Biomedicine & Pharmacotherapy 140 (2021) 111763

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Original article

Sensitive identification of silibinin as anticancer drug in human plasma

samples using poly (β-CD)-AgNPs: A new platform towards efficient
clinical pharmacotherapy
Rana Ansari a, b, Mohammad Hasanzadeh b, *, Maryam Ehsani c, Jafar Soleymani b, d,
Abolghasem Jouyban b
a
Student Research Committee, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
b
Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
c
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
d
Liver and Gastrointestinal Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: Silibinin is effective in significantly inhibiting the growth of cancer cells which shown significant anti-neoplastic
Silibinin effects in a variety of in vitro and in vivo cancer models, including skin, breast, lung, colon, bladder, prostate and
advanced Pharmaceutical Analysis kidney carcinomas. So, development of a new method to its biomedical analysis in clinical samples in highly
Biomedical Sensor
demanded. In this study, an innovative electroanalysis method for the accurate, sensitive and rapid recognition
Nano-biomedicine
Clinical pharmacotherapy
of silibinin in human plasma samples was proposed and validated. The sensing platform was designed using
silver nanoparticles (AgNPs) dispersed on the polymeric layer of β-cyclodextrin (β-CD). AgNPs with cubic shape
providing a large effective surface area for β-CD electropolymerization. So, a layer with high electron conduc­
tivity boosting the detection electrochemical signals. Also, poly(β-CD) providing an efficient substrate with
cavities to interact with silibinin and its oxidation. Differential pulse voltammetry technique was conducted to
measure silibinin concentration in human real samples. Under optimized conditions, proposed sensor indicated
linear relationship between the anodic peak current and concentration of silibinin in the range of
0.0103–10.3 µM on the standard and human plasma samples. Based on obtained results, proposed sensor is an
efficient platform to efficient therapy of cancer based on recognition of silibinin in clinical samples.

1. Introduction silibinin (50–60%), silychristin (20%), silidianin (10%), isoSilibinin

Cancer is reported as a far fatal disease according to its worldwide
incidence and mortality rate reported by the World Health Organization
[1]. In the past few decades, herbal extracts have been playing an un­
deniable role in either prevention or therapy of various cancer types [2].
Statistics represent that near 60% of the common drugs in cancer
treatment or prevention have been derived from plants [3]. Among
different categories of natural compounds, flavonoids have attracted
researchers’ attention since they are an outstanding antioxidant,
cell-growth inhibitor, apoptosis inducer and anti-proliferative agents [3,
4]. Amongst several naturally-occurring flavonoids, silymarin drawn out
from milk thistle plant (scientifically named Silybum marianum) has
positively contributed to treatment of hepatitis, cirrhosis, poisoning
caused by herbicides and cancer. Silymarin is typically constituted of
(5%) and trace amounts of taxifolin and quercetin [5]. The major
bioactive ingredient of silymarine named as silibinin (also known as
silybin) is a polyphenolic compound with two diastereomers (silybin A
(2R, 3R, 10R, 11R) and silybin B (2R, 3R, 10S, 11S)) containing hydroxyl
and carboxyl groups [6].
The US Food and drug administration (FDA) has proved silibinin
consumption in tablet or capsule formulation as a drug for liver disor­
ders treatment [7]. Considering recent investigations, silibinin has
several other therapeutic applications such as hormonal [8], oxidative
stress control [9], fibrosis inhibitor [10], anti-inflammatory [11], hep­
atoprotective [12] and anticancer [3] effects. Thus, it is of great
importance to develop precise, accurate, rapid, simple and cost effective
analysis method for silibinin detection in biological samples. According
to its hydrophobic structure, silibinin has a low solubility in aqueous

* Corresponding author.
E-mail address: hasanzadehm@tbzmed.ac.ir (M. Hasanzadeh).

https://doi.org/10.1016/j.biopha.2021.111763
Received 26 April 2021; Received in revised form 15 May 2021; Accepted 20 May 2021
Available online 24 May 2021
0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
R. Ansari et al. Biomedicine & Pharmacotherapy 140 (2021) 111763

Table 1
Analytical figure of merits of Silibinin detection by different methods.
Detection method Matrix Linear range (µM) LOD (µM) Refs.

