ISSN 10231935, Russian Journal of Electrochemistry, 2012, Vol. 48, No. 8, pp. 1–8.

© Pleiades Publishing, Ltd., 2014.

Published in Russian in Elektrokhimiya, 2012, published in Elektrokhimiya, 2012, Vol. 48, No. 8, pp. @@@–@@@.

Electrochemical Measurement of the Antidiabetic Potential

of Medicinal Plants Using MultiWalled Carbon Nanotubes
Paste Electrode1
M. Mohiuddina, z, D. Arbaina, A. K. M. Shafiqul Islama, b, M. Rahmanc, M. S. Ahmada and
M. N. Ahmadb
a
School of Bioprocess Engineering
b
Centre of Excellence for Advanced Sensor Technology, Universiti Malaysia Perlis, 01000, Kangar, Perlis, Malaysia
c
School of Chemical Science and Food Technology, Universiti Kebangsaan Malaysia (UKM), 43600Bangi, Selangor
D.E., Malaysia Received October 02, 2013
Abstract—A sensor for the determination of antidiabetic potential of medicinal plants based on the inhibi
tion of the αglucosidase (AG) activity is developed using multiwalled carbon nanotubes (MWCNTs) paste
electrode. The fabricated electrode was used to measure the amount of paranitrophenol released through the
hydrolysis of paranitrophenylαDglucopyranoside (PNPG) catalyzed by AG enzyme. The enzymatic
reaction is inhibited by bioactive compounds in medicinal plant extracts, which indicates the antidiabetic
potential of these extract. The inhibition of the enzymatic reaction by medicinal plant extracts and Acarbose
was studied by cyclic voltammetric method using the developed electrode in phosphate buffer at pH 6.8. The
results show that the inhibition is higher in the presence of Tebengau (Ehretia laevis) than that in the presence
of Acarbose, Cemumar (Micromelum pubescens) and Kedondong (Spondias dulcis). A good correlation was
obtained between the spectrophotometric and the cyclic voltammetric methods for the measurement of the
inhibition achieved with the medicinal plant extracts. Therefore, the fabricated MWCNTs paste electrode is
useful for the measurement of the antidiabetic potential of medicinal plants.

Keywords: Cyclic voltammetry (CV), αglucosidase, medicinal plant extracts, inhibition activity, paranitro
phenylαDglucopyranoside (PNPG)
DOI: 10.1134/S1023193514120027

1. INTRODUCTION These drugs, however, have significant gastrointes

Diabetes mellitus (DM) is a chronic disease char
acterized by increasing of glucose concentrations in
the blood stream and is commonly known as hyperg
lycaemia. DM causes serious damage of many systems
in the body, particularly the nerves systems and blood
vessels. Diabetic patients are increasing globally and
around 346 million people have diabetes according to
the World Health Organization [1] Most of diabetic
patients suffer from type 2 DM which is associated
with the inability of insulin to perform glucose uptake
from the blood stream, thus results in elevated blood
glucose levels. This type of DM can be controlled
through the use of some drugs with different modes of
action. These include the Biguanide class of drugs,
which reduces carbohydrate absorption from the gas
trointestinal tract, the Sulphonylureas class, which
increases endogenous insulin production [2], DPP4
inhibitors, which delay the clearance of natural incre
tins [3], and AG inhibitors, which delay the digestion
and absorption of ingested carbohydrates [4].
1 The article is published in the original.
z Corresponding author: mohi8090@yahoo.com (M. Mohiuddin).
tinal side effects, such as flatulence, diarrhea, and
abdominal discomfort [5]. These side effects have
prompted researchers to search for alternative medi
cines for the treatment of diabetes. These alternatives
include natural herbal medicines, which are locally
available, affordable, and cause minimum side effects
[6]. In fact, there are many medicinal plants world
wide that have been traditionally used for diabetes
treatment with various effectiveness such as, Misai
Kucing (Orthosiphon stamineus) in Malaysia [7], sor
ghum, foxtail millet, and proso millet in South Korea
[8], Spergularia rubra in Portugal and Spain [9],
Emblica officinalis gaertn, Allium sativum, Gymnema
sylvestre, Citrullus colocynthis, Trigonella foenum
greacum, and Momordica charantia in India [10, 11],
and Ramulus cinnamomi, Cinnamomum cassia, and
Eucommia ulmoides in China [12].
In the body, the glucose is produced from carbohy
drate through digestion by αamylase and AG
enzymes. Thus, inhibition of any of these enzymes
would reduce the production of glucose. For this rea
son, in the laboratory, the antidiabetic potential of
medicinal plants is commonly measured by the capa

