Differences in antioxidant potential of chalcones in human serum: In vitro study

Article abstract of DOI:10.1016/j.cbi.2020.109084

Introduction: An imbalance between oxidants and antioxidants in favour of oxidants, potentially leading to damage, is termed oxidative stress. Antioxidants (AO), either enzymatic or non-enzymatic, are the ones that can reduce diverse effects of pro-oxidan

Full text of DOI:10.1016/j.cbi.2020.109084

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Differences in antioxidant potential of chalcones in human serum: In vitro study
Tamara Janković, Nemanja Turković, Jelena Kotur-Stevuljević, Zorica Vujić, Branka
Ivković
PII:
S0009-2797(19)32129-5
https://doi.org/10.1016/j.cbi.2020.109084
CBI 109084
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 28 December 2019
Revised Date: 23 March 2020
Accepted Date: 2 April 2020
Please cite this article as: T. Janković, N. Turković, J. Kotur-Stevuljević, Z. Vujić, B. Ivković, Differences
in antioxidant potential of chalcones in human serum: In vitro study, Chemico-Biological Interactions
(2020), doi: https://doi.org/10.1016/j.cbi.2020.109084.
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Author statement
Tamara Janković: Corresponding Author, Formal Analysis, Validation, Writing Original Draft
and Review and Editing.
Nemanja Turković: Formal Analysis, Resources, Data Curation.
Jelena Kotur-Stevuljević: Methodology, Validation, Writing First Draft and Review and
Editing.
Zorica Vujić: Conceptualization, Methodology, Software, Writing-Review and Editing, Funding
Acquisition.
Branka Ivković: Conceptualization, Methodology, Software, Investigation, Writing First Draft
and Review and Editing.

Differences in antioxidant potential of chalcones in human serum: in vitro study
a
b
c
d
Tamara Janković , Nemanja Turković , Jelena Kotur-Stevuljević , Zorica Vujić , Branka
d
Ivković
a
Institute for the Application of Nuclear Energy, INEP, University of Belgrade, Banatska 31b,
1
1080 Belgrade, Serbia
b
Agency for Medicines and Medical Devices of Montenegro, Podgorica, Montenegro
c
Department for Medical Biochemistry, Faculty of Pharmacy, University of Belgrade,
VojvodeStepe 450, 11010 Belgrade, Serbia
d
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Belgrade,
VojvodeStepe 450, 11010 Belgrade, Serbia
tamara.jankovic@inep.ac.rs, nemanja.turkovic@gmail.com,
jelena.kotur@pharmacy.bg.ac.rs, zorica.vujic@pharmacy.bg.ac.rs,
branka.ivkovic@pharmacy.bg.ac.rs
Corresponding author:
Tamara Janković, Institute for the Application of Nuclear Energy, INEP, University of Belgrade,
Banatska 31b, 11080 Belgrade, Serbia, Tel: +381 65580 66 26; E-mail:
tamara.jankovic@inep.ac.rs

Abstract
Introduction: An imbalance between oxidants and antioxidants in favour of oxidants, potentially
leading to damage, is termed oxidative stress. Antioxidants (AO), either enzymatic or non-
enzymatic, are the ones that can reduce diverse effects of pro-oxidants such as DNA, proteins
and lipids damage. Chalcones (1,3-diaryl-2-propen-1-ones) are open chain flavonoids that are
widely biosynthesized in plants. Aim of this study was to test antioxidative potency of 15
chalcones (Chs) in in vitro model in serum (native conditions), so as with exogenously induced
oxidative stress.
Material and Methods: Oxidative stress was induced in serum samples of healthy individuals
with 0.25 mmol/L terc-buthyl-hydroperoxide (TBH), and then we monitored the effects of
various concentrations of chalcones on oxidative stress parameters: total antioxidative status
(TAS), total oxidative status (TOS), total concentration of sulfhydryl group (SHG) and
prooxidative-antioxidative balance (PAB), and calculated prooxidative score, antioxidative
score, and oxy score (OS). Nonparametric repeated measures ANOVA (Friedman’s test) was
used for comparison of antioxidative potency of samples with 15 different chalcones, in a native
state and upon TBH influence. Spearman’s nonparametric correlation analysis was used for
estimation of relation between different parameters.
Results: Negative Oxy Score (OS) values for Chs11-15 showed significantly stronger
antioxidative potency compared to other investigated chalcones (p<0.05). Ch11, Ch13 and Ch14
remained with negative OS even after TBH addition, whereas OS of Ch12 and Ch15 became
positive, with small nominal values. Samples with Ch11 and Ch13 showed significant negative
correlation between TAS and TOS (p<0.05 for both), but in Ch14 sample the negative
correlation existed between TAS and PAB (p<0.05).