HPLC- UV Plasma 0.014–0.228 0.0058 [32]

HPLC-EC 0.011–0.466 0.0005 [33]
HPLC-UV 0.011–0.466 0.011 [33]
TLC Serum 0.420–1.447 – [34]
UPLC-UV Standard Methanol Solution – 1.587 [35]
EC Britton–Robinson buffer 0.2339–336 0.07 [36]
RP-LC Standard Methanol Solution 333.769–3354.02 – [37]
LC-MS Commercial milk thistle extracts 233.404– 233,404 – [38]
capillary zone electrophoresis (CZE) Standard Methanol Solution 23,340.491– 466,809.821 1167.024 [39]
UHPLC–IM–QTOF milk thistle-based food supplements 0.116–11.670 – [40]
HPLC-DAD Standard Methanol Solution 70.021–583.512 2.567 [40]
Quantitative nuclear magnetic resonance (qNMR) Standard Solution 23.340–5448.01 1.400 [41]
uHPLC-DAD Standard Solution 58.351–5448.01 9.336 [41]
EC Biological samples 0.002–0.123 0.001 [42]
Fluorescence Aqueous solutions 0.023–1.167 0.079 [43]

environments resulting in a limited bioavailability [13]. So, it is neces­
sary to propose new analysis methods with a high sensitivity and
without any pre-concentration steps. To date, some of analytical
methods have been reported for silibinin monitoring in diverse matrices
which are listed in Table 1.
Whether chromatography-based methods or spectroscopy tech­
niques are labor-intensive and time-consuming demanding sophisti­
cated bench top instruments. On the other hand, according to growing
importance and consumption of silibinin in therapeutics, there is an
urgent need to develop robust analytical methods that are affordable,
portable, user-friendly, and miniaturized with reduced analysis time,
high selectivity, and sensitivity. Electrochemical methods possessing
some advantages, such as high sensitivity, ability to miniaturize, rapid
and simple analysis which suggest an efficient alternative for the se­
lective detection of silibinin in real samples.
Glassy carbon electrode (GCE) as an electrochemically inert surface
in a wide potential window with proper chemical stability and electrical
conductivity has been employed in many electrochemical bioanalysis
assays [14]. To improve its performance, electrode surface modification
has been strongly recommended [15]. In situ potentiodynamic electro­
polymerization of monomers containing functional groups to form
conductive polymeric films is the extensively applied strategy for elec­
trode surface modification [16]. Electropolymerization of conductive
polymers on GCE surface does not need any oxidative substance or
catalyst and leads to a surface with more electroactive sites, consider­
able stability, reproducibility, homogeneity and rapid electron transfer
[14,17]. β-cyclodextrin (β-CD) is a cyclic macro-saccharide composed of
seven D-glucose subunits with α-1,4 glycosidic links [18]. The molecular
structure of β-CD contains two kinds of functional groups: primary and
secondary alcohols with functional –OH groups in the outer side
resulting in hydrophilic character and C–H bonds in the inner side
inducing a significant hydrophobic character [19]. These two functional

Fig. 1. Schematic view of fabrication of the modified electrode.

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R. Ansari et al. Biomedicine & Pharmacotherapy 140 (2021) 111763

