Synthesis, crystal structure, antioxidation and DNA-binding studies of a zinc(II) complex with the bis(N-allylbenzimidazol-2-ylmethyl)aniline

Article abstract of DOI:10.1002/jccs.201100456

A complex of zinc(II) picrate (pic) with bis(N-allylbenzimidazol-2- ylmethyl)aniline (abba), with composition [Zn(abba)₂](pic) ₂, was synthesized and characterized by elemental analysis, electrical conductivity, IR and UV/Vis spectra

Full text of DOI:10.1002/jccs.201100456

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Electrochemical Behaviors of Baicalin at an Electrochemically Activated Glassy
Carbon Electrode and Its Determination in Human Blood Serum
Fei Wang, Ming-Xiu Lv, Kui Lu,* Lin Gao and Jian Liu
Department of Material and Chemistry Engineering, Henan Institute of Engineering, Zhengzhou, 450007, P. R. China
(Received: Jul. 23, 2011; Accepted: Oct. 4, 2011; Published Online: ??; DOI: 10.1002/jccs.201100456)
Electrochemical behavior of baicalin (BA) at an electrochemically activated glassy carbon electrode
(GCE) had been investigated in Britton-Robinson (B-R) buffer solution (pH = 2.87) by cyclic voltam-
metry (CV) and square wave voltammetry (SWV). The experimental results suggested that the electrode
exhibited an electrocatalytic activity toward the redox of BA. The electron transfer coefficient (a) and the
standard rate constant (ks) of BA at the electrochemically activated glassy carbon electrode were calcu-
lated. The reaction mechanism was proposed and discussed in this work. Under the selected conditions,
the reduction peak current was linearly dependent on the concentration of BA in the range of 5.0 × 10-8 to
3.0 × 10-6 mol L-1 (r = 0.9990), with a detection limit of 2.0 × 10-8 mol L-1. The relative standard deviation
(R.S.D.) for five times successful determination of 1.0 × 10-6 mol L-1 baicalin were 2.9%. The proposed
method was also successfully applied for the determination of BA in human blood serum.
Keywords: Baicalin; Square wave voltammetry; Adsorptive voltammetric behaviors; Blood serum
samples.
INTRODUCTION
Surface treatment of a glassy carbon electrode has
function and also can relieve fever by affecting the central
nervous system (CNS).15,16 To quantify BA in biological
samples, in addition to analytical methods based on UV-vis
spectrophotometry,17 thin layer chromatography (TLC),18
high performance liquid chromatography (HPLC)19-25 and
capillary electrophoresis (CE)26-28 have been developed.
However, these measurements are hindered by low sensi-
tivity, a long retention time, low throughput or require ex-
pensive devices. Electrochemical detection of analyte is a
very elegant method in analytical chemistry.29,30 Addi-
tional application of electrochemistry includes the deter-
mination of electrode mechanisms. And redox properties
of organic molecules can give insights into their meta-
bolic fate or their in vivo redox processes or pharmaco-
logical activity. To our knowledge, by far a few literatures
have been published concerning electrochemical behav-
ior of BA at solid electrode. Recently, Zi-yi Sun and his
co-workers investigated BA-DNA interaction through
electrochemical and spectroscopic techniques, but did not
establish relevant method to determine the BA concentra-
tion.31
been used extensively to improve the electrochemical per-
formance of the electrode. Especially, electrochemical pre-
treatment has been established as one of the widely used
pretreatment techniques to clean and activate electrode sur-
faces.1-4 The surfaces of glassy carbon electrodes (GCE)
can be oxidized, and thus various kinds of oxygenous
groups, such as phenolic, quinoidal, and carboxyl function-
alities, can be added on the surfaces. The existences of
these active groups can adsorbe aromatic cations and neu-
tral species and catalyze some reactions.5-7
Baicalin (7-glucuronic acid-5,6-dihydroxy-flavone,
BA, structure shown in Fig. 1), is predominant flavone
glucuronide of the commonly used traditional Chinese
medicine (TCM) Scutellariae radix. A mass of work indi-
cated that BA exhibited various pharmaceutical effects
such as anti-viral,8 anti-inflammation,9 anti-oxidation10,11
and anticancer activities.12-14 Studies have also revealed
that BA can act on the dopamine system, influence cerebral
In this approach, a new, simple, fully validated, rapid
and selective voltammetric method to directly determine
the BA concentration in blood serum samples without any
separation steps prior to drug assay is developed. At the
same time, our aim of this study was also to establish the
Fig. 1. The chemical structure of BA.