Conclusion: Lower value of OS (i.e. better antioxidative potency) was noticed in samples with
Ch11-Ch15. Electron-donor effects of substituent groups as a structural part of these chalcones
could explain its antioxidative capability. Phenolic and methyl groups are responsible for
antioxidative ability enhancement of five chalcones with the best activity.
Key word: chalcones; oxidative stress; antioxidants; oxy score

1
. Introduction
Free radicals are widely believed to contribute to the development of several age-related diseases
as well as cancer, atherosclerosis, neurodegenerative diseases and diabetes, by causing
“oxidative stress” and thus “oxidative damage”. Antioxidants, either enzymatic or non-
enzymatic, can decrease different harmful effects that prooxidants lead to, such as damage of
different biomolecules i.e. DNA, proteins and lipids (1). Chalcones (1,3-diaril-2-propen-1on
compounds) are flavonoid derivates consisting of two aromatic rings that are interconnected by a
highly electrophilic α,β-unsaturated carbonyl system. They are natural compounds that are
largely distributed in plants and some of them exhibit numerous biological activities, such as:
anti-tumor, anti-microbial, anti-inflammatory, antibacterial and finally anti-oxidant activity
which is already reported in different studies (2–5). Their antimicrobial activity is already well
documented by our team in Bozic et al. (6) and Usjak et al. articles (7). The results showed
chalcones’ strong antihemolytic activity towards distinct bacterial strains (6) and substantial
antimicrobial activity combined with their synergistic activity with antibiotics (7). Those studies
were in vitro studies with several bacterial strains. Recent reports indicate preventive role of
chalcone derivate against oxidative stress in cultured cardiomyocytes exposed to high glucose
(5). Ilieva Batovska review (8) explains in detail chalocones’ electron donor properties as the
rationale for its antioxidative activity towards lipid peroxidation during proinflammatory events.
This chalcones’ neutralizing capability towards prooxidants’ harmful activity could prevent
different tissues damage. Computational analysis confirmed that chalcones with higher energy
of the lowest unoccupied molecular orbital and smaller ionization potential are better
hydroperoxides inhibitors (9). Chalcones differ in functional groups and heterocyclic system
included in its structures. Naturally occurring chalcones, mostly have hydroxyl- and metoxy-

groups, while semi-synthesized or totally synthesized chalcones often have amino-, nitro-, alkyl-,
aryl- or heteroaryl- groups (8).The goal of our study was to develop in vitro biological model
system which is different from the majority of that presented in other studies, because all
reactions are measured in biomolecules’ rich environment (human blood serum). This bio-system
enables testing and comparison of the antioxidative activity of 15 newly synthesized chalcone’s
compounds with different substituents. Chemical structure and biological activity of the tested
compounds analysis explains structure-activity relationship of the novel pharmacophores with
potential biological activity and makes starting point for further, deeper investigation of different
chalcone compounds.
2
. Material and methods
.1. General procedure for the synthesis of 1,3-diphenylpropenone derivatives Ch1-Ch15
2
In the present study, the synthesis of title compounds has been carried out by the application of
the reaction is known as Claisen-Schmidt condensation and represents the base-catalysed
condensation on 2-hydroxy acetophenone and ortho-, meta-, para- monosubstituted or
disubstituted benzaldehyde (Scheme 1).