groups give β-CD a donut-shaped structure with an inward cavity of

0.65 nm in diameter. Thus, β-CD can construct a host-guest complex
with target molecules through hydrophobic interactions and hydrogen
bonds [20].
Silver nanoparticles (AgNPs) are mostly applied noble metal as sur­
face modifiers owing to their properties including: low cost, accessi­
bility, biocompatibility, high chemical and physical stabilities, high
surface to volume ratio, considerable electrical conductivity and ho­
mogenous morphology with narrow size distribution [21,22]. Electro­
deposition of Ag NPs on GCE surface increases the active surface area
and dense loading which improves the sensitivity and detection limit of
the sensor [23].
The favorable properties AgNPs and poly (β-CD) persuaded us to
prepare a novel electrochemical sensor for silibinin detection in real
sample. The sensor assembly steps are demonstrated in Fig. 1. GCE
Fig. 2. CVs for AgNPs electrodeposition on the GCE surface at − 0.6 to 0.2 V,
electrode surface was modified with AgNPs and poly (β-CD) to gain a n = 60 cycles, sweep rate = 0.05 mV/s.
sensitive platform or biomedical analysis. These substances were
employed to form conductive polymeric layers on electrode surface
microscope (FE-SEM, Hitachi-SU8020, Czech) with an operating voltage
through electropolymerization technique to increase the electron
of 3 kV.
transfer rate and active surface area. The effect of diverse factors in
electropolymerization and target analysis were investigated and opti­
mized. Cyclic voltammetry (CV) and differential pulse voltammetry 2.3. Sensor fabrication
(DPV) techniques were conducted to measure silibinin concentration in
samples. 2.3.1. Electrode cleaning
Bare GCE was cleaned both physically and electrochemically to be
2. Experimental prepared for modifications. Firstly, electrode was polished on a polish­
ing pad containing alumina powder followed by sonication in piranha
2.1. Chemicals and reagents solution. So, the electrode was plunged in 0.1 M H2SO4/0.1 M HNO3
and 1:1 water/acetone mixtures for 15 min and finally cleaned with
Silver nitrate, silibinin, nitric acid, sodium phosphate monobasic deionized water and allowed to dry at room temperature. Secondly, the
dehydrate (NaH2PO4.2H2O), disodium hydrogen phosphate heptahy­ GCE was chemically pretreated. By immersing in H2SO4 0.1 M and CV
drate (Na2HPO4.7H2O), acid ascorbic, Arginine, Methionine, Tyrosine, technique was performed for 30 cycles between − 0.2 and 0.8 V until
dopamine, serine, sodium acetate, proline, aspartic acid, leucine, repetitive cycles were observed.
glycine, sodium chloride, cysteine, glucose, potassium chloride were
procured from Merck (Darmstadt, Germany) and utilized without 2.3.2. Electrogeneration of AgNPs on GCE
further refinement. Deionized water obtained from Shahid Ghazi phar­ The cleaned bare GCE was put in electrochemical cell containing
maceutical Co. was used to prepare solutions. Human plasma samples AgNO3 (0.005 mmol) and HNO3 (21.1 mmol). CV technique was applied
were provided by the Iranian Blood/Plasma Transfusion Research Cen­ at − 0.6 to 0.2 V and the sweep rate of 50 mV/s vs. Ag/AgCl with 30
ter (Tabriz, Iran). cycles to electrochemically generation of AgNPs on GCE surface (Fig. 2).
Phosphate buffer saline 0.1 M (PBS) employed as supporting elec­
trolyte was prepared by dissolving 0.62 g of NaH2PO4 and 2.8 g of 2.3.3. β-CD electrodeposition
Na2HPO4 in deionized water. The Ag Solution used for AgNPs electro­ AgNPs/GCE was soaked in the electrochemical cell containing 5 mM
deposition on electrode surface was provided by mixing 0.001 g AgNO3 solution of β-CD with pH = 5. Then, CV technique at − 2.0 to 2.0 V for 20
and 60 µL HNO3 and diluting with 10 mL deionized water. β-CD stock cycles was performed to form a polymeric layer of β-CD on AgNPs/GCE.
solution was prepared by dissolving 0.0567 g β-CD in PBS buffer (0.1 M
and pH = 5.7) and stored at 4 ◦ C. 2.3.4. Analytical approach
We did not used any patient samples. The analytical performance of the sensor was evaluated using CV and
DPV techniques. The sensor AgNPs-P(β-CD)/GCE was incubated in
2.2. Apparatus standard solutions containing different concentrations of silibinin and
then placed in the cell to record cyclic and differential pulse
All Electrochemical experiments were conducted in a common three- voltammograms.
electrode cell (from Metrohm) empowered by an electrochemical system
including AUTOLAB system with PGSTAT302 N (Eco Chemie, Utrecht, 3. Results and discussion
the Netherlands). NOVA1.11 software was employed to run system. Ag/
AgCl-Sat’d KCl (from Metrohm) and platinum wire were utilized as the 3.1. Electrode surface modification
reference and counter electrodes, respectively. GCE from Azar Electrode
Co. (Urumia, Iran) was used as working electrode. CV technique was As mentioned in Section 2.3.1 GCE surface was polished and cleaned
performed for surface modification and experiment optimization. DPV via physical and chemical methods. AgNPs were electrochemically
technique was carried out for silibinin monitoring in both standard and deposited on GCE surface. For this purpose, Ag+ ions are reduced and
plasma samples. The modified electrodes’ surface morphology was AgNPs are generated in situ and directly formation method. Electro­
investigated using high-resolution field emission scanning electron generation through CV technique allows us to control the aggregation of

