A combination risk assessment of paracetamol: Electrochemical oxidation behavior and cytotoxic effect evaluation of paracetamol in the presence of: N 1, N 2-dibenzylethane-1,2-diamine

Article abstract of DOI:10.1039/c5nj03250d

The cytotoxic effect of paracetamol in the presence of a diamine derivative was evaluated in liver cells. In this study, hydropyrazinoquinoxalinylidene-acetamide (HPQA), as an agent that is toxic to the liver, was synthesized in an electrochemical cell as a simulated body environment by an electrooxidation reaction. A direct electron transfer (DET) mechanism occurred during the process on the surface of the carbon anode. The electrochemical oxidation of paracetamol was studied using cyclic voltammetry and controlled-potential coulometry (CPC) techniques. The product was characterized by FT-IR, ¹H NMR, ¹³C NMR and ESI-MS² after purification. The cytotoxicity of the final compound was evaluated using an MTT assay on the CCL-13 liver cell line. The results indicate that the presence of amine derivatives leads to an increase in the toxic effects of paracetamol in the human body. The cell viability at a concentration of 500 μg mL^-1 was 78% for paracetamol, whereas the viability of liver cells in the presence of the product was 18% at 168 μg mL^-1. A cycloaddition mechanism was suggested according to the overall results that were obtained.

Full text of DOI:10.1039/c5nj03250d

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DOI: 10.1039/C5NJ03250D
A combination risk assessment of paracetamol: electrochemical oxidation behavior
1
2
and cytotoxicity evaluation effects of paracetamol in the presence of N ,N -
dibenzylethane-1,2-diamine
*[a]
[a]
[a]
[b]
Bahram Dowlati , Mohamed Rozali Othman , Azizan Ahmad , Elham Safaei
[
a]
School of Chemical Sciences and Food Technology, Faculty of Science and
Technology, Universiti Kebangsaan Malaysia 43600 UKM Bangi Selangor, Malaysia
[
b]
Department of Chemistry, Faculty of Sciences, Kurdistan University, Pasdaran St.,
6
6177 Sanandaj, Iran
Corresponding author: Bahram Dowlati. Tel: +60178751250
Email: bahram.dowlati@hotmail.com
1

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DOI: 10.1039/C5NJ03250D
Abstract
The cytotoxicity effect of paracetamol in the present of a diamine derivative was evaluated
in the liver cell. In this study hydropyrazino-quinoxalinylidene-acetamide (HPQA) as agent
toxicity of liver was synthesized in electrochemical cell as the simulated body environment
by electrooxidation reaction. A direct electron transfer (DET) mechanism occurred during the
process on the surface of the carbon anode. The electrochemical oxidation of paracetamol
was studied using cyclic voltammetry and controlled-potential coulometry (CPC) techniques.
1
13
2
The product was characterized by FT-IR, H NMR, C NMR and ESI-MS after purification.
Cytotoxicity of final compound was evaluated using MTT assay on the CCL-13 line cell of
liver. The results indicate that the presence of amine derivatives lead to increased toxicity of
paracetamol toxicity effects in the human body. Cell viability at concentration of 500µg/mL
was 78% for paracetamol while viability of liver cells in present of product was 18% for 168
µg/mL. The cycloaddition mechanism was suggested according to the overall obtained
results.
Key words: Cytotoxicity effect; Diamine; Electroorganic synthesis; Paracetamol
1
. Introduction
Paracetamol is a valuable drug in widespread use in pharmaceutical industries. It is
extensively used as analgesic and antipyretic drug. It is mainly used for the relief of mild to
1
–3
moderate pain associated with arthritis, head ache, back ache, and postoperative pain . It is
4
also used for treatment of fevers of bacterial or viral origin . Electrochemical methods are
widely used for the study of electro active compounds in pharmaceutical forms and
5
physiological fluids due to their simple, rapid, and economical properties . As an electro
2