Scheme 1 Preparation of chalcone derivatives
A mixture of
10 ml of 96% ethanol, 2-hydroxiacetophenone (0.012 mol), benzaldehyde
derivatives (0.01 mol) was stirred continuously at 25°C. Immediately, 20 g of 30% NaOH
solution was added in small portions. After becoming homogeneous, mixture was left at room
temperature to stir overnight. The reaction mixture was left to stand in the refrigerator at 4-8 ° C
for 24 h occasionally stirring by vigorous shaking. The precipitated solid was then filtred off,
washed with water and acidified with cold 1mol / l HCl until the pH was about 3. A precipitate
of yellow colour was collected by vacuum system and washed with ice water to neutral reaction
(on litmus) and then poured over with cooled 96% ethanol, and air-dry, in a dark place. The raw
chalcone is left to. After drying, it was purified by TLC and column dry flash
chromatographyand crystallized from ethanol. The pure trans chalcone isomer was obtained in
1
the form of a yellow crystalline powder (confirmed H NMR; J
= 15-16 Hz). The purity of
vinyl H

the newly synthesized compounds was established by HPLC and melting point.The stuctures of
compounds were determined by infrared spectroscopy (IC), nuclear magnetic resonance (NMR)
and mass spectroscopy (MS) (results are not shown). Chalcones showed the IR absorptions
characteristics of phenolic -OH (3200-3350 cm-1), carbonyl >C=O (1685-1600 cm-1) and
1
aromatic C=C (1580-1400 cm-1) functionalities. The H NMR spectra of chalcones displayed
multiplet due to aromatic protons at 6.92-8.00 δ (m, Ar-H) and singlet due to phenolic –OH at
1
1.94-12.75 δ (s, H, OH). The characteristics signals for a chalcone moiety appeared as two
doublets at 7.55-7.79 δ (d, 1H, α-H) and 7.82-7.95 δ (d, 1H, β-H).
2
.2. Sample collection
We collected serum samples from 10 healthy individuals, in order to make serum pool. Healthy
volunteers who had attended their regular medical check-up at the Military Medical Academy in
Belgrade and had given written approval that any serum left over after biochemical analyses
planned by physicians could be used for this study. The investigators didn’t know and didn’t use
any of the data connected with subjects whose serum leftover was used for the serum pool
formation and subsequent analyses. The Faculty of Pharmacy, Belgrade, Ethical Committee
decision (No. 797/2, 11.05.2012) have verified this procedure for samples collection. Only
samples with basic biochemical parameters within metabolite reference ranges were selected.
Serum pool’s aliquots were frozen at -80°C and analyzed within 2 weeks after collection. All
samples were incubated in duplicate for 2h, at 37°C. Serum volume was 450 µL and added
volume of every chalcone dilution was 50 µL (1:10 ratio). We added serial dilutions (range of
concentration 0.0625-1 mg/mL) of newly synthesized chalcones, dissolved in dimethyl sulfoxide
(DMSO), alone or in combination with well-known prooxidant terc-buthyl-hydroperoxide (TBH,