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R. Ansari et al.
Fig. 3. CVs for β-CD electropolymerization on the AgNPs/GCE surface at − 2 to
2 V, N = 20 cycles, sweep rate = 0.1 mV/s.
particles and consequently decrease the thickness and uniform distri­
bution of AgNPs on electrode surface.
After multiple CVs for the electrodeposition of Ag NPs on the GCE
surface, both oxidation and reduction peaks current showed an upward/
downward trend with an increase in the number of CV scans resulting in
an increase/decrease in the area under the voltammogram curves. This
depicts the formation of electro conductive layer of AgNPs on the
working electrode.
In the next step, a polymer film of β-CD was formed on AgNPs/GCE
surface. Electropolymerization was performed via CV technique. As this
technique allows us to control the scanning parameters including
number of cycles, potential range and speed, the morphological char­
acteristics of the film can be controlled and determined [24]. While
conducting CV, an increase in the anodic and cathodic currents with
increase in the number of cycles was observed. Moreover, as the β-CD
polymer chain grows, a more extended network of delocalized electrons
are formed. Thus, the oxidation potential decreases gradually.
Electropolymerization mechanism comprises of several consecutive
steps[25]: I) The β-CD is oxidized to form radical cation on the carbon
atom possessing –OH functional group. This carbon atom covalently
links to the GCE surface while forming a ketone. Thus, establishment of
β-CD monomers on GCE surface happens in this step [26]: II) while
potential is applied, primary hydroxyl groups become delicate to
oxidation and conversion to carboxylic acid functional group [27]. Two
cation radicals interact with each other to make a dimer structure
through esterification reaction [28]. In other words, oligomers couple to
radical cations through C-O-C bonds. III) The oxidized dimer links to
another radical cation monomer to form a trimer structure. IV) The
polymer chain growth proceeds with coupling of oligomers and oxidized
monomers.
As shown in Fig. 3, the primary and secondary cycles of the β-CD
electropolymerization depict distinct behavior from the other cycles
because it corresponds to the onset of the electropolymerization process
and does not provide indications of polymer chain growth [29,30]. So,
the β-CD monomers commence to be polymerized on AgNPs/GCE
surface.
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Biomedicine & Pharmacotherapy 140 (2021) 111763
3.2. Field emission scanning electron microscopy (FE-SEM) and energy
dispersive X-ray spectroscopy (EDS)
FEG-SEM images of GCE, AgNPs/GCE, and AgNPs-P (β-CD)-GCE are
represented in Fig. 4A. According to Fig. 4A, the surface morphology of
bare GCE after cleaning steps is plain and smooth. After AgNPs depo­
sition on the GCE surface, cubic AgNPs are formed. Fig. 4B confirms the
successful electrodeposition of AgNPs. Fig. 4C indicates macromolecules
of β-CD with an arranged and orderly interwoven structure. This
compact layer containing cavities acts as a host for silibinin molecules
entrapment.
3.3. Electrochemical characterization of electrode surface modification
CV has been designated as an ordinary technique for exploring the
electrode surface properties while surface modification. In this study,
CVs were recorded in PBS solution (0.1 M, pH = 7.4) as the supporting
electrolyte to gain information about the electrochemical features of the
modified electrodes.
Fig. 5A depicts CVs of bare GCE, AgNPs/ GCE, and AgNPs-P (β-CD)-
GCE in the absence of silibinin in the supporting electrolyte at − 1 to
+1 V with sweep rate of 100 mV/s.
According to Fig. 5A, bare GCE does not have efficient electro cat­
alytic activity. Electrodeposition of AgNPs gives a noticeable conduc­
tivity to electrode surface. Sharp anodic and cathodic peaks at 0.45 V
and 0.11 V are emerged. Electrodeposited AgNPs also raise the active
surface area and provide greater area for polymeric layer of β-CD for­
mation. After electropolymerization of β-CD on AgNPs/GCE surface,
anodic and cathodic peaks at 0.18 V and − 0.70 V are emerged. Weaker
peak current in comparison with AgNPs/GCE are perceived since β-CD is
a macromolecule with poor electron conductivity. Table 2 shows anodic
and cathodic peaks position and current. In order to evaluate the elec­
trochemical behavior of the designed sensor in the presence and absence
of the silibinin. Fig. 5B shows CVs of AgNPs-P (β-CD)-GCE in the absence
and presence of 103.6 µM silibinin (PBS, pH = 7.4) with a potential scan
rate of 100 mV/s. The results indicate that, using AgNPs-P (β-CD)-GCE
in the presence drug a new anodic peak appears at 0.48 V.
3.4. Effect of waiting time
As aforementioned, β-CD has a donut-shaped structure owning an
internal cavity of 0.65 nm in diameter. So, it is possible to develop hy­
drophobic interactions and hydrogen-bonding with silibinin molecules
causing to a host-guest complex [31]. AgNPs-P (β-CD)-GCE was
immersed in 500 µL of silibinin solution with concentration of 50 ppm
and incubated for a certain time. Then, CVs were recorded in PBS so­
lution. Incubation time and pH of supporting electrolyte were optimized
as they were effective parameters.
Fig. 6 shows the CVs of AgNPs-P (β-CD)-GCE incubated with silibinin
solution for 5, 10, 15, 20 min. Considering the peak currents in Table 3,
the optimum incubation time is 5 min. It can be attributed to over­
loading of silibinin molecules during longer time periods in P (β-CD)
cavities and steric hindrance preventing efficient electro catalytic ac­
tivity of electrode surface.
Table 3 also provides information about the peak currents of AgNPs-
P (β-CD)-GCE incubated with Silibinin solution for 5, 10, 15, 20 min
recorded via CV technique.
R. Ansari et al. Biomedicine & Pharmacotherapy 140 (2021) 111763