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active substance, paracetamol has also attracted much interest. Paracetamol is considered a
DOI: 10.1039/C5NJ03250D
safe drug at therapeutic level but in overdose states it produces renal necrosis in both humans
6
and experimental animals . In experimental animals, it causes hepatic depletion of
7
glutathione at overdose level . The first report was elucidated mechanisms of paracetamol-
induced toxicity. It showed that a minor route of paracetamol metabolism involved its
conversion by monooxygenases (cytochrome P450s) to a reactive arylating metabolite,
known as N-acetyl-p-benzoquinone imine (NAPQI), which may cause acute tissue necrosis
8
,9
with toxic doses of this agent . NAPQI causes a depletion of both the mitochondrial and
cytosolic pools of reduced glutathione, thus cellular proteins are directly oxidized by the
1
0
reactive metabolite . NAPQI also inhibition of the antioxidant enzyme-glutathione
1
1
peroxidase . Because electrochemical oxidation very often parallels the cytochrome P450s
catalyzed oxidation in liver microsomes, it was interesting to study the anodic oxidation of
paracetamol in presence of a diamine derivative. A few studies have evaluated the effects of
diamines on paracetamol. This encourages us to study electrochemical oxidation of
1
2
paracetamol in the presence of N ,N -dibenzylethane-1,2-diamine as nucleophile agent. The
synthesized product was characterized by different techniques. Then, the CCL-13 line was
used as a model of liver progenitor cells to examine the cytotoxicity effects of paracetamol
and the corresponding oxidized product (HPQA) in the presence of the diamine derivative.
The best of our knowledge, this is the first report of electrochemical oxidation of paracetamol
in the presence of diamine. Additionally, the electrochemical synthesis and the cytotoxicity
effects of final product were simulated in the electrochemical cell.
2
2
2
. Experimental
.1. Chemical and electrochemical synthesis
.1.1. Materials and methods
3

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Reagent: The paracetamol (ACOP) was reagent grade material from Aldrich. The
DOI: 10.1039/C5NJ03250D
ammonium chloride and other acids and bases were of pro-analysis grade from E. Merck.
Benzaldehyde, 99% and ethylenediamine were purchased from Aldrich. The reagents and
solvents used in this study were of analytical grade and were used without further
purification.
Apparatus: All electrodes for cyclic voltammetry experiments were from France
Radiometer Analytical. Controlled-potential coulometry and cyclic voltammetry were
performed using an Autolab model PGSTAT 302N potentiostat/galvanostat. The working
electrode used in the voltammetry experiments was a glassy carbon disc (1.8 mm diameter).
The glassy carbon was polished with polishing cloth before each measurement. A platinum
wire was used as a counter electrode and the reference was a saturated calomel electrode
1
2
(
SCE). An undivided cell was used for controlled-potential coulometry . The working
electrode potentials vs. SCE were measured. The SCE reference and carbon rods were placed
together, and their distance from the counter electrode was approximately 20 mm. A
magnetic stirrer was used during electrolysis.
1
2
2
.1.2. Chemical synthesis of N ,N -dibenzylethane-1,2-diamine (3)
Benzaldehyde (4.24 g, 40 mmol) and ethylenediamine (1.20 g, 20 mmol) were mixed in an
appropriate beaker in 100 mL MeOH. The obtained mixture was stirred overnight at room
temperature, and then sodium borohydride (6.04 g, 160 mmol) was added. The mixture was
refluxed for 2-3 hours, cooled and poured into 250 mL of H O. The solution was removed by
2
filtration off and evaporated to dryness. The residue was then extracted with water-
chloroform. The organic layer was separated and dried over anhydrous CaCl . The solvent
2
was evaporated to yield yellow oil. The product (3) was characterized as a pure compound by
1
13
2 13
FT-IR, H NMR, C NMR, and ESI-MS
.
4