1
2 MOHIUDDIN et al.
bility of the medicinal plants to inhibit the AG enzyme
reaction. This inhibition is normally determined
through several conventional analytical methods, such
as colorimetric [13], titration [14], highperformance
liquid chromatography (HPLC) [15], and gas chro
matographymass spectroscopy (GCMS) [16]. How
ever, these conventional methods involve some extrac
tion, intensive solvent usage, expensive devices and are
time consuming. In addition, these methods require
welltrained operators. Hence, it would be beneficial
and practical to find an alternative method for fast
measurement of the antidiabetic potential of medici
nal plants.
ElRies et al. (2008) determined the antidiabetic
drugs Repaglinide using carbon paste and glassy car
bon electrodes by CV and DPV methods [17]. The
measurement relies on the electroactive properties of
Repaglinide, thus not necessarily suitable for mea
surement of others medicinal plants antidiabetic prop
erties. Suna Timur and Ulku Anik (2007) developed a
biosensor using AG enzyme based on bismuth film
electrode for inhibition detection of commercial
antidiabetic drugs [18]. The authors have not shown
the measurement method of antidiabetic potential of
medicinal plants using their sensors.
In this study, MWCNTs were used to fabricate the
paste electrode to measure the antidiabetic potential
of medicinal plants using electrochemical method.
MWCNTs are new types of carbon nanostructure
materials which are widely used for modification of
electrodes due to their fascinating electronic, chemi
cal and mechanical properties, such as electrocatalytic
outcome, rapid electron transfer and broad working
surface area [19–21]. Additionally, the paste electrode
has some special benefits such as easy construction,
renewability and individual polarizability [22–25].
Therefore, MWCNTs paste electrodes were
selected to measure the antidiabetic potential of
medicinal plants. The measurement is based on the
inhibition of AG enzyme activity in conversion of
PNPG into pNP according to the reaction in
scheme.
ParanitrophenolαDglucopyranoside + H2O
AG enzyme
αDelucopyranose+ Paranitrophenol.
Scheme. Enzymatic reaction of PNPG.
PNPG is an electroactive substance because of the
presence a nitrophenyl Oglycoside group and release
paranitrophenol (pNP) upon hydrolysis in the pres
ence of AG enzyme. The released pNP is measured
by the CV method. In the presence of a medicinal
plant extract as an inhibitor, the liberated pNP will be
reduced. Thus, the difference of the released pNP
defines as the antidiabetic potential of a medicinal
plant.
RUSSIAN JOURNAL OF ELECTROCHEMISTRY
2. EXPERIMENTAL SECTION
2.1. Reagents and Chemicals
The AG enzyme, paranitrophenol (granular),
anhydrous sodium carbonate (Na2CO3), and PNPG
were obtained from Sigma Aldrich. The MWCNTs
with an average diameter of 10–40 nm and a length of
1–25 μm was purchased from Fibermax Composites
(Greece). Potassium chloride (ICQ) was used as the
supporting electrolyte. Dipotassium hydrogen phos
phate (K2HPO4) and potassium dihydrogen phos
phate (KH2PO4) were used for the preparation of
0.1 M phosphate buffer. All chemicals were used
according to their analytical grade. Acarbose tablets
(50 mg) were obtained from a local pharmacy.
2.2 Apparatus
The cyclic voltammetric studies were performed
using a portable potentiostat with threeelectrode
configuration (DropSens, Spain) consisting of
Ag/AgCl and Pt as reference and counter electrodes,
respectively. The working electrode was MWCNTs
paste electrode. The colorimetric measurements were
performed using a Lambda25 UVVIS spectrometer
(PerkinElmer, USA). The pH of the solutions was
measured using a Starter300 pH meter (Ohaus Corpo
ration, USA)
2.3. Preparation of MWCNTs Paste Electrode
The working electrode was prepared by hand mix
ing the MWCNT powder and mineral oil at a ratio of
60 : 40% w/w in a mortar to produce a uniform mix
ture. The paste was firmly packed into one end of a
plastic tube (3mm diameter, 0.5mm depth), and a
copper wire was inserted through the other end of the
tube to establish the electrical connection. The freshly
prepared MWCNTs paste electrode was kept at room
temperature for 24 hours for stabilisation [26]. The
electrode was polished using weighing paper prior to
each measurement.
2.4. Preparation of Medicinal Plant Extracts
Tebengau (Ehretia laevis), Cemumar (Micromelum
pubescens), and Kedondong (Spondias dulcis) plant
leaves were collected from the Agrotechnology
Research Station in Sungai Chuchuh, Perlis, Malay
sia. The plant leaves were airdried and then crushed