Fig. 4. FE-SEM images of A) Bare GCE, B) AgNPs/GCE, C) AgNPs-P (β-CD)/GCE in different magnifications.

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Table 3
Peak currents and potentials of AgNPs-P (β-CD)-GCE incubated with Silibinin
solution for 5, 10, 15, 20 min.
Incubation Anodic peak Anodic Cathodic Cathodic
time (min) potential (v) peak peak peak current
current potential (v) (µA)
(µA)

5 0.11 8 -0.01 -3.1

0.71 6 0.46 -5
10 -0.58 3.7 -0.37 -4
0.71 1.1 0.47 -8
15 0.10 1.9 -0.29 -1.6
0.71 1.4 0.46 -6
20 0.10 3 -0.33 -2.2
0.72 0.8

Fig. 5. A) CVs of Bare GCE, AgNPs/GCE and AgNPs-P (β-CD)-GCE in PBS so­
lution (0.1 M, pH 7.4), Sweep rate 100 mV/s B) CVs of AgNPs-P (β-CD)-GCE in
the absence and presence of Silibinin (103.6 μM) in PBS solution (0.1 M,
pH = 7.4) Sweep rate 100 mV/s.

Table 2
Potential and current of modified electrodes in different stops.
Electrode Anodic peak Anodic Cathodic Cathodic
potential (v) peak peak peak current
current potential (v) (µA)
(µA)

Bare GCE -0.013 6.4 0.8139 -1.63

AgNPs/ GCE 0.23 13 -0.37 -7
0.45 98 0.11 -48
P(β-CD)/Ag 0.18 1.4 -0.70 -0.46
NPs/GCE
Fig. 7. A) CVs of AgNPs-P(β-CD)-GCE in PBS solution (0.1 M) in pH range 2–10
at − 1 to 1 V, sweep rate 100 mV/s, B) calibration curve CV of silibinin in
different pH of 0.1 M PBS.

3.5. The effect of pH

After determining the appropriate incubation time (5 min), sup­

Fig. 6. CVs of AgNPs-P (β-CD)-GCE incubated with Silibinin solution for 5, 10,
15, 20 min at − 0.8 to 0.1 V in PBS (0.1 M, pH 7.4), sweep rate 100 mV/s.
porting electrolyte pH optimization to record CVs was conducted.
Fig. 7A illustrates the CVs of AgNPs-P (β-CD)-GCE in PBS solution with
pH range of 2–10. Considering the peak currents in Table 4, the optimum
pH for supporting electrolyte is 2. It is mainly because of the relation
between pKa of NaH2PO4 and Na2HPO4 and pH. When pH < pKa, the
amount of H+ to be transferred is considerable. So, the ion transfer rate
in the solution and electron transfer rate on electrode is greater. In
addition, it is found that the oxidation peak potential of silibinin shifted
to the negative potentials with increase of pH, The pH dependence of
Epa can be explained by the linear regression equation as Epa(V) = –
0.0442 pH + 0.5683 (R2 = 0.96) (Fig. 7B). Table 4 gives information
about the peak currents of AgNPs-P (β-CD)-GCE in PBS solution with pH
range of 2–10 recorded via CV technique.

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Table 4 4. Analytical method validation

Anodic and cathodic peaks of β-CDP/AgNPs/GCE in PBS solution with pH range
of 2–10. 4.1. Inter-day repeatability
pH Anodic peak potential (v) Anodic peak current (µA)