conc. 0.25 mmol/L) in samples, at same volume (25 µL chalcone + 25 µL TBH), so that
exogenous addition was exactly 10% compared to total samples volume. This is important
because of samples’ natural bio-matrix preservation, because it seems that maximal 10% of
dilution will not substantially change the bio-matrix quality. We analyzed 15 different chalcones,
which were synthesized in the laboratory of our Department of Pharmaceutical Chemistry and
signed as Ch1-Ch15.
2
.3. Biochemical parameters
a. Total Antioxidative Status (TAS)
Serum TAS was determined in serum according to Erel's method (10) and optimized in our
laboratory (11). Reduced 2,2-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) is
oxidized using hydrogen peroxide in acidic medium. The antioxidants present in the sample
leads to reagent discoloration, up to a degree proportional to their concentrations that we
measured using an ILAB 300 Plus analyzer (Instrumentation Laboratory, Milan, Italy). The
reaction was calibrated with Trolox (a water-soluble analogue of vitamin E, 6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) and the TAS value of the samples tested is expressed as
μmol Trolox equivalent/L.
b. Total Oxidative Status (TOS)
Serum TOS was determined using the method developed by Erel (12) and modified in our
laboratory (11). Oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to
ferric ion. Intensity of color is proportional to the total amount of oxidant molecules present in
sample. The assay was calibrated with hydrogen peroxide (water solution, 10–200 μmol/L H O )
2
2
and was performed using an ILAB 300 Plus analyzer. Results are expressed as μmol H O
2
2
equivalent/L.

c. Prooxidative-Antioxidative balance (PAB)
Serum PAB was measured according to a previously published method (11, 13). 3,3’-5,5’-
Tetrrametylbenzidine (TMB) reacts with hydrogen peroxide and antioxidant simultaneously.
Reaction of hydrogen peroxide and chromogen is enzymatically catalyzed with peroxidase,
whereas reaction of antioxidants presented in serum and chromogen is non-enzymatically
catalyzed reaction. Standard solutions were prepared by mixing varying proportions (0–100%) of
1
mmol/L H
Pharmacia LKB, Wien, Austria) at 450 nm. PAB values are expressed in arbitrary units, which
correspond to the percentage of H O in the standard solution.
2 2
O with 6 mmol/Luric acid. Absorbance was measured on ELISA reader
(
2
2
d. Total sulphydril groups content (SHG)
The levels of SHG were measured by Ellman’s method (14) modified in our laboratory (11),
using 10 mmol/L dinitrodithiobenzoic acid (DTNB) as a reagent. DTNB reacts with aliphatic
thiol compounds in a basic environment (pH 9.0) and this reaction generates 1mol p-nitrophenol
anion per mol of thiol. The absorbance was measured at 412 nm. The calibration of the method
was achieved with the reduced glutathione in the concentration range from 0.1–1.0 mmol/L.
e. Prooxidative Score, Antioxidative Score, and Oxy Score
Oxy Score (OS) was calculated as the difference between Prooxidative Score (average value of Z
scores of all measured prooxidants and their products, i.e. TOS and PAB) and Antioxidative
Score (average value of Z scores of all measured antioxidants, i.e. TAS and SHG). Z score is the
difference between the original value and the control value divided by SD of controlvalues (or
population means and SDs). Higher OS of substance means weaker antioxidative protection and
higher prooxidantscontent (15,16).

2
.4. Statistical analysis
Statistical analysis was obtained with SPSS 18.0 (SPSS, INC. Chicago, Illinois USA).Data are
presented as medians± interquartile range. To compare OS between chalcones, we used ANOVA
repeated measures analysis (non-parametric variant i.e. Friedman’s test) because we performed
analysis on the same material (the same serum pool from healthy donors).In order to determine
possible correlations between parameters of oxidative stress status in serum, Spearman's
correlation analysis was employed.
3
. Results
After the 2h incubation at 37°C, we have measured several oxidative stress status parameters as
follows: TAS and SHG (antioxidants), TOS and PAB (prooxidants).