into a powder. Aqueous extracts of the plant leaves
were prepared through an infusion method [27].
Essentially, 0.5 g of each type of leaf powder was dis
solved in 100 ml of boiling (distilled) water, and the
mixtures were allowed to infuse for 15 min without
additional heating. All of the samples were filtered
through Whatman filter paper to obtain the extracts.
Vol. 48 No. 8 2012
 ELECTROCHEMICAL MEASUREMENT OF THE ANTIDIABETIC POTENTIAL
HO
O Antidiaoetic
αglucosidase
OH
HO O NO2
Herbal extracts
OH
αglucosidase
Fig. 1. Principle of the measurement of the antidiabetic potential through the CV method.
2.5. Preparation of Acarbose Sample
Acarbose is a commercial antidiabetic drug used
to treat type 2 diabetes mellitus. Acarbose slows the
digestion of carbohydrates in the body and thus helps
to control the blood sugar levels. Ten tablets were
weighed and ground into a homogeneous fine powder
in a mortar, and the average weight of each tablet was
calculated. Then, a 1 mg/mL acarbose solution was
prepared by dissolving the appropriate amount of
powdered acarbose in 50 mL of distilled water.
2.6. Cyclic Voltammetric Measurement Method
The PNPG sample solution was prepared in 0.1 M
phosphate buffer of pH 6.5 with 0.1 M KCl as the sup
porting electrolyte. The AG enzyme solution
(1.79 unit/mL) was prepared in 0.1 M phosphate
buffer at pH 6.5 and used for the enzymatic hydrolysis
of the PNPG solution. The plant extracts were used to
inhibit the enzymatic hydrolysis of the PNPG solution
in order to measure the antidiabetic potential of the
medicinal plants.
The cyclic voltammetric measurements were con
ducted using the fabricated MWCNTs paste electrode
in a stirred PNPG sample solution for a given period of
time. The stirring was then stopped for 15 s to allow the
solution to settle, and the cyclic voltammograms were
recorded at potential range between –0.6 to +0.6 V. The
enzymatic hydrolysis reaction was performed at room
temperature. The deposition potential (Edep) and time
(tdep) were set to –0.8 V and 20 s for each of the mea
surements to obtain maximum peak current.
2.7. Colorimetric Measurement Method
The colorimetric measurements were performed
using a Lambda25 UVVIS spectrometer (PerkinElmer,
USA). The 0.1 M PNPG and enzyme solution were
mixed through stirring to allow enzymatic hydrolysis
reaction indicating by the formation of yellowish pNP.
After a certain period of time, 0.1 M sodium carbonate
solution was added to stop the enzymatic reaction and
pNP was measured at 400 nm [28].
RUSSIAN JOURNAL OF ELECTROCHEMISTRY
3
potential
αDglucopyranose + pnianitrophenol
a1
αDglucopyranose + pnianitrophenol a2
2.8. Measurement Method of the Antidiabetic Potential
of Medicinal Plants
The antidiabetic potential measurement of medic
inal plants is based on the inhibition of AG enzyme
activity in conversion of PNPG into pNP according
to the Fig. 1.
The produced pNP will exhibit oxidation or
reduction peak (а1) using the CV method. In the
presence of the medicinal plant extract, the peak
would decrease (а2) inversely with the concentra
tion of the medicinal plant extract. Thus, the differ
ence between these two oxidation or reduction
peaks define as the antidiabetic potential of the
medicinal plants.
3. RESULTS AND DISCUSSION
3.1. Cyclic Voltammetric (CV) Behaviour of PNPG
at MWCNTs Paste Electrode
The voltammetric response of PNPG at
MWCNTs paste electrode was studied at the scan rate
50 mV/s with 0.1 M KCl solution using CV method.
PNPG is a synthetic electroactive chemical com
pound due to presence of the nitrophenyl Oglyco
sides group. The effect of scan rate at MWCNTs paste
electrode for PNPG was observed using CV method.
The cyclic voltammograms of PNPG at various scan
rates (10 to 60 mV/s) are depicted in the Fig. 2a and
it shows that the oxidation and reduction peak cur
rents increase linearly with the increasing of scan rate
from 10 to 60 mV/s. The peak currents increase lin
early to the square root of the scan rate for reversible
electron transfer. The result from the plotting of ip
versus v1/2 in the Fig. 2b showed that the anodic and
cathodic peak currents varied linearly with the square
root of scan rate, suggesting that the redox reaction of
PNPG follows a diffusion controlled mechanism
[29]. In addition, the peak potential shifted with the
increasing of the scan rate. The larger separation in
the peak potentials and the shift of the peak potential
with the scan rate indicate the quasireversible nature
of the process [30].
PNPG is hydrolysed by AG enzyme into para
nitrophenol and αDglucopyranose, as depicted in
Vol. 48 No. 8 2012
4 MOHIUDDIN et al.