2 -0.73 5.2
The inter-day stability assessment to prove the precision of the
-0.62 0.3 proposed analysis method is conducted since it is significantly important
0.08 3.6 for practical applications. For inter-day stability investigation, AgNPs-P
0.48 6.3 (β-CD)-GCE was prepared and DPV technique was conducted in PBS
0.72 0. 3
(0.1 M, pH = 2) for 5 consecutive days with 24 h intervals. Fig. 10A
3 -0.80 7
0.46 1.1 illustrates the recorded DPV at − 1 to 1 V.
0.66 1.3 Considering changes in peaks current shown in Fig. 10B, it is obvious
4 -0.81 2 that the reported sensor is stable just for 24 h and as time passes, it loses
0.38 3.3 the effective functionality for silibinin detection.
0.65 4
5 -0.93 1
-0.75 1 4.2. Stability analysis
0.10 6
0.32 2.4
0.61 3
To evaluate the stability of electrodeposited layers on GCE surface,
6 -0.80 2 CV technique was conducted for 1, 5, 50 and 100 successive cycles and
0.10 6 immediately after each CV, DPVs of sensor were recorded (Fig. 11A). As
0.25 2.4 it is observed in Fig. 11B, the peak current of voltammograms have
0.56 3
minor change which represents the excellent stability of coated layers.
7 -0.92 4
0.14 1 In this research, the freeze thaw cyclic stability was also studied
0.19 2 because it is likely the patients’ plasma samples could be freeze-thawed
8 -0.91 4 until analyses with the proposed method. Fig. 12 indicates the Freeze-
-0.15 4 thaw stability of the silibinin analysis method in human plasma sam­
0.19 1.7
9 0.16 1.2
ples after three freeze and thaw cycles. This experiment was done using
-0.23 3 plasma aliquots comprising low, medium and high concentrations of
10 -0.95 2 silibinin (0.082, 2 and 10.3 µM, respectively). These solutions were
-0.19 4 stored at − 4 ◦ C for 3 days and thawed at room temperature. Then, DPVs
0.18 1.1
at potential range of 1 to 1 V were recorded.
Moreover, bench-top stability of the method was studied by keeping
3.6. Analytical approach silibinin plasma samples at room temperature for 12 h and 24 h and then
DPVs were recorded. Fig. 13 depicts the changes in the recorded vol­
3.6.1. Silibinin detection in standard samples tammograms. As it is shown in Table 5, only plasma sample containing
Fig. 8A presents DPVs of AgNPs-P (β-CD)-GCE of silibinin in PBS 2 µM of silibinin indicates bench-top stability (Fig. 14).
solution (0.1 M, pH = 2) as supporting electrolyte. As shown in Fig. 8, as
silibinin concentration increases, the peak current goes up. Calibration 4.3. Selectivity studies
curve (Fig. 8B) is drawn using peaks currents in respect of silibinin
concentration range. The corresponding regression equation is Selectivity of a method is described as the sufficiency to detect the
y = 0.7675x + 0.1115, where y is Ipa (µA) and x is silibinin concentra­ target molecule in the proximity of various species usually existing in the
tion (µM) with a determination coefficient of R2 = 0.967. biological samples without their interference. The selectivity of the re­
ported sensor toward silibinin was also evaluated by DPV technique in
3.6.2. Determination of silibinin in human plasma samples the presence of biological molecules and ions. Ascorbic Acid, arginine,
The developed electrochemical sensor was also employed for silibi­ methionine, tyrosine, dopamine, serine, acetate, proline, aspartic acid,
nin detection in human plasma samples. No additional steps were leucine, glycine, sodium chloride, cysteine, glucose, potassium chloride,
attended to determine silibinin in human plasma samples. Silibinin so­ Cu+, Al3+, Fe3+, Zn2+, NH were selected as interfering agents. The
lution with different concentrations were added to human plasma electrochemical sensor was employed to measure the peak current in the
samples, then incubated with AgNPs-P (β-CD)-GCE for 5 min. DPVs at absence and presence of these molecules in the plasma samples. The
− 1 to 1 V was applied for silibinin measurement in human plasma peak currents are reported in Table 6.
samples. The recorded voltammograms are depicted in Fig. 9.
DPVs of AgNPs-P (β-CD)-GCE for the silibinin determination in 5. Conclusion
standard and plasma samples are consistent. Both demonstrate a growth
as silibinin concentration increases. Also, according to calibration curve In summary, a novel sensing platform consisting of AgNPs and P
in Fig. 9, it is evident than the sensor can accurately and precisely detect (β-CD) was developed to detect silibinin in untreated plasma samples.
silibinin in untreated plasma samples with a linear function of CV technique was implemented to modify bare GCE surface. Under
y = 0.4881x + 0.0267 (where y is Ipa (µA) and x is silibinin concentra­ optimized conditions, a good linear relationship between the anodic
tion (µM)) and correlation coefficient is 0.9903 (R2 = 0.9903). peak current and silibinin concentration in the range of 0.0103–10.3 µM
with correlation coefficients of 0.9715 and 0.9906 for standard and
untreated human plasma samples, respectively. Employing the

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Fig. 8. A) DPVs of AgNPs-P(β-CD)-GCE with different concentrations of silibinin (0.0103, 0.02, 0.041, 0.082, 0.124, 0.165, 0.2, 2, 4.1, 6.2, 10.3 µM) in PBS solution
(0.1 M, pH = 2), B) Calibration curve of AgNPs-P(β-CD)-GCE.

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Fig. 9. A) DPVs of AgNPs-P (β-CD)-GCE with different concentrations of silibinin in untreated whole plasma (0.0103, 0.02, 0.041, 0.082, 0.124, 0.165, 0.2, 2, 4.1,
6.2, 10.3 µM), B) Calibration curve of silibinin in untreated (whole) plasma.