The obtained OS results for different chalcones and its structural formulae are presentedin Table
1
.
Table 1. OS value of chalcones with and without TBH
Significant
OS value of
Significant
difference
vs.
OS valueof
No.
Structural Formulae
difference
Chalcone + TBH
*
Chalcones
*
vs.
combination
H8, H15
(p=0.010,
O
F
H5, H8-
H15
5
8.19
72.41
Ch1
0.014) H5-
H7, H9-H14
(
37.45-61.11)
(63.58-76.79)
(
p<0.001)
OH
(
p<0.001)
H4, H8-
H10, H15
p=0.043,
.007,
0.016,
.020,
.010),
H11-H14
(
O
F
0
4
7.98
36.54
Ch2
ns
(
6.13-56.24)
(9.72-68.95)
0
OH
0
(
p<0.001)
H6 (p=0.037)
H5,H7,H10,
H13,H14
O
F
3
C
H5
5
3.83
53.72
(
p=0.016)
H8-H15
p<0.001)
Ch3
(
52.40-56.58)
(47.69-62.59)
(p=0.001)
OH
(
H9,H11,H12
(
p<0.001)
H10,H11,H1
,H13,H14
p=0.003,
0.004, 0.006,
0.001
.002)
H2, H5
2
O
CF
3
(
p=0.043,
0.010),
5
4.61
53.88
(
Ch4
Ch5
(
(
51.83-58.74)
(30.27-69.66)
H8-H15
OH
<
(
p<0.001)
0
O
Cl
H3,H4
H1,H3,H11,
H12,H13
(p<0.001,
p=0.001,
46.06
24.01
(
p=0.016,
0.010)
H1,
23.07-51.24)
(19.07-27.48)
OH

H8-H15
0.001, 0.002,
0.006)
(
p<0.001)
H1,H3,H10,
H11,H12,H1
3
,H14
O
H
3
C
F
2
1.23
28.56
(
p<0.001,
Ch6
ns
(
19.06-21.77)
(24.91-40.43)
p=0.007,
OH
0
0
.025, 0.001,
.003, 0.001,
0
.024)
H1,H3,H11,
H12,H13
O
3
H CO
1
6.65
22.65
(
p<0.001,
Ch7
ns
(
14.19-19.11)
(18.26-26.22)
p=0.001,
OH
0
.001, 0.001,
0
.007)
H2
O
H
3
CO
(p=0.007)
H1, H3-
H5, H11-
H14
1
4.25
12.36
Ch8
H1 (p=0.010)
(
11.53-16.86)
(-17.01-29.99)
OH
OCH
3
(
p<0.001)
H2, H5,
H15
(
p=0.016,
.002,
0.037)
H1,H3,H4
H12,H13
(p=0.001,
0.008)
O
H3C
0
1
4.71
19.27
Ch9
(
5.87-19.82)
(17.09-22.08)
H1,H3,H11
(p<0.001)
OH
,
H11-
H14
(
p<0.001)
H2, H15
p=0.020,
CH
3
(
H1,H3,H4,H
6 (p<0.001,
p=0.001,
O
1
7.54
-2.46
0
.022)
Ch10
(
12.25-19.27)
H1,
(-6.17-7.06)
H3- H5,
0.003, 0.025)
OH
H11-H14

(
p<0.001)
H13, H14
p=0.019,
.008),
H1-H5,
H4,H5,H6,H
7 (p= 0.004,
0.001, 0.001,
0.001)
(
O
CH
3
-
12.33
-2.68
0
Ch11
(
-15.29 – (-1.47)
(-4.37– (-)0.32)
OH
H8-H10
H1,H3,H9
(p<0.001)
H4,
(
(
p<0.001)
H14
H5,H6,H7
(p=0.006,
0.002, 0.003,
0.001)
O
2
O N
p=0.002)
-
10.46
0.30
Ch12
, H1-H5,
H8-H10
(
-12.72– (-)8.60)
(-1.78-1.50)
OH
(
p<0.001)
H1,H3(p<0.0
0
1)
H3,H5,H6,H
7,H9
H11, H14
NO
2
(
p=0.019,
.001),
H1-H5,
O
(p=0.001,
0.006, 0.001,
-
3.70
-12.37
0
Ch13
(
-7.56 – (-)2.94)
(-36.17– (-5.53)
0
.007, 0.008)
H1,H4
H8-H10
OH
(
p<0.001)
(
p<0.001)
H11, H12,
H13, H14
(
p=0.008,
.002,
0.001,
H1,H3,H4,H
6,
O
HO
0
-
31.13
-5.59
Ch14
(p<0.001,p=0
.001, 0.002,
0.024)
(
-35.84 – (-)16.28)
(-20.31– (-0.86)
0
.001),
OH
H1-H5,
H8-H10
(
p<0.001)
H2, H9,
OH
H10, H14
p=0.010,
.037,
0.022,
.001),
O
-
3.23
6.63
(
Ch15
H1 (p=0.014)
(
-9.69-10.09)
(3.28-15.15)
0
OH
0