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DOI: 10.1039/C5NJ03250D
2
.1.3. Electro-organic synthesis of final product (11)
For the electrodeposition experiments, in typical procedure, an aqueous solution of
ammonia buffer (pH 9.0, 0.1mol/L) was pre-electrolyzed at 0.42 V versus SCE.
1
2
Subsequently, 0.35 mmol of paracetamol (1) and 0.35 mmol of N ,N -dibenzylethane-1,2-
diamine (3) were added to the cell, and the electrolysis was performed using the same
potential at room temperature (Scheme 1). To reactivate the graphite anode the process was
cut off during the electrolysis and the anode washed in acetone. After completion the
electrolysis, the precipitated solids that separated from the setup, were filtered, washed with
distilled water and dried with sodium sulfate. The product was purified and characterized by
1
3
2 1
several techniques including C NMR, ESI-MS , H NMR and FT-IR.
Scheme1 diagram of preparation process for the final product using electrooxidation reaction.
2
2
.2. Characterization of products
1
2
.2.1. N ,N -dibenzylethane-1,2-diamine (C H N ) (3)
1
6
20
2
Isolated yield = 67.6%, IR (KBr): 3304, 3084, 3061, 3027, 2924, 2829, 1950, 1877, 1811,
603, 1584, 1494, 1452, 1407, 1358, 1202, 1108, 1074, 1054, 1028, 982, 911, 820, 736, 698
1
5

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-1
1
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DOI: 10.1039/C5NJ03250D
and 591 cm . H NMR, δ ppm (600 MHz CDCl ): 2.01, 2.76, 3.77, 7.24-7.25, 7.27-7.31,
3
1
3
7
.32-7.34. C NMR, δ ppm (600 MHz CDCl ): 48.6, 53.9, 126.9, 128.2, 128.45, 140.3. ESI-
3
2
+
MS : m/z, calcd. 240.16; found 241.1 (M +1)
.2.2. N- (1,4,6,9-tetrabenzyl-10-oxo-1,2,3,4,6,7,8,9-octahydropyrazino [2,2 g]
guinoxalin 5 (10H)-ylidene) acetamide (C H N O ) (11)
2
4
0
39
5
2
Isolated yield = 94.3%, IR (KBr): 3027, 2924, 2376, 1528, 1296, 1240, 1161, 1074, 1028,
-
1 1
9
2
7
7
15, 827, 735 and 700 cm . H NMR, δ ppm (600 MHz CDCl ): 2.121, 2.494, 2.497, 2.505,
3
.511, 2.519, 2.521, 3.171, 3.179, 3.186, 3.200, 3.221, 3.559, 3.63, 3.785, 3.806, 3.850,
.093, 7.105, 7.121, 7.133, 7.181, 7.195, 7.202, 7.209, 7.215, 7.227, 7.253, 7.260, 7.270,
13
.285, 7.295, 7.39, 7.312, 7.318, 7.327, 7.337, 7.349 and 7.361. C NMR, δ ppm (600 MHz
CDCl ): 50.64, 56.92, 58.73, 76.84, 77.05, 77.26, 89.04, 126.61, 126.80, 127.00, 128.12,
3
2
1
6
28.24, 128.31, 128.59, 128.92, 129.13, 129.54, 139.19, 140.27. ESI-MS : m/z, calcd.
+
21.31; found 622.28 (M +1).
2
2
.3. Cytotoxicity assays
.3.1. Reagents and apparatus of cytotoxicity assay
Reagent: Purified water, phosphate buffered saline solution (calcium and magnesium
free), trypan blue (0.4%) solution, thiazolyl blue tetrazolium bromide (SIGMA), dimethyl
sulfoxide (R&M Chemicals) and hydrogen peroxide (Univar). These chemicals were used
without further purification.
Cell culture: American type culture collection Chang liver cell, (Homo sapiens, epithelial,
CCL-13), with passage number 8.
2
Apparatus: Sterile plastic containers of issue culture grade: 75 cm tissue culture flask
(NUNC), 96-well tissue culture plate (NUNC) and 5 mL conical tubes (Falcon),
haemocytometer counting chamber (Hirschmann Laborgerate ), single and multichannel
6

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pipettor (Eppendorf and Treff Lab), sterile 0.20 µm syringe filter unit (Sartorius), inverted
DOI: 10.1039/C5NJ03250D
microscope with phase contrast (Nikon Eclipse TS 100), carbon dioxide heat-coil-jacketed
incubator (RS Biotech, model Galaxy R), biological safety cabinet class II (Bioair
TM
Instruments, model Aura 2000), microplate spectrophotometer (BIO-RAD, model xMark ).
2
.3.2. Preparation of cell culture and exposure of test material
American type culture collection Chang liver cell was prepared for this study. Cells were
grown as a monolayer at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air.
The confluent monolayer was removed by trypsinisation and number of viable cells was
calculated. Cells were seeded into a 96-well plate and incubated at 37°C for at least 12 hours
or until attaining 80% confluency. The test material was tested at 15.63, 13.25, 62.5, 125, 250
and 500 µg/mL concentration. Growth medium from each well of a 96-well plate containing
healthy culture was replaced with 200 µg of the test material solution. The cultures were then
incubated for 24 hours at 37°C in a humidified atmosphere of 5% carbon dioxide and 96%
air.
2
.3.3. Cytotoxicity assays for paracetamol
In this study, the cytotoxic potential of the material paracetamol was assessed by
determining the rate of cell proliferation. Cells were treated with varying concentration of test
material and cytotoxicity was determined by assessing the rate of cell proliferation through
the reduction of tetrazolium salts (MTT). Cell proliferation rate was obtained by dividing the
mean optical density (OD) values of the test material with the mean OD of medium control.
2
.3.4. Staining with MTT solution
5
mg/mL MTT solution was added into each well and incubated for 4 hours at 37°C in a
humidified atmosphere of 5% carbon dioxide and 95% air. The optical density (OD) of test
material and controls were determined by colorimetric method.
2
.3.5. Statistically analysis
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The rate of cell proliferation of test material is the percentage of cell viability calculated
DOI: 10.1039/C5NJ03250D
by dividing the mean optical density (OD) values of material with the mean OD absorbance
of negative control, and multiplied by 100. Low cell viability value represents evidence of
cytotoxic response.
2
.3.6. Cytotoxicity assays of hydropyrazino-quinoxalinylidene-acetamide (HPQA)
The toxicity test of hydropyrazino-quinoxalinylidene-acetamide (HPQA) that synthesized
from electrochemical step was carried out as a test material. Cells were treated with varying
concentrations of the test material. Additionally, cytotoxicity was determined by assessing
the rate of cell proliferation through the reduction of tetrazolium salts (MTT). Cell
proliferation rate was obtained by dividing the mean optical density (OD) values of the test
material with the mean OD of medium control.
3
3
. Results and discussion
.1. Chemical synthesis of N , N -dibenzylethane-1, 2-diamine (3)
1
2
1
3
As previously mentioned, conventional method for the synthesis of Schiff bases is
included reaction of aldehyde/ or ketone and amine in the presence of a catalyst under reflux
conditions. In this work a simple method for the preparation of heterocyclic compounds
based on Schiff base was provided without any catalyst in mild condition. This type of
reaction condition can be suggested as a two-step mechanism. The first step is reversible,
progressing through a carbinolamine intermediate and requires the removal of water by
azeotropic distillation with benzene to achieve high yields. Although the reaction is acid
catalyzed in the presence of amines, catalysts are not required generally. In the next step, the
adduct containing two azomethine groups (C=N) reduced using sodium borohydride to
provide of compound 3. According to the results, the analytical and spectral data are
completely consistent with the proposed formulation (supporting information).
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3
.2. Electrochemical oxidation of paracetamol in the presence of N ,N -
DOI: 10.1039/C5NJ03250D
dibenzylethane-1,2-diamine
1
2
The electrochemical behavior of paracetamol in the absence and presence of N ,N -
dibenzylethane-1,2-diamine has been investigated using cyclic voltammetry during the
electrosynthesis on the electrode of a glassy carbon in an aqueous solution (pH 9.0) at room
temperature.
Fig.. 1 (curve a) shows cyclic voltammetry of paracetamol 1. This cyclic voltammogram
shows one anodic peak (A ) at +0.22 V .VS. SCE and the corresponding cathodic peak (C )
1
1
at +0.148 V .VS. SCE, which correspond to the transformation of paracetamol 1 to N-(4-
methylenecyclohexa-2,5-dienyl) acetamide 2 and vice versa within a quasi-reversible two-
electron process. The oxidation of 1.0 mmol/L solution of paracetamol 1 in the presence of
1
2
1
.0 mmol/L N ,N -dibenzylethane-1,2-diamine 3 as a nucleophile in aqueous solution
containing 0.1 M in the buffer solution NH /NH Cl (pH 9.0) was studied in some detail (Fig..
3
4
1
. Curve b). Under these conditions, the anodic peak (A ) shifts to +0.34 V and the cathodic
1
counterpart of it (peak C ) disappears and the voltammogram shows an irreversible feature.
1
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Fig. 1. Cyclic voltammograms of: (a) 1.0 mmol/L paracetamol; (b) 1.0 mmol/L paracetamol in the
1
2
presence of 1.0 mmol/L N ,N -dibenzylethane-1,2-diamine at a glassy carbon electrode (1.8 mm
diameter) in the buffer solution NH /NH Cl (0.1 mol/L, pH 9). Scan rate: 50 mV/s; T=25 ± 1 ºC.
3
4
The effect of intensification of the potential sweep rate exhibits in Fig. 2 (curves a-g). It
shows that the anodic peaks (A ) have been increased by increasing of potential sweep rate
1
while cathodic peak have been disappeared with increasing of potential sweep rate up to 250
mV/s. However, the cathodic peaks have slightly increasing at higher scan rates of 250-1000
mV/s (Fig. 2, inset). The variation of the peak current ratio ( ꢀꢂꢃꢄ ꢀꢅ ꢂ) versus the scan rate for
ꢁ
1
2
a mixture of paracetamol (1) and N ,N -dibenzylethane-1,2-diamine (3) confirms the
reactivity of 2 toward 3.
1
0