* Corresponding author. Tel: +86 0371 67718913; Fax: +86 0371 67718923; E-mail: luckyluke@haue.edu.cn
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Wang et al.
experimental conditions, to investigate the adsorptive
voltammetric behavior and redox mechanism of BA on the
surface of an electrochemically activated glassy carbon
electrode using cyclic voltammetric techniques.
GCE and the GCE(ox), BA showed a pair of redox peaks,
with oxidation peak potential (Epa) of 0.525 V and reduc-
tion peak potential (Epc) of 0.489 V. However, the peak cur-
rent of BA at the GCE(ox) was much larger than that at the
alumina slurry freshly polished GCE, and it was about 10
times of that at the alumina slurry freshly polished GCE.
This result testified the GCE(ox) exhibited an electrocata-
lytic activity toward the redox of BA, which is beneficial to
the determination. Thus, in the following discussion, the
GCE(ox) is used.
RESULTS AND DISCUSSION
1. Characterization of the slurry polished and acti-
vated GCE
Potassium ferricyanide was selected as a probe to
evaluate the performance of an alumina slurry freshly pol-
ished GCE and the GCE(ox). Fig. 2 showed the cyclic
voltammograms of the alumina slurry freshly polished
GCE and the GCE(ox) in 1.0 × 10-3 mol L-1 K3[Fe(CN)6] +
0.1 mol L-1 KCl solution, respectively. At the alumina
slurry freshly polished GCE, the peak-to-peak potential
separation (DEp) was about 0.103 V (Fig. 2a). While at the
GCE(ox), the DEp was decreased to 0.085 V, indicating the
quick charge transfer kinetics of the electrons on the acti-
vated electrode.32 On the other hand, the peak currents of
K3[Fe(CN)6] at the GCE(ox) were much bigger than that at
the alumina slurry freshly polished GCE. This phenome-
To elucidate the electrode reaction of BA, the repeti-
tive cyclic voltammograms of BA in B-R buffer (pH =
2.87) supporting electrolyte were recorded, see Fig. 4. The
peak current decreases with increasing number of cycles,
and after the second cyclic sweep, the peak current de-
creases slightly. This phenomenon may be caused by the
adsorption of BA at the GCE(ox).
Effect of scan rate
To further estimate the electrode reaction of BA, the
influence of potential scan rate (v) on ip of 2.0 × 10-6 mol L-1
BA at the GCE(ox) was studied by CV at various sweep
rates. As shown in Fig. 5A, the peak currents of BA grow
with the increasing of scan rates and there are good linear
relationships between ip and v (Fig. 5B). The regression
equation is ipc (mA) = 0.456 + 11.074v (r = 0.9997); ipa (mA)
= -1.852 – 20.803v (r = 0.9963), indicating the redox pro-
cess of 2.0 × 10-6 mol L-1 BA at the GCE(ox) was adsorp-
tion-controlled. According to the above results, it could be
2+
non illuminates that Fe(CN)63+/Fe(CN)6 can reach the
surface of the activated electrode more easily than that of
the alumina slurry freshly polished GCE.
2. Electrochemical behavior of BA at the GCE(ox)
Fig. 3 showed electrochemical responses of the alu-
mina slurry freshly polished GCE and the GCE(ox) in 2.0 ×
10-6 mol L-1 BA + Britton-Robinson (B-R) solutions (pH =
2.87), respectively. At the alumina slurry freshly polished
Fig. 3. CV curves of 2.0 × 10-6 mol L-1 BA in B-R
buffer solution (pH = 2.87) at the alumina
slurry freshly polished GCE (a) and the
GCE(ox) (b); the accumulation step under open
circuit for 300 s, and scan rate 0.05 V s-1.
Fig. 2. CV curves of a alumina slurry freshly polished
GCE (a) and the GCE(ox) (b) in 1.0 × 10-3 mol
L-1 K3[Fe(CN)6] + 0.1mol L-1 KCl solution,
scan rate 0.1 V s-1.
2
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JOURNAL OF THE CHINESE
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Electroanalysis
0.9992), see Fig. 6. According to the Eqs. (1) and (2) the
values of a and n were calculated to be 0.49 and 2, respec-
tively. Based on the Eq. (3), the value of ks was further cal-
culated to be 3.37 s-1, which is much higher than the re-
ported value for baicalein in the DNA Langmuir–Blodgett
film modified glassy carbon electrode (0.246 s-1).34 Baicalein
is an also kind of flavonoid, and is similar structure and
character with BA. This result indicated that the GCE(ox)
possessed a high electrocatalytic activity toward the redox
of baicalin.
deduced reasonably that BA was firstly adsorbed on the
surface of the electrode, and then the electrode reaction was
followed.
The influence of v on the peak potentials of BA was
also investigated. With the increase of v the oxidation peak
potential was positively shifted, and the reduction peak po-
tential was negatively shifted, indicating that the redox re-
versibility of BA was impaired. The relationship of the
peak potentials with a scan rate was further constructed,
which could be used for the calculation of the electrochem-
ical parameters of BA. According to the Laviron’s equa-
tion:33
Effect of supporting electrolyte and pH value
The types of supporting electrolytes played a key role
in the voltammetric responses of BA. The voltammetric be-
havior of BA at the GCE(ox) was investigated over a wide
pH range (2.0-10.0) in HCl, H2SO4, phosphate, NaOH, ace-
tate, B-R and borate buffer solution by using cyclic voltam-
metry. Experiments indicated that only the measurements
in acidic media produced stable responses, and the best de-
RT
Epc = E0 -
Epa = E0 +
ln n
(1)
(2)
anF
RT
(1-a)nF
ln n
RT
logks = alog(1-a)+ (1-a)loga - log
nFn
(1-a)anFDEp
-
(3)
2.3RT
where a is the charge transfer coefficient, n the number of
electron transfer, ks the electrode reaction constant, v the
scan rate, E0 the formal potentials and F the Faraday’s con-
stant. A linear relationship between the Ep with the lnv was
established, and two straight lines were gotten with two lin-
ear regression equations as Epa (V) = 0.03246 lnv + 0.5469
(r = 0.9902) and Epc (V) = -0.03389 lnv + 0.4502 (r =
Fig. 4. The voltammograms of the background (a) and
the solution (b) containing 2.0 × 10-6 mol L-1
BA in B-R buffer solution (pH = 2.87) for
multi-cycles with scan rate of 0.05 V s-1 at the
GCE(ox), other experimental conditions are the
same as those described in Fig. 3.
Fig. 5. CV curves (A) and linear relationship of ip vs. v
(B) of 2.0 × 10-6 mol L-1 BA in B-R buffer solu-
tion (pH = 2.87) at the GCE(ox). Scan rate (in-
ner to outer): 0.10, 0.20, 0.30, 0.40, 0.50, 0.60,
0.70, 0.80 V s-1.
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fined peak was obtained in B-R buffer. Therefore, in this
work B-R buffer was chosen as the supporting electrolyte.
The effect of the buffer pH on the formal peak poten-
tial (E0) was also investigated, see Fig. 7. In the pH range
from 2.0 to 7.0, the value of E0 shifted to the negative direc-
tion with the increase of the buffer pH. A linear regression
equation was obtained as E0 (V) = -0.057 pH + 0.615 (r =
0.9988). The slope of -0.057 was close to the theoretical
value of -0.059 V/pH at 25 °C. According to the equation:
-0.054 m/n = -0.059, where n is the electron transfer num-
ber and m is the number of hydrogen ion participating in
the reaction, so the uptaking of electron was accompanied
by an equal number of hydrogen ion and m = n = 2.
Reaction mechanism of BA
ton process, and the possible electrode reaction mechanism
was expressed as shown in Scheme I.
The electrochemical redox reaction mecha-
nism of BA
Scheme I
3. Analytical applications and methods validation
Square wave voltammetry (SWV) is effective and
rapid electroanalytical techniques with well-established
advantages, including good discrimination against back-
ground currents and low detection limits.35 Therefore, in
this study SWV was used as a determinate technique. At the
same time, for the analytical determination of BA, the
redution peak was chosen, since it is much smaller back-
ground currents and better stable responses.