H1,
H3-H5
p<0.001)
(
*
th
th
Data are presented as medians and 25 -75 percentile values in brackets; p according to
Friedman’s test, ns-not significant (p>0.05)
We have initially used 5 different dilutions of the every chalcone tested in our current study in
order to test if there is some dose dependence in the chalcones’ reactivity in biological samples.
We didn’t get consistent change in redox status parameters which we have measured, so we
presented here results as average value of all 5 concentrational level.
By comparing the OS values between chalcones without addition of oxidant (TBH), we can
notice significantly lower OS values in chalcones Ch11-15. Negative OS value of this chalcones
is due to Antioxidative score being higher than Prooxidative score, indicating that Ch11-15 have
better antioxidative potential compared to Ch1-5 and Ch8-10. In fact, large positive OS
especially in Ch1-5 (from 46-58) indicates their prooxidative potency, which could be useful as
protective function like cytostatic, bacteriostatic, antivirotic etc.
The results remained the same even after TBH prooxidant addition, i.e. chalcones Ch11-15
showed superiority in antioxidative protection upon prooxidant influence, even it was obvious
that TBH addition slightly decreased antioxidative potency of these substances, i.e. their OS
remained negative in some (Ch11, Ch13 and Ch14), while became somewhat positive in Ch12
and Ch15, although with small nominal values. Preserved OS negativity in samples with
chalcones 11, 13 and 14 is a sign of its antioxidative strength, even in prooxidant presence.
Moreover, Ch13 showed significant improvement in OS (became more negative compared to
sample without TBH), which indicates complex chemical reaction between hydro-peroxide and
chalconic structure. Small change in OS towards positive values in samples with Ch12 and Ch15
speaks in favor its lesser capability to respond at oxidative stress challenge.

Ch11-without TBH
32.0
31.5
31.0
30.5
30.0
29.5
29.0
28.5
28.0
27.5
ρ = -0.724
p<0.05
2200
2220
2240
2260
2280
2300
TAS (µmol/L)
Ch13-without TBH
3
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ρ = -0.671
p<0.05
3
3
3
3
3
3
3
3
3
2040
2050
2060
2070
2080
2090
2100
TAS (µmol/L)

Ch14-with TBH
1
1
20.0
00.0
ρ = -0.765
p<0.05
8
6
4
2
0.0
0.0
0.0
0.0
0.0
2040
2080
2120
2160
2200
TAS (µmol/L)
Figure I. Spearman nonparametric correlation of separate oxidative stress parameters in samples
of the chalcones (Ch11-15) with the strongest antioxidative potential
Regarding separate oxidative stress parameters, TAS, SHG, TOS and PAB (data presented in the
Table 1 as supplementary data) measured in the samples with best antioxidants (Ch11-15), we
have analyzed its relation by Spearman’s nonparametric analysis (Figure I). Our results showed
significant negative correlation between TAS and TOS parameters in Ch11 chalcone, sample
without prooxidant (ρ = -0.724, p<0.05) and in Ch13 chalcone, sample without prooxidant
(ρ = -0.671, p<0.05). In addition, significant negative correlation was observed between
parameters TAS and PAB in chalcone Ch14, sample with TBH prooxidant (ρ = -0.765, p<0.05).
These strong opposite correlations between antioxidative-prooxidative pair of parameters
indicate their mutual connection and suggest chalcone induced chemical reaction influence on
the totally antagonized redox status measures.