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Fig. 12. The Freeze-thaw stability of the Silibinin in plasma samples: DPVs of
AgNPs-P (β-CD)-GCE in different conditions.

Fig. 10. A) DPVs of AgNPs-P(β-CD)-GCE in different day in 0.1 M PBS pH 2, B)

Influence of different day on peak currents.

Fig. 13. Bench-top stability of the Silibinin in plasma samples after A) 12 h and
B) 24 h by recording DPVs.

Table 5
Details of the stability of the proposed method in whole plasma samples.
Concentration (µM) Short time temperature (detected)

12 h 24 h

0.082 0.027 0.0104

2.00 0.314 0.315
10.3 0.162 1.98

fabricated electrochemical sensor for silibinin detection offers several

Fig. 11. A) DPVs of AgNPs-P(β-CD)-GCE in different cycle numbers (1–100) in
0.1 M PBS pH 2, B) Effect of different CV’s cycle numbers of AgNPs-P(β-CD)-
GCE on variations of peak current.
advantages such as fast and in situ electrode surface modification, good
stability of electrode surface substrate, high selectivity, and rapid elec­
tron transfer process and analyte detection. Thus, these features portend
that the proposed sensor can be utilized as an appropriate sensor for the
sensitive and selective analysis of silibinin in both standard and plasma
samples. It is also prognosticated that this research will open the door to
new opportunities for the development of pioneer series of electro­
chemical sensors for rapid, selective and sensitive analysis of natural and
herbal compounds in biological fluids.

10
R. Ansari et al. Biomedicine & Pharmacotherapy 140 (2021) 111763

Fig. 14. Influence of detection in presence of interfering agents in 0. 1 M PBS pH 2.

Table 6
Maximum tolerable concentration of interfering species on 1 ppm of Silibinin in whole plasma matrices.
Interfering Concentration of the interfering agent/ Concentration of Silibinin/ With interfering agent Without interfering Interfering
agent ppm ppm (µA) agent percent

Ascorbic acid 1.0 1.0 0.452 0.481 5.86

Arginine 1.0 1.0 0.439 0.481 8.61
Methionine 1.0 1.0 0.416 0.461 9.72
Tyrosine 1.0 1.0 0.657 0.562 -17.10
Serine 1.0 1.0 0.499 0.562 11.04
Proline 1.0 1.0 0.540 0.562 3.88
Aspartic acid 0.01 1.0 0.329 0.417 20.86
Dopamine 0.1 1.0 0.545 0.431 -26.58
Leucine 0.1 1.0 0.457 0.431 -6.22
Glycine 0.1 1.0 0.468 0.417 -12.40
Cysteine 0.1 1.0 0.564 0.481 -17.40
Glucose 1.0 1.0 0.433 0.481 9.93
NaCl 0.1 1.0 0.452 0.461 1.86
Kcl 0.1 1.0 0.404 0.431 6.02
Cu+ 0.1 1.0 0.430 0.481 10.48
Sodium acetate 1.0 1.0 0.491 0.562 12.50
NH+ 4 1.0 1.0 0.421 0.461 8.54
Fe3+ 0.1 1.0 0.563 0.481 -17.21
Zn2+ 1.0 1.0 0.533 0.562 5.00
Al3+ 0.1 1.0 0.445 0.481 7.50

CRediT authorship contribution statement Appendix A. Supporting information

Rana Ansari: Experimental works, Writing - original draft.
Mohammad Hasanzadeh: Conceptualization, Writing - original draft,
Writing - review & editing. Maryam Ehsani: Experimental works, Data
analysis. Jafar Soleymani: Experimental works, Methodology. Abol­
ghasem Jouyban: Writing - review & editing.
Conflict of interest statement
The authors have no conflict of interest to declare relevant to this
article.
Acknowledgments
The authors gratitude the financial supports from Pharmaceutical
Analysis Research Center of Tabriz University of Medical Sciences
(Tabriz, Iran). The results presented in this work was part of thesis of
Rana Ansari (Project No. 63994).
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.biopha.2021.111763.
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