According to the above results, the electro-oxidation
reaction of BA at the GCE(ox) was a two-electron two-pro-
Effect of instrumental parameters
The SWV was used for the determination of BA. In
order to obtain a much more sensitive peak current, the op-
timum instrumental conditions (pulse-amplitude Esw, fre-
quency f) were studied using anodic square wave voltam-
metry for 2.0 × 10-6 mol L-1 BA solution following tacc of
300 s and open circuit. The results indicated that the peak
current ip increased with the increasing of square wave am-
plitude from 5 to 50 mV or square wave frequency in the
range of 5-45 Hz, but the peak potential shifted to more
positive values, and the peak changed unshapely. So 35 mV
was chosen as the optimum amplitude and 25 Hz was cho-
sen as the optimum frequency.
Fig. 6. Relationship between Ep and logarithm of po-
tential scan rate.
Effect of accumulation potential and accumulation
time
Accumulation step is usually a simple and effective
way to enhance the determining sensitivity. Accumulation
potential (Eacc) and time (tacc) are two crucial parameters for
the accumulation step. The reduction peak current of 2.0 ×
10-6 mol L-1 BA was compared after 300 s accumulation at
different potential and open circuit by SWV. The peak cur-
rent almost does not vary, revealing that the accumulation
potential has no influence on the reduction peak current of
BA at the GCE(ox). Thus, the accumulation of BA was
carried out under open circuit.
Fig. 7. The cyclic voltammograms of 2.0 × 10-6 mol L-1
BA at different pH values with scan rate of 0.10
V s-1, pH: (1) 1.98, (2) 2.87, (3) 4.10, (4) 5.02,
(5) 6.09, (6) 7.00.
Unlike the accumulation potential, accumulation
time severely influences the oxidation peak current re-
sponses. The reduction peak current increases significantly
4
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Table 1. Determination results of BA in human blood serum
samples
in the first 210 s, and then levels off. This may be caused by
the fact that the adsorption of BA on the electrode surface
becomes saturated. Thus, the accumulation step in this
study was performed under open circuit for 210 s.
Calibration curve
Standard solution
added
Standard
Recovery a RSD
No
solution found
(10-7 mol L-1)
(%)
(%)
(10-7 mol L-1)
1
2
3
4
5
6
1.00
2.00
4.00
6.00
8.00
10.00
0.98
2.05
4.10
5.89
7.83
9.68
98.0
102.5
102.5
98.2
2.5
1.9
1.7
2.3
3.0
2.8
Series concentrations of standard solutions of BA
were detected under the optimized working conditions de-
scribed above. Fig. 8 showed that the peak currents in-
creased linearly with increasing concentration of BA. The
inserted calibration plot highlights a linear relationship be-
tween peak currents and increasing concentration with a re-
gression coefficient of 0.9990. The peak currents re-
sponded were linearly relationships with BA concentra-
tions in the range of 5.0 × 10-8 to 3.0 × 10-6 mol L-1. The
linear equation was:
97.9
96.8
a Average of five determinations
the good repeatability of the method.
Interference studies
For the possible analytical application of the pro-
posed method, the effect of various species that likely to be
in biological samples were evaluated by analyzing sample
solutions containing a fixed amount of BA (2.0 × 10-6 mol
L-1) spiked with various excess amounts of the species un-
der the same experimental conditions. The tolerance limit
for a foreign species was taken as the largest amount yield-
ing a relative error < 5% for the determination of BA. The
results show that no serious interference occurred from the
classical additives tested. The influence of ascorbic acid,
which is a potentially interfering compound present in bio-
logical samples, was investigated. It was found that an
equimolar concentration or even at higher molar excess
(2:1) of ascorbic acid had no distinct effect on the peak re-
sponse of BA. Also interference of some metal ions was
tested under the same conditions. It was observed that
100-fold excess of Al(III), Cu(II), Fe(II), Zn(II) and Mg(II)
metal ions had no effects on BA determination. Therefore,
the proposed method can be used as a selective method.
Determination of BA in human blood serum
ip(mA) = 1.588 + 1.597 ´ 107c (r = 0.998)
Based on the signal-to-noise ratio of 3 (S/N),36 the de-
tection limit was obtained as 2.0 × 10-8 mol L-1. These val-
ues confirmed the sensitivity of the proposed method for
the determination of BA. To estimate the repeatability of
the proposed method, the R.S.D. of five times successful
measurement of peak current of 2.0 × 10-6 mol L-1 BAat the
GCE(ox) was calculated to be 2.9%, which demonstrates
The applicability of the SWV to the determination of
BA in spiked urine was investigated. The direct determina-
tion of BA in human blood serum samples was found to be
possible by employing a high dilution of the sample with
the supporting electrolyte. The repeatability of a total ana-
lytical process was determined from multiple measure-
ments at each of the human blood serum samples (Table 1).
An average deviation of 2.4% was obtained.
Fig. 8. Square wave anodic stripping voltammograms
and their associated calibration plot (insert) for
increasing concentrations of BA at the GCE(ox)
under optimum conditions; BA concentration:
1) 0 mol L-1, 2) 5.0 ´ 10-8 mol L-1, 3) 6.0 ´ 10-8
mol L-1, 4) 8.0 ´ 10-8 mol L-1, 5) 1.0 ´ 10-7 mol
L-1, 6) 2.0 ´ 10-7 mol L-1, 7) 4.0 ´ 10-7 mol L-1,
8) 6.0 ´ 10-7 mol L-1, 9) 8.0 ´ 10-7 mol L-1 , 10)
1.0 ´ 10-6 mol L-1, 11) 2.0 ´ 10-6 mol L-1.
EXPERIMENTAL
Apparatus and Reagents
Model 650A electrochemical system (CHI Instru-
ment Company, Shanghai, China) was employed for elec-
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trochemical techniques. Astandard three-electrode electro-
chemical cell was used for all electrochemical experiments
with a bare GCE or activated GCE (d = 3 mm) as working
electrode, a platinum (Pt) wire as an auxiliary electrode and
a saturated calomel electrode (SCE) as a reference elec-
trode. All the pH measurements were carried out with a
pHS-2C Digital pH meter (Shanghai Lida, Shanghai, China)
equipped with a combined glass electrode, which was cali-
brated regularly with standard buffer solutions (pH 4.00
and 6.86) at 25 0.1 °C.
tion curve method from the related calibration equation.
CONCLUSIONS
The electrochemical voltammetric behaviour of BA
at the GCE(ox) in B-R buffer (pH = 2.87) solution was in-
vestigated. The experimental results suggested that the pro-
cess at the GCE(ox) was adsorption controlled and in-
volved two-electron two-proton transfer. An analytical
method for the determination of BA in human blood serum
samples was successfully developed on the basis of the
voltammetric studies. Under the selected conditions, the re-
duction peak current was linearly dependent on the concen-
tration of baicalin in the range of 5.0 × 10-8 to 3.0 × 10-6 mol
L-1 (r = 0.9990), with a detection limit of 2.0 × 10-8 mol L-1.
The method is highly sensitive and has a reasonable selec-
tivity and good precision. Additional, the oxidation mecha-
nism was also proposed and discussed in this work.
All reagents were of analytical grade and were used
as received. BA was purchased from National Institute for
the control of pharmaceutical and biological products
(Beijing, China). Double distilled water was used for all
preparations. Stock solutions (5.0 × 10-4 mol L-1) of BA
were prepared with ethanol and stored at 4 °C in the dark.
Dilutions were done just prior to use. Each assay was per-
formed at room temperature.
Working voltammetric procedure
ACKNOWLEDGEMENTS
GCE was polished progressively with finer emery-
paper, then thoroughly with 0.5 mm Al2O3 (CHI Company)
on a polishing cloth. The working electrode was cleaned in
an ultrasonicating bath for 1 min, and then the electrode
was activated by performing 50 cycles from 0.5 to 1.3 V in
0.2 mol L-1 NaOH with a scan rate of 100 mV s-1 before use.
This electrochemically activated glassy carbon electrode
was named GCE(ox).
The authors would like to thank the financial supports
from the Chinese National Science Foundation (No.
21172054), Innovation Scientists and Technicians Troop
Construction Projects of Henan Province (No. 104200510022)
and Natural Science Foundation of Henan Province (No.
2010B150007).
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or 0.1 and 0.8 V.
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www.jccs.wiley-vch.de
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