4
. Discussion
Here we present the results of testing the antioxidant potential of fifteen new synthetic chalcones
in biological material in vitro, in the natural state and in exogenously induced oxidative stress
conditions. Similar model of testing antioxidant potential of newly synthesized molecules we
have already published (16).
Of particular relevance to the present work is the fact that the antioxidant properties of chalcones
are highly influenced the substitution on two aryl rings of chalcone molecule and their
substitution patterns. The hydroxyl group is proven one of the key groups that greatly enhance
the antioxidant activity of chalcone because of its conversion to the corresponding phenoxy
radicals through the hydrogen atom transfer mechanism. The role of the double bond and the
carbonyl group in the antioxidant activity is also importance (17).
The OS values can be explained by electronic and steric effects of functional groups in the
structure of the chalcone. Number of groups are also important for theirs antioxidative activity,
more groups should have better antioxidative activity. That is not always the case, as 2-hidroxy
chalcones α,β-unsaturated carbonyl systems at unfavorable electron donor influence of phenol
group.
As far as electronic effects of the substituents on an aromatic ring are concerned, the measure
used is known as the Hammett type substituent constant or inductive sigma constant (σ I ). This
constant is a measure of the inductive electron withdrawing and electron donating effects for the
introduced groups (in our case -F, -CH , -OCH , -Cl, - NO , -OH and -CF ). The positive values
3
3
2
3
for the σ I indicate electron withdrawing effect and vice versa. To explain the steric effects of the
groups introduced in the ortho, meta or para position of the ring B of the phenylpropiophenone,

3 3 2
we used the calculated values for the molar refractivity of the -F, -CH , -OCH , -Cl,- NO , -OH
and -CF groups. [Reference: http://www.wiredchemist.com/chemistry/data ].
3
Table 2. Substituent inductive sigma constant (σ ) and molar refractivity (MR) for the different
I
introduced substituents
substituent -F
-CH3
-0.04
5.65
-OCH3
0.27
-Cl
- NO2
0.76
-OH
0.29
2.85
-CF3
0.42
5.02
*
σI
0.52
0.92
0.47
6.03
*
MR
7.87
7.36
*Reference: http://www.wiredchemist.com/chemistry/data
Increased electronic density in the molecule, which a group with positive resonant and inductive
effects exhibit (phenolic-Ch14 and Ch15, methyl-Ch10 and Ch11 and methoxy-Ch-7 and Ch-8),
will enhance antioxidant abilities.
Chemical groups that enhance electron density of α,β-unsaturated carbonyl systems contribute to
antioxidative activity. However, one must bear in mind the fact that steric effects affect OS
value, because they influence formation of transition states in electron and proton transfer
processes. If it is a voluminous substituent (regardless of the electronic effect) ortho positioning
can interfere with the formation of transition states and thus reduce the antioxidative potential of
the molecule, which is the case with methyl and methoxy groups (MR 5.65 and 7.87). If the
steric effect is overcomed by positioning the substituent in the para position, the antioxidative
effect will be correlated with the electronic effect, which was confirmed in our case by Ch11. On
the other hand, the methyl group in the para position of the Ch11 has the property of toluene
hydrogen which itself acts as an antioxidant. Groups with pronounced negative inductive effect,
such as halogen groups (-F, -Cl and CF ) could reduce the electron density in the enone system
3
and thus the antioxidant potential of chalcones. In the case of the halogen group, the electronic
effect is far stronger than the steric one and the steric effect which is not very pronounced, as