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DOI: 10.1039/C5NJ03250D
Fig. 2. Typical cyclic voltammograms of 1.0 mmol/L paracetamol in the presence of 1.0 mmol/L
1
2
N ,N -dibenzylethane-1,2-diamine in water containing of 0.1 mol/L NH /NH Cl as the buffer and
3
4
supporting electrolyte (pH=9); at a glassy carbon electrode and at various scan rates. Scan rate from
a) to (d) are 10, 25, 50, and 100 mV/s, respectively. Inset: cyclic voltammograms of 1.0 mmol/L
(
1
2
paracetamol in the presence of 1.0 mmol/L N ,N -dibenzylethane-1,2-diamine at faster various scan
rates. Scan rate from (e) to (g) are 250, 500 and 1000 mV/s, respectively. T=25 ± 1 ºC.
Fig. 3 shows the cyclic voltammograms of 1.0 mmol/L paracetamol in absent (curve a)
1
2
and the presence (curve b) of 1.0 mmol/L N ,N -dibenzylethane-1,2-diamine in acidic
buffered solution pH 4. Results indicate that the height of cathodic peak do not disappears
with decreasing of pH. This behavior is related to the low intermolecular Michael addition
reaction rate of 2 toward 3 at acidic pHs. Because of the low reactivity at acidic pHs and the
increase in the rate of Michael addition reaction between 3 and NAPQI (2) at alkaline pHs,
the solution containing buffer NH /NH Cl (pH 9.0) was selected as the medium for a detailed
3
4
electrochemical study.
1
1

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1
2
Fig. 3. Cyclic voltammograms of 1.0 mmol/L paracetamol in the presence of 1.0 mmol/L N ,N -
dibenzylethane-1,2-diamine in acidic buffered solution pH=4. Scan rate: 50 mV/s; T=25 ± 1 ºC.
To investigate the electrochemical properties of this reaction, controlled-potential
coulometry was carried out in aqueous solution containing 0.35 mmol paracetamol and 0.35
1
2
mmol N ,N -dibenzylethane-1,2-diamine in 0.1 M NH /NH Cl buffer solution during
3
4
controlled-potential coulometry at 0.42 .V. VS. SCE. The electrolysis was monitored by
cyclic voltammetry. As can be observed, Fig. 4 (curves a-e) shows the advancement of
ꢅꢂ
ꢀ
coulometry. Comparison of the current of peak A ( ) shows a decrease in the current of
1
peak A during charge consumption linearly (Fig. 4, inset).
1
1
2

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1
2
Fig. 4. Cyclic voltammograms of 0.35 mmol paracetamol in the presence of 0.35 mmol N ,N -
dibenzylethane-1,2-diamine in 0.1 mol/L NH /NH Cl buffer solution during controlled-potential
3
4
coulometry at 0.42 V .VS. SCE. After consumption of: (a) 0, (b) 25, (c) 45, (d) 90 and (e) 130 C. Scan
rate: 100 mV/s. T=25 ± 1 ºC. Inset: Variation of A peak current versus consumed charge of 0.35
1
1
2
mmol paracetamol in the presence of 0.35 mmol N ,N -dibenzylethane-1,2-diamine.
3
.3. In vitro cytotoxicity
The results of MTT assay showed that there were no significant differences in viability of
the cells incubated with paracetamol and control during the period of incubation when the
concentrations of paracetamol were 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL. Cell
viability compared to the control for liver cells that incubated with 250 and 500 µg/mL of
paracetamol was 84% and 78% respectively (Fig. 5 and Table 1). Paracetamol did not
demonstrate a cytotoxic effect at concentration (500 µg/mL) under the condition of this test.
1
3

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Table 1 Optical density values and Chang liver cell viability obtained after 24-hour exposure
to the test material Paracetamol and controls.
Negative Positive
Control
Control
Paracetamol (µg/mL)
15.63
31.25
62.5
125
250
500
OD
550 nm)
1.449
1.633
0.400
0.421
0.403
0.408
0.011
23.59
1.332
1.868
1.318
1.506
0.314
96.11
1.537
1.441
1.711
1.563
0.137
99.74
1.612
1.487
1.382
1.494
0.115
95.32
1.214
1.679
1.464
1.452
0.233
92.68
1.258
1.452
1.259
1.323
0.112
84.43
1.190
1.371
1.141
1.234
0.121
78.75
(
1
.619
Mean
SD
1.567
0.102
100.00
Viability
%)
(
Fig. 5. Viability of Cheng liver cells at various concentration of the test material paracetamol.
1
2
Cytotoxicity of paracetamol in the presence of N ,N -dibenzylethane-1,2-diamine was
studied in the biological environment. The results show that there were considerable
difference in viability of the liver cells incubate with hydropyrazino-quinoxalinylidene-
acetamide (HPQA) and control during the period of incubation when the concentrations of
HPQA were 42, 84, and 168 µg/mL (Table 2). The Fig. 6 shows the cell viability for liver
cells with 168 µg/mL of HPQA were 18%. The test material hydropyrazino-
quinoxalinylidene-acetamide demonstrated a cytotoxic effect at 168 µg/mL under the
1
4

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condition of this study. According to the results, concurrent use of paracetamol with amine
DOI: 10.1039/C5NJ03250D
derivatives causes toxicity of liver cells.
Table 2 Optical density values and Chang liver cell viability obtained after 24-hour exposure
to the test material Aceta 1 and controls.
Negative
Control
Positive
Control
HPQA (µg/mL)
4
2
84
168
0.243
0.245
0.250
0.246
0.004
18
OD
550 nm)
1.300
1.388
0.472
0.517
0.452
0.408
0.033
33.00
1.242
1.137
0.931
1.103
0.158
82
0.868
0.897
1.079
0.948
0.114
70
(
1
.340
Mean
SD
1.343
0.044
100.00
Viability
%)
(
Fig. 6. Viability of Cheng liver cells at various concentration of the test material HPQA.
The proposed mechanism reaction of paracetamol in the presence of the mentioned amine
derivative was suggested in Scheme 2.
1
5

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Scheme 2 The proposed mechanism for the electrooxidation of paracetamol (1) in the presence of
1
2
N ,N -dibenzylethane-1,2-diamine (3).
1
6

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Based on the obtained results, it seems that the anodic electrooxidation of paracetamol in
DOI: 10.1039/C5NJ03250D
the presence of diamine 3 follows the ECECECECE mechanism. Diagnostic criteria of cyclic
voltammograms, coulometry, and spectroscopy allow us to propose the pathway illustrated in
Scheme 3. According to Scheme 2, the diamine derivative 3 as nucleophile attacked to N-(4-
methylenecyclohexa-2,5-dienyl) acetamide 2 and the intermolecular Michael addition
reaction leads to intermediate 4. This reaction is faster than other secondary electro reaction.
Then, the secondary Michael addition reaction is occurred that leads to the formation of 5.
The final compound 11 was prepared by intermolecular Michael addition and cycloaddition
-
reactions in the next steps. Moreover the oxidation take place through a 10 e solution
electron transfer (SET) reaction.
1
2
Scheme 3 Overall proposed the pathway illustrated reaction of paracetamol in the presence of N ,N -
dibenzylethane-1,2-diamine.
1
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DOI: 10.1039/C5NJ03250D
4
.Conclusion
Since the aqueous media of electrochemical synthesis is similar to the biological
environment, the toxicity of paracetamol in present of an amine derivative was evaluated on
the liver cells by MTT assay in this study. The cytotoxicity measurement showed that the
paracetamol has not significantly toxicity on liver cells and cell viability at concentration of
5
00µg/mL was 78%. While combination of paracetamol with a benzyl-ethylene-diamine
derivative led to cytotoxicity effects on the liver cell. Based on the results, viability of this
product was 18% for 168 µg/mL liver cells. The results indicate that existence of amine
derivatives led to dramatically increasing of paracetamol toxicity effects in the human body.
From the point of view of this method, the electro-synthesis method has some significant
benefits. The use of electricity as energy instead of oxidative reagents, clean synthesis, one-
step reaction, technical applicability, work in room temperature and pressure, and use of
aqueous media instead of organic solvents, are of preeminent green advantages.
This study opens a new doorway to development of paracetamol electrochemical toxicity
studies in the present of variety amine groups.
5
. Supporting informations.
1
13
2
FT-IR spectra, H-NMR spectrum, C-NMR spectrum and ESI-MS spectrum of 3 and 11
products, also cytotoxicity test of paracetamol and HPQA are provided in supporting
information.
Acknowledgments
We would like to acknowledge the School of Chemical Sciences and Food Technology,
Faculty of Science and Technology for the financial support and research facilities. Thanks
are also due to the Makmal Bioserasi, Center for Research and Instrumentation Management,
The National University of Malaysia for providing the cytotoxicity test reports.
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8

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DOI: 10.1039/C5NJ03250D
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New Journal of Chemistry
Page 20 of 20
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DOI: 10.1039/C5NJ03250D
Simulation of paracetamol reaction in the presence of diamine in the liver using electrochemical cell