(a) (b)

Oxidation peak
10 10
8
Oxidation peak
6 6
60 mV/s
Current, µA

4 40 mV/s R 2 = 0.987
40 mV/s
2 30 mV/s 2
20 mV/s
0 10 mV/s

–2 –2 R 2 = 0.995

–4
Reduction peak Reduction peak
–6 –6
–0.4 –0.2 0 0.2 0.4 0.6 0 2 4 6 8 10
Potential, V Square root of scan rate

Fig. 2. (a) Cyclic voltammograms of PNPG in 0.1 M phosphate buffer of pH 6.5 at the MWCNTs paste electrode with different
scan rates (10, 20, 30, 40, 50, and 60 mV/s), (b) the corresponding plot of the oxidation and reduction peak current as a function
of the square root of the different scan rates.

(a)
40 PNPG
(b)
PNPG + Enzyme Growing released pNP
20
reduction peak current
20
Current, µA

16
Current, µA

0 Growing released 12
pNP reduction
peak current
8
–20 Decreasing attached pNP
Decreasing attached pNP 1 reduction peak current
reduction peak
–40 0
–0.6 –0.2 0.2 0.6 5 15 25 35
Potential, V Time, min

Fig. 3. (a) Cyclic voltammograms of PNPG before (red) and after the addition of enzyme (black), (b) Decrease in the PNPG
peak current (attached pNP peak) and increase in the reduction peak current of released pNP as a function of time.

the scheme. The decrease in the amount of PNPG
and the release of pNP yielded by the above reaction
can be followed on the MWCNTs paste electrode
through the cyclic voltammetric method, as shown in
the Fig. 3a. The corresponding plots of the decrease
in the PNPG peak (attached pNP peak) and the
growth of the released pNP peak as a function of
time are indicated in the Fig. 3b. Thus, either
increase value of pNP as well as the deterioration of
PNPG reduction peak currents obtained in this sys
tem can be used to measure the AG enzyme activity.
However, the value of the oxidation peaks cannot be
used as it appears to be constant due to the overlap
ping of PNPG (attached pNP) and released pNP
peaks at around 0.16 V.
3.2. Measurement of the Antidiabetic Potential
of Medicinal Plants and a Commercial Drug, Acarbose
In the presence of an inhibitor, such as the bioactive
compounds in medicinal plants extracts, both the deteri
oration of PNPG reduction peak and the increasing rate
of the pNP are slower. The results in Figs. 4a and 4b show
that the reduction peak current of the released pNP from
the enzymatic reaction inhibited by the Tebengau plant
extract is lower than that obtained with Acarbose, Cemu
mar and Kedondong plant extracts. This suggests that the
inhibition of AG by Tebengau is higher than that obtained
by Acarbose, Cemumar and Kedondong.
The percentage of inhibition by medicinal plant
extracts and acarbose was calculated according to the
Eq. (1) [31] which is shown in Fig. 4c.

RUSSIAN JOURNAL OF ELECTROCHEMISTRY Vol. 48 No. 8 2012

 ELECTROCHEMICAL MEASUREMENT OF THE ANTIDIABETIC POTENTIAL 5

60 (c)

% of inhibition
40

20
(a)
0
10

ar ar
be e
em g

au
on ol

Te bos
C don
Ac um
ed tr

ng
4.50 (b)

K on
C
Control
3.50
Current, µA

Kedondong
PNPG Cemumar

Current, µA
0 Tebengau
Acarbose 2.50
Cemumar Acarbose
Kedondong
Tebengau
Control
1.50
–10
0.50

–0.6 –0.2 0.2 0.6 0 10 20 30

Potential, V Time, min

Fig. 4. (a) Cyclic voltammograms of PNPG in the presence of the enzyme and the medicinal plant extracts, (b) Reduction peak
current of released pNP over time in the presence of Tebengau (Ehretia laevis), Cemumar (Micromelum pubescens), Kedondong
(Spondias dulcis), and acarbose, (c) % of inhibition of Tebengau, Cemumar Kedondong and acarbose.

% of inhibition = ip/Abs. of control

 – ip/Abs.
 of sample × 100.
 (1)
ip/Abs. of control

The inhibition kinetics of AG enzyme by the in the presence of the Tebengau plant extract is shown
medicinal extracts was analyzed using the Lin in Fig. 6. There was a good correlation (R2 = 0.987)
eweaver–Burk equation which is shown as Eq. (2): between these two methods, which indicates the high
accuracy of the presented CV method. The correlation
1 = ⎛ 
Km ⎞ ⎛ 1 ⎞ 1 curves for acarbose (R2 = 0.977), Cemumar (R2 =
  + . (2)
V0 ⎝ V max⎠ ⎝ [ S ]⎠ V max 0.966) and Kedondong (R2 = 0.971) are not shown.
Fig. 5 shows the Lineweaver–Burk plot of the
effect of different concentrations of Tebengau plant 20 μL (2 mg/mL)
extracts on the activity of the AG enzyme at different 35 Tebengau herbal extracts
concentrations of PNPG. The results are shown in
table. It is noticeable that the apparent Km value
25 20 μL (1 mg/mL)
increases with an increase in concentration of the
1/V, µA/min

Tebengau herbal extracts

Tebengau plant extracts, whereas the Vmax values
remain almost unchanged. This is a typical behaviour 1/Vmax
of competitive inhibition [32], the inhibitor reversibly 15
–1/Km
binds to the same site of enzyme as the substrate.
Based on this mechanism, the inhibition can be Control
5
entirely overcome using a very high concentration of
substrate. In the present case, the inhibitor, which is 0
represented by the Tebengau plant extract, competes –2 2 4
–5
with PNPG for the active sites of the AG enzyme. This 1/[S], mM–1
competition is attributed to the hydroxyl groups of the
phenolic compounds within the extract that competes –15
with the hydroxyl groups of the PNPG substrate.
–1/Km
The correlation between the reduction peak cur
rent measured using the CV method and the absor Fig. 5. Effect of different concentrations of the Tebengau
bance measured using spectrophotometer found for plant extract on the activity of the AG enzyme at different
the pNP released by the enzymatic reaction of PNPG concentrations of PNPG.

RUSSIAN JOURNAL OF ELECTROCHEMISTRY Vol. 48 No. 8 2012

6 MOHIUDDIN et al.
Km and Vmax values at different concentrations of the Teben
gau plant extracts
Concentration
of Tebengau plant Km value, mM
extract, µL
Control 1.55
20 µL (1 mg/mL) 3.85 (apparent)
20 µL (2 mg/mL) 4.78
The above discussion not only confirms the reli
ability of the antidiabetic measurement method but
also indicates that the success of this method is contin
gent on the development of AG inhibition activity
based electrochemical sensor that can be used to mon
itor the antidiabetic potential of medicinal plants and
commercial drugs. Thus, it can be concluded that the
antidiabetic potential of Tebengau is significantly
higher than that of the other plant extracts. Therefore,
Tebengau plant extracts may be used as a good medic
inalbased candidate for the development of antidia
betic drugs.
3.3. Interferences Study
In order to study the selectivity of the method,
1 mg/mL of Acarbose solution were added in the con
trol solution and recorded the voltammograms of CV.
Then, the common interferences such as methanol,
ethanol, Na+, K+, Ca2+, Mg2+ were mixed with Acar
bose and control solution and recorded voltammo
Tebengau
14
pNitrophenol (µg/mL)
R 2 = 0.987
by CV method
10
6 based PNPG system for the measurement of the inhi
2 enzyme activity is not convenient due to the colour
0 2 4 6
pNitrophenol (µg/mL)
by Spectrophotometric method
Fig. 6. Correlation of the reduction peak current measured
using the CV method and the absorbance measured
through a UVvisible spectrophotometer for the pNP
released by the hydrolysis of PNPG in the presence of the
Tebengau plant extract.
grams. There was no other peak response appeared
and same inhibition was observed as without interfer
ences which is shown in Fig. 7. Thus indicating the
excellent selectivity of the MWCNTs paste electrode
Vmax value,
to measure the antidiabetic potential of medicinal
µA/min
plants as well as commercial antidiabetic drugs.
0.98 3.4. Repeatability and Reproducibility
of the MWCNTs Paste Electrode
0.88 To study the repeatability of the fabricated
MWCNTs paste electrode, 1.0 mM PNPG solution
0.81 and the fabricated electrode were used every two hours
within a day. The electrode showed an almost stable
peak current during every measurement obtained
using the cyclic voltammetric method at constant
temperature. After 15 days, the voltammograms of
PNPG were measured under the same conditions with
the same electrode and recorded. The MWCNT paste
electrode showed 1.7% less activity after 15 days com
pared to the initial current response. The relative stan
dard deviation (R.S.D.) of the peak current was in the
range of 1.0 to 3.5%. Therefore, it can be concluded
that the MWCNT paste electrode exhibits a good
response to repeatability and stability for the electro
chemical analysis of PNPG.
The reproducibility of the MWCNT paste elec
trode was investigated through cyclic voltammetric
using 1.0 mM PNPG. Five fabricated electrodes were
prepared on five consecutive days under the same con
ditions, and the peak current value was measured for
each electrode. It can be observed that the five elec
trodes showed almost the same current peak on five
consecutive days at constant temperature. The relative
standard deviation (RSD) of the cyclic voltammetric
currents obtained less than 4.0%.
4. CONCLUSIONS
PNPG has been widely used as a colourproducing
substrate for assaying the activity of AG spectrophoto
metrically. Although this substrate is sensitive and use
ful for the enzyme assays, the application of a spectral
bition potential of medicinal plant extracts on the
interferences exerted by the natural colour of the
8 10 12 extracts. Unlike the spectralbased PNPG system, the
electrochemicalbased PNPG system is not affected
by the colour of the inhibitor. However, attempts to
develop the electrochemicalbased system have been
hindered by the barely detectable currents of PNPG
and nitrophenol obtained using conventional elec
trodes. The use of a MWCNT electrode markedly
improved the peak currents of PNPG and its enzy
matic product nitrophenol, which demonstrates that
this electrode can be used to assay the enzymatic activ
RUSSIAN JOURNAL OF ELECTROCHEMISTRY Vol. 48 No. 8 2012
 ELECTROCHEMICAL MEASUREMENT OF THE ANTIDIABETIC POTENTIAL
10
Current, µA
0 Acarbose
–5
–10
–0.4
Fig. 7. Interferences study for Acarbose to measure the AG enzyme inhibition.
ity and the inhibition efficiency of the medicinal plant
extracts.
The fabricated PNPGbased MWCNT paste elec
trode exhibited good sensitivity for the measurement
of the inhibition of the AG enzyme reaction by medic
inal plant extracts. The MWCNT paste electrode also
showed good repeatability and reproducibility. The
electrode is useful for determining the antidiabetic
potential of medicinal plant extracts. The electrode
construction was simple and utilises no unstable or
toxic materials. Thus, this electrode can be regarded as
green based on economic and environment concerns.
The extension of this work for the development of a
reagentless and disposable biosensor for the practical
and easy measurements of the antidiabetic potential of
medicinal plants is reported separately.
ACKNOWLEDGMENTS
The researchers would like to thank the University
Malaysia Perlis (UniMAP) for their financial support
and the grant provided for this research work.
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