confirmed by the results obtained with Ch3 and Ch4 (18). Kumar et al. also suggested that
electron-withdrawing groups reduce the efficiency of hydrogen release from the amino group of
pyrazoline, so if halogens were the substituents on the 3-phenyl group of pyrazoline, antioxidant
activity is greatly reduced (22).
The results obtained by examining the antioxidant potential of Ch 12 and Ch13 with the –NO2
group are interesting. The nitro group has a pronounced negative inductive but also a resonant
effect (Table 2.) which reduces the electron density in the ketenone system; on the other hand –
2
NO is a voluminous group, which collectively favors a pro-oxidative effect. However, the nitro
group is planar and attracts ketenone hydrogen by its electronegativity by building an
intramolecular hydrogen bond, which affects the reactivity of the ketenone system and increases
proton mobility to C ketenone. The intramolecular hydrogen bond intensities are weakened by
positioning the –NO group at the meta position. An interesting observation is also the fact that
2
the results of the antioxidant activity of Ch13 in the presence of TBH, we suppose because
NO group consumes TBH, and thus minimizing its prooxidative effects.
2
However, it is not clear if antioxidative activity of chalcones is expressed in bio-environment,
because of their ability to scavenge free radicals or because of interaction with xanthine oxidase
enzyme or some other system, which catalyzes the synthesis of superoxide anion (23). Vogel and
associates synthesized several natural and synthetic chalcones that showed significant activity in
capturing free oxygen radical (24). Study of natural chalcones, derivates of fruit Artocarpus
nobilis, has shown stronger 2,2-diphenyl-1-picryhydrazyl (DPPH) scavenging activity then well-
known antioxidant substance, α-tocopherol (8).
A high intake of flavonoids has been associated with a lower incidence of cardiovascular disease
(25). One explanation leys in his ability to inhibit cyclooxygenase, which is important enzyme

2
+
that mediates inflammation, thrombosis (26), but also in ability to chelate Cu (27). LDL
oxidation, can be initiated by catalytic transition metal ions such as copper and during that kind
of reactions, the polyunsaturated fatty acids in lipoproteins are rapidly converted to lipid
hydroperoxides and aldehyde breakdown products (28). In line with this observation we could
suggested that chalcones could have protective potential. Namely, flavonoids and structurally
2
+
similar chalcones, have the ability to chelate Cu ions and to inhibit copper-mediated LDL
oxidation (29, 30).
Chalcones and chalcone-like (chalconoid) compounds represent an important class of small
useful molecules in cancer chemotherapy, because of their clear cytotoxic activity (31). The one
of the main advantages of chalcones as potential drugs,are their low propensity to interact with
DNA and thus decreased risk of mutagenicity as a common side effect of current
chemotherapeutic agents (32).
Nowadays, chalcones are used as food additives and cosmetic products ingredients much more
rather than in clinical practice as drugs with particular activity. The reason for this decreased
chalcones clinical application, contrary to former expectations lie in its complicated and not
enough revealed mechanism of action, so as its low selectivity, which should be crucial
characteristic for an adequate drug effect (33). This in vitro study could be a contribution for the
chalcones antioxidant activity estimation in biological system, with a purpose of their selection
for further investigation dealt with its antioxidative potency and prospective use in clinical
practice.
5
. Conclusion
The results of the redox status examination of synthesized chalcones and its attitude in biological
medium (human serum) differs because of distinct substituents and its positions within

pharmacophore’s structure. We can notice that chalcones which have methyl-, phenol- and nitro-
groups as substituents, are better antioxidants compared to other analyzed compounds, under
applied experimental conditions. The investigated chalcones can serve as starting compounds for
the synthesis of structurally similar compounds, which could be used in the treatment of various
pathological conditions induced by free radicals.
Acknowledgment
This work was supported by the Ministry for Education, Science and Technological
Development of the Republic of Serbia, under Grant 175035 and under Grant 172041.
Disclosure statement: The authors report no conflicts of interest.

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Differences in antioxidant potential of chalcones in human serum: in vitro study
Highlights
-
-
-
In vitro biological model system.
The best antioxidative properties showed methyl-, phenol- and nitro- chalcones.
Further investigation should lead to chalcones use in human medicine.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: