s
Fig. 1. (a) CV voltammograms of salicylic acid at the concentration of 100 µg/ml measured at various scan rates (25, 50, 75, 100,
and 125 mV/s; 1, 2, 3, 4 and 5, respectively), (b) influence of various scan rates on the height of SA peak at (1) 25 µg/ml,
(2) 50 µg/ml, and (3) 100 µg/ml SA.
wave voltammetry (SWV) on the surface of a graphite
pencil electrode with the diameter of 500 µm. SA gave
a well-developed signal at a potential of 1 V (Fig. 2a).
The height of SA signal was proportional to its concentration above detection limit of 1.7 ng/ml (Fig. 2b).
We used methanol for extraction of SA. Organic solvents, such as methanol and acetonitrile, can negatively
influence electrochemical analysis [16, 24]. We
checked how a volume of samples from S. viminalis
could influence the determination of SA measured in
the presence of 2.0 ml of Britton–Robinson buffer
(pH 1.8). A current produced by SA linearly rose with
increasing volume of the sample up to 60 µl (3 µl of
methanol in the buffer), after that the current response
slightly decreased, which was associated probably with
the higher amount of methanol in the electrochemical
cell (Fig. 3). The SA peak potential was shifted by
about 20 mV to more positive potential after addition of
100 µl of the sample (inset in Fig. 3). It is evident that
the most suitable volume of the plant sample for determination of SA was 60 µl (Fig. 3).
The detection limit of the method is 1.7 ng/ml, reliable range lies between 100 ng/ml and 80 µg/ml.
Recovery was checked for the compound of interest by
addition of known amounts of SA working standard to
homogenates (Table 2). Recoveries of SA were from 98
to 103% (Table 2). Reproducibility of the procedure
was tested by analyzing representative samples in six
replicates during 5 days (Table 3). The precision for SA
was measured in S. purpurea, cv. Nana branch and bark
samples, with coefficients of variation ranging from 2.0
to 4.2% in the intra-assay and from 3.1 to 5.4% in the
interassay. Overall recoveries were from 98 to 105%
(n = 30). Accuracy of the method was about ± 5%.
As willow bark contains SA producing an antipyretic effect recognized for more than 200 years [25],
we were interested how much SA is contained in the
barks and branches of thirteen willow species. Contents
of free SA in the analyzed samples were calculated
from the calibration curve and evaluated by standard
addition of known amounts of SA (relative standard
deviation varied from 1.5 to 3.5%) (Fig. 4). The highest
contents of free SA in the barks were observed in the
following willow species S. laponum (3.0 mg/g fr wt),
S. purpurea cv. Nana (1.1 mg/g fr wt), and S. triandra
(0.6 mg/g fr wt). The highest contents of SA have been
determined in the branches in S. planifolia (2.2 mg/g fr wt),
S. purpurea cv. Nana (2.1 mg/g), and S. foetida ×
S. hastata (1.4 mg/g). In contrast, the content of SA in
the bark and branch of a unique willow species Salix cv.
Table 2. Recovery of SA amount in S. purpurea, cv. Nana, sample analysis (n = 5) by SWV method
Plant tissue
Branch
Bark
Homogenate, µg
Spiking, µg
Homogenate + spiking, µg
Recovery, %
2.38 ± 0.05 (2.1)
1.45 ± 0.03 (2.1)
2.51 ± 0.06 (2.4)
2.48 ± 0.05 (2.0)
5.06 ± 0.11 (2.2)
3.84 ± 0.12 (3.1)
103
98
Notes:SA amounts are expressed per electrochemical cell containing 2 ml of Britton–Robinson buffer, pH 1.8, and represent the means ±
standard deviation. In brackets, the coefficient of variation is shown (%).
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PETREK et al.
Square wave voltammetry
Salicylic acid
100
Relative peak height, %
(‡)
2 µÄ
Electrolyte
1.0
Potential, V
4
Peak height, µÄ
(b)
3
40
1.19
1.3
20
y = 0.1787x + 0.2414
R2 = 0.9908
2
0
1
0
60
Potential, V
0.7
80
20
1.16
1.13
1.10
0
50
Sample volume, µl
40
60
Sample volume, µl
100
80
100
Fig. 3. Influence of sample volume of the methanolic
extract from S. viminalis on SA peak height and potential
(in inset) measured by SWV method.
10
15
5
Concentration of SA, µg/ml
20
Fig. 2. The measurements of SA by square-wave voltammetry.
(a) SWV voltammogram of SA at 20 µg/ml measured on the
surface of graphite pencil electrode in the presence of Britton–Robinson buffer (pH 1.8), R2—regression coefficient.
(b) Standard curve for dependence of SA peak height on
concentration. Volume of supporting electrolyte and working solution of salicylic acid did not exceed 1.1 µl.
Aegma Brno occurring only in Arboretum of Mendel
University of Agriculture and Forestry (Brno) was low,
particularly, 42 and 129 µg/g fr wt, respectively.
DISCUSSION
The SWV method in combination with a pencil lead
electrode is likely to be useful for measuring SA.
Recently Torreiro et al. [12] published the paper on
determination of SA with a glassy carbon electrode.
Detection limit of their technique was 90 ng/ml. They
proved that there is no need for any extraction procedure before electrochemical analysis, because no
change of the peak potentials in the presence of the
interfering substances (derivates of salicylic acid) was
observed. We confirmed these results by means of
cyclic voltammetry (Table 1), because peak potential of
SA was different enough to distinguish salicylic acid
from its derivates. We also checked the recovery and
accuracy of the suggested technique and obtained satisfactory results (Tables 2, 3). The detection limit for SA
was low. The electrochemical technique has been used
for determination of SA in the barks and branches of
thirteen willows species (Fig. 4). We found out that the
highest content of SA in the branches of S. planifolia,
Table 3. Precision and recovery of SA for S. purpurea, cv. Nana, sample analysis by SWV method
Plant tissue Repetitive analyses
Homogenate, µg
Spiking, µg
Homogenate + spiking, µg
Recovery, %
Branch
intra-day (n = 6)
2.41 ± 0.07 (2.9)
2.50 ± 0.05 (2.0)
4.96 ± 0.18 (3.6)
101
inter-day (n = 30)
2.40 ± 0.09 (3.8)
2.54 ± 0.08 (3.1)
5.18 ± 0.22 (4.2)
105
intra-day (n = 6)
1.42 ± 0.06 (4.2)
2.48 ± 0.06 (2.4)
3.81 ± 0.14 (3.7)
98
inter-day (n = 30)
1.48 ± 0.08 (5.4)
2.56 ± 0.08 (3.1)
4.19 ± 0.18 (4.3)
104
Bark
Notes:SA amounts are expressed per electrochemical cell containing 2 ml of Britton–Robinson buffer, pH 1.8, and represent the means ±
standard deviation. In brackets, the coefficient of variation is shown in %.
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ANALYSIS OF SALICYLIC ACID IN WILLOW BARKS AND BRANCHES
3.0
5.
2.5
6.
2.0
1.5
1.0
7.
S. laponum
S. planifolia
S. foetida
S. foetida × S. hastata
S. clix cv. Aegma Brno
S. purpurea cv. Nana
S. reinii
S. repens
S. erythrotoflexuosa
S. caterii
S. fragilis
0
S. triandra
0.5
S. viminalis
Content of SA, mg/g fr wt
3.5
8.
9.
Fig. 4. Content of SA in willow —branches and —barks
measured by SWV method. The results are expressed as the
means and standard deviations.
S. purpurea, cv. Nana, and S. foetida × S. hastate
appears to correspond to their higher ability to resist
pathogen attacking. The technique utilized here for
determination of SA in willow tissue has a much
broader range and lower limit of detection in comparison to both linear sweep and cyclic voltammetry
because of its efficient discrimination of the capacitance current.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Frantisek Jelen for
correction and peer reviewing of the manuscript. This
work was supported by Grant Agency of the Czech
Republic (grant no. 525/04/P132) and the Ministry of
Education, Youth, and Sports of Czech Republic (grant
no. 1M06030).
10.
11.
12.
13.
14.
15.
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