Synthesis and biological activities of a nitro-shiff base compound as a potential anti-inflammatory agent

Article abstract of DOI:10.1016/j.ejps.2020.105300

In order to discover a new compound having anti-inflammatory activity, a nitro-Schiff base was evaluated. The compound was synthesized and characterized by ¹H NMR and ¹³C NMR. The cytotoxic activity was evaluated in vitro by hemolysi

Full text of DOI:10.1016/j.ejps.2020.105300

Journal Pre-proof
Synthesis and Biological Activities of a Nitro-Shiff Base Compound
as a Potential Anti-Inflammatory Agent
Beatriz Cardoso Roriz , Danieli Fernanda Buccini ,
Beatriz Fuzinato dos Santos , Suellen Rolon de Sousa Silva ,
Nelson Lu´ıs de Campos Domingues , Susana Elisa Moreno
PII:
S0928-0987(20)30089-0
DOI:
Reference:
https://doi.org/10.1016/j.ejps.2020.105300
PHASCI 105300
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
31 December 2019
2 March 2020
7 March 2020
Please
cite
this
article
as:
Beatriz Cardoso Roriz ,
Danieli Fernanda Buccini ,
Beatriz Fuzinato dos Santos , Suellen Rolon de Sousa Silva , Nelson Lu´ıs de Campos Domingues ,
Susana Elisa Moreno , Synthesis and Biological Activities of a Nitro-Shiff Base Compound as
a Potential Anti-Inflammatory Agent, European Journal of Pharmaceutical Sciences (2020), doi:
https://doi.org/10.1016/j.ejps.2020.105300
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.

Synthesis and Biological Activities of a Nitro-Shiff Base Compound as a Potential
Anti-Inflammatory Agent
Beatriz Cardoso Roriza, Danieli Fernanda Buccinib, Beatriz Fuzinato dos Santosc,
Suellen Rolon de Sousa Silvab, Nelson Luís de Campos Dominguesc, Susana Elisa
Morenob*
aPrograma de Pós-Graduação em Biotecnologia e Biodiversidade, Universidade Federal
de Mato Grosso do Sul, Campo Grande, MS, Brazil
bPrograma de Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco,
Campo Grande, MS, Brazil
cPrograma de Pós-Graduação em Química em Associação, Universidade Federal da
Grande Dourados, Dourados, MS, Brazil
*Correspondence
Beatriz Cardoso Roriz
Address: Cidade Universitária, Avenida Costa e Silva - Pioneiros, Zip code: 79070-900
Campo Grande – MS, Brazil
E-mail: beatrizroriz@hotmail.com
Tel: +5567996377616
Susana Elisa Moreno
Address: Avenida Tamandaré, 6000 - Jardim seminário, Zip code: 79117-010
Campo Grande – MS, Brazil
E-mail: smoreno@ucdb.br
Tel: +556733123473
1

Abstract
In order to discover a new compound having anti-inflammatory activity, a nitro-Schiff
1
base was evaluated. The compound was synthesized and characterized by H NMR and
13C NMR. The cytotoxic activity was evaluated in vitro by hemolysis and MTT cell
viability assay. To evaluate genotoxicity, the micronucleus assay was performed in vivo.
The anti-inflammatory effects of the compound were examined using in vivo models of
inflammation such as neutrophil migration assay, paw edema, and exudation assay. The
production of NO was also estimated in vivo and in vitro. The data showed that the
compound did not induce hemolysis at all the tested concentrations. Similarly, the
compound did not induce cytotoxicity and genotoxicity to the cells. The neutrophil
migration assay showed that the compound reduced the number of neutrophils recruited
to the peritoneal cavity by approximately 60% at all the tested concentrations. In the
exudation assay, the compound showed a reduction in extravasation by 24%. The paw
edema model demonstrated a significant reduction in the paw volume at all the
evaluated time points. The production of NO was decreased both in vitro and in vivo.
These results suggest that the nitro-Schiff base compound efficiently inhibited
inflammation and might be a good candidate for the treatment of inflammatory-
associated conditions.
Keywords: Inflammation, Schiff bases, Nitro group.
2

1. Introduction
Inflammation is a mechanism of self-protection of the body against injurious
agents that can damage the tissues (Medzhitov, 2010). It provides support to the body to
eliminate harmful stimuli and helps in initiating the process of healing (Alessandri et al.,
2013). Swelling, pain, leukocyte migration, and loss of function are the main signs of
inflammation and are caused by the release of various inflammatory mediators (Serhan,
2017). Prolonged and severe inflammation contributes to the development of various
diseases such as arthritis, chronic obstructive pulmonary disease, asthma, cancer,
multiple sclerosis, and cardiovascular diseases (Freire and Van Dyke, 2013; Nathan and
Ding, 2010).
Inflammatory response involves the recruitment of leukocytes, especially
neutrophils (Mayadas et al., 2014) and macrophages (Nagatoshi and Kazuo, 2005).
Neutrophils are the first cells to be recruited to the inflammatory sites and are
responsible for the production of several inflammatory mediators (e.g. cytokines and
chemokines) and reactive oxygen species (ROS) (Halade and Ma, 2016). These cells
play a major role in the host defense through phagocytosis; however, neutrophils can
play dual roles in inflammation. An increased neutrophil count contributes to the
damage of tissues in inflammatory diseases, whereby they become inappropriately
activated (Wright et al., 2010). In this context, numerous anti-inflammatory strategies
are under investigation to control increased neutrophil migration in inflammatory
diseases.
The main pharmacotherapy for inflammatory diseases comprises nonsteroidal
anti-inflammatory drugs (NSAIDs). This class of drugs has antipyretic, anti-
inflammatory, and analgesic properties, and is among the most commonly prescribed
drugs in the world (Dwivedi et al., 2015). Despite good efficacy, their use, especially in
high doses and for a prolonged period, is associated with several adverse effects such as
gastrointestinal damage including gastric lesions, bleeding (Sostres et al., 2010; Tanaka
et al., 2001), and cardiovascular disorders (Grosser et al., 2017).
Imines, also called azomethines or Schiff bases, are compounds containing the
functional group –C=N–, which were first described by Hugo Schiff in the 19th century.
Schiff bases form an important class of organic compounds having several applications
in synthetic organic processes, organometallic chemistry, and also as pharmaceutical
targets (Kajal et al., 2013; Qin et al., 2013). The functional group –C=N– brings a
spectrum of biological activities to these compounds (Hameed et al., 2017). Schiff bases
3

have importance in the medical and pharmaceutical fields due to their reported
biological activities including antitumor (Ceyhan et al., 2015; Hu et al., 2012), anti-
inflammatory (Sondhi et al., 2006), analgesic (Pandey et al., 2012), nematicide (Al-
Kahraman et al., 2010), and antimicrobial (Abirami and Nadaraj, 2015) activities. The
present study evaluated the anti-inflammatory activities of a nitro-Schiff base derivate,
(E)-2-(((4-nitrophenyl)imino)methyl) phenol (ENIMF).
2. Material and methods
2.1 Reagents
The following substances were used: salicylaldehyde (Sigma, China), 4-
nitroaniline (Sigma, China), dimethyl sulfoxide (DMSO) (Vetec, Brazil), saline (JP
Farma, Brazil), Triton X-100 (Vetec), thioglycolate (Himedia, India), Trypan blue
(Gibco, USA), 3-[(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT)
(Sigma, USA), isopropyl alcohol and hydrochloric acid (solubilizing solution) (Vetec),
saline phosphate buffer (PBS) (Sigma, USA), ethylenediamine acid (EDTA) (Dinâmica,
Brazil), colchicine (Geolab, Brazil), May-Grunwald stain (NewProv, Brazil), Giemsa
stain (RenyLab, Brazil), dexamethasone (Aché, Brazil), carrageenan (Sigma, USA),
ketamine (Syntec, Brazil), xylazine (Agener União, Brazil), rapid staining in
hematology kit (Instant Prov, Brazil), Evans blue dye (Vetec), and lipopolysaccharide
from Escherichia coli (LPS) (Sigma, USA).
2.2 Synthesis of ENIMF
The compound was synthesized with slight modifications of a reported
procedure (Shahnaz et al., 2016). An ethanolic solution of salicylaldehyde (0.252 g,
2.06 mmol, 15 mL EtOH) was added drop-wise to an ethanolic solution of 4-
nitroaniline (0.285 g, 2.06 mmol, 15 mL EtOH) and the mixture was stirred at 80 oC.
After 3 h, the solvent was evaporated and the orange liquid was extracted with a
chloroform/water mixture (1:1). The organic layer was then dried over anhydrous
Na2SO4 and the solvent evaporated to obtain an orange crystalline solid (E)-2-(((4-
nitrophenyl)imino)methyl)phenol (Figure 1). ENIMF solubilization was performed
using saline and DMSO (2%). The NMR conditions were as follows: 1H NMR (300
MHz, CDCl3): δ ppm 11.02 (s, 1H), 9.91 (s, 1H), 8.10−8.05 (m, 2H), 7.46−7.44 (t, J =
4.5 Hz, 1H), 7.40−7.35 (m, 1H), and 7.08−7.00 (m, 2H), 6.65−6.60 (m, 2H); and 13C
NMR (75 MHz, CDCl3): δ ppm 196.83, 165.60, 137.22, 133.94, 133.21, 126.56,
125.44, 122.10, 120.06, 117.82, and 113.58.
4

2.3 Animals
Male and female Swiss mice weighing 20−22 g obtained from the Dom Bosco
Catholic University (UCDB) (Campo Grande, MS, Brazil) were maintained at room
temperature (23−25 °C) and controlled light/dark cycle, with free access to feed and
water. The experimental protocol adhered to the Brazilian National Council for the
Control of Animal Experimentation (CONCEA) and was approved by the Committee
for Ethics in Animal Experimentation (CEUA-UCDB, protocol no. 031/2017).
2.4 Hemolytic assay
The hemolytic assay was performed according to a previous report (Park et al.,
2004) with minor modifications. The murine erythrocytes were washed with saline
(0.9%) and centrifuged at 580 g at 4 °C for 2 min. The cells were distributed on a 96-
well plate (KASVI) and incubated with different concentrations of ENIMF (100, 50, 25,
12.5, and 6.25 μg/mL). The positive control cells were treated with Triton X-100 (2%).
The material was incubated at room temperature for 1 h and then centrifuged at 1400 g
for 2 min at 10 °C. The reading was recorded at 415 nm (SpectraMax F3, Molecular
Devices). All concentrations were evaluated in triplicate. The percentage of hemolysis
was compared to the positive control and calculated according to the following formula:
2.5 Cell viability assay
The macrophage culture was performed according to a previous report (Zhang et
al., 2008) with modifications. The macrophages were obtained from the peritoneal
cavity of the Swiss mice (n = 10) 48 h after intraperitoneal (i.p) injection of
thioglycolate (4%) (3 mL i.p.). After euthanasia (deepening anesthesia with ketamine
150 mg/kg and xylazine 7.5 mg/kg), the exudate was collected, washed (twice), and re-
suspended in incomplete RPMI 1640 medium. Subsequently, the cells were counted in a
Neubauer chamber. The cell viability was determined by the Trypan blue exclusion
method (Strober, 1997). The samples with cell viability >90% were considered suitable
for subsequent experiments. The cytotoxicity of ENIMF was determined by the salt dye
MTT colorimetric assay (Sieuwerts et al., 1995; Takeuchi and Kobata, 1991). A
suspension of peritoneal macrophages (1 × 105 cells/mL) was placed on 96-well plates
and incubated with incomplete RPMI 1640 medium with different concentrations of
ENIMF (200, 100, 50, 25, 12.5, 6.25, and 3.12 µg/mL) and kept in the presence of 5%
CO2 at 37 ºC. The cell viability was evaluated after 24 h and 48 h incubation with
ENIMF. Following that, all medium was aspirated and the MTT solution (5 mg/mL
5

0.01 mL) was added and incubated for 4 h. After the incubation period, a solubilizing
solution (0.01 mL) was added to solubilize the formazan crystals. The plates were
shaken slightly at room temperature for 5−10 min and the reading was recorded at 540
nm (SpectraMax F3, Molecular Devices). Experiments in all the groups were performed
in triplicate. The cell viability percentages were calculated relative to the untreated cell
controls.
2.6 Micronucleus assay
The micronucleus assay was performed with the peripheral blood of the Swiss
mice according to (Schmid, 1975) and (Heddle, 1973). The mice (n = 6) were treated
with a concentration of 5 mg/kg of ENIMF (0.2 mL i.p.), vehicle (0.2 mL i.p.), or
colchicine (5 mg/kg, 0.2 mL i.p.). After 24 h, the mice were anesthetized (ketamine 150
mg/kg and xylazine 7.5 mg/kg) and blood samples were obtained by caudal vein
puncture for making blood smears. The slides were fixed and stained with May
Grunwald and Giemsa stain (10%). The erythrocytes (2,000/slide) were counted, and
the polychromatic cells were identified with or without micronuclei. The slides were
analyzed under an optical microscope (Nikon® Eclipse 80i, oil immersion lens,
100/1.25). The data are expressed as the number of micronucleus/2000 cells.
2.7 Neutrophil migration
The mice (n = 6) were pretreated with subcutaneous (s.c.) vehicle (0.1 mL s.c.),
dexamethasone (0.4 mg/kg, 0,1 mL s.c.), or ENIMF (5, 2.5, and 1.25 mg/kg s.c.) 15 min
prior to the application of the inflammatory stimulus (carrageenan, 1.5% 0.1 mL i.p.).
The negative control group received saline solution (0.9%, 0.1 mL i.p.). After 6 h, the
animals were euthanized (deepening anesthesia with ketamine 150 mg/kg and xylazine
7.5 mg/kg) and the peritoneal cavity was washed with PBS/EDTA (0.3%, 3 mL). The
total and differential cells present in the exudate were assessed. The total count was
performed in a Neubauer chamber. The differential count was performed with slides of
the exudate cell smear, which were stained and examined under an optical microscope
(Nikon® Eclipse 80i, oil immersion lens, 100/1.25). The results are expressed as the
number of cells × 105/mL (Moreno et al., 2006).
2.8 Exudation assay
The effects of ENIMF on the increase in carrageenan-induced permeability were
performed according to a previous report (Graesser et al., 2002) with modifications. The
mice (n = 5) were pretreated with vehicle (0.1 mL s.c.) or ENIMF (5 mg/kg s.c.) 15 min
prior to the inflammatory stimulus (carrageenan, 1.5% 0.1 mL i.p.). After 45 min, Evans
6

blue dye (20 mg/kg) was intravenously (i.v.) administered (0.05 mL). After 4 h, the
animals were euthanized (deepening anesthesia with ketamine 150 mg/kg and xylazine
7.5 mg/kg) and the peritoneal cavity was washed with PBS/EDTA and centrifuged at
220 g for 10 min at 10 °C. The absorbance of the supernatant was determined at 650 nm
(SpectraMax F3, Molecular Devices). The results were compared to a nitrite standard
curve (100–0.001 μM) and expressed as μM/mL.
2.9 Carrageenan-induced paw edema
The paw edema model was established according to a previous report (Winter et
al., 1962). The mice (n = 5) were pretreated with ENIMF (5 mg/kg 0.1 mL s.c.), vehicle
(0.1 mL s.c.), or dexamethasone (0.4 mg/kg, 0.1 mL s.c.). After 30 min, carrageenan
(1.5%, 0.05 mL) (inflammatory stimulus) was injected sub-plantar (s.p.) into the left
paw of all the mice, except the vehicle group that received the saline solution (0.9%,
0.05 mL s.p.). The paw volume was measured before and at 5, 10, 15, 30, 60, 120, and
240 min after carrageenan injection to assess the edema. The feet volumes were
measured with a plethysmometer (Insight, Ribeirão Preto, Brazil). At the end of the
experiment, the mice were euthanized (deepening anesthesia with ketamine 150 mg/kg
and xylazine 7.5 mg/kg). The results are expressed in variation (Δ) of the increase in
paw volume.
2.10 Nitric oxide (NO) production
The production of NO was quantified according to the Griess reaction method
(Green et al., 1982; Stone et al., 2005) with some modifications. The peritoneal
macrophages (2 × 105 cells/mL) were incubated with RPMI 1640 medium for 24 h for
adhesion. Thereafter, the cells were incubated with different concentrations of ENIMF
(200, 100, 50, 25, 12.5, 6.25, and 3.25 μg/mL), and after 12 h, LPS (200 ng/well) was
added. After 24 h, the supernatant was collected and quantified with the Griess reaction
method. To evaluate the production of NO in vivo, an aliquot of peritoneal exudate of
ENIMF-treated mice was centrifuged at 400 g for 10 min at 10 °C and then incubated
with Griess reagent as described above. The reading was obtained at 570 nm
(SpectraMax F3, Molecular Devices). The results were compared to a nitrite curve (200
to 1 μM) and expressed as μM/mL.
2.11 Statistical analyses
All data analyses and graphical representations were performed by GraphPad
Prism 6.0 program. The data are expressed as the means ± standard errors of the means
(SEM). The means of the different treatment groups were compared by analysis of
7

variance (ANOVA). If significance was detected, individual comparisons were
subsequently tested with Bonferroni’s t-test for unpaired values. A p-value of < 0.05
was considered significant.
3
Results
3.1 Cytotoxic activity
The hemolysis assay was used to assess whether ENIMF could induce direct
damage to the cell membrane. Table 1 demonstrates a weak hemolytic activity of
ENIMF, even at the highest concentrations, as compared to that of the positive control
(Triton X-100). The cellular toxicity of ENIMF was evaluated by MTT assay in murine
macrophages (Table 1). ENIMF did not induce cytotoxicity after 24 h and 48 h of
macrophage incubation. The cell viability was similar to that in the non-treated cells.
However, the cells treated with DMSO 2% showed a reduction in viability at 24 h (50.6
± 4.1%) and 48 h (54.1 ± 5.8%) as compared to that of the non-treated group. The data
suggested the possibility of using ENIMF in in vivo experiments.
3.2 Genotoxic activity
To evaluate the genotoxic activity of ENIMF in murine erythrocytes, the
micronucleus assay was performed. Figure 2 shows that ENIMF (5 mg/kg) did not
induce an increase in micronucleus formation in the polychromatic erythrocytes as
compared to that in the colchicine treated group. ENIMF-treated mice showed that the
number of micronucleated cells (0.8 ± 0.4) was similar to that of the vehicle-treated
group.
3.3 Anti-inflammatory activity
3.3.1 Cellular signs of inflammation
The effects of ENIMF on the cellular signs of inflammation were investigated.
Figure 3 shows that ENIMF, in all the tested doses, could prevent carrageenan-induced
leukocyte migration to the peritoneal cavity of mice (Figure 3A). The inhibition was
64%, 63%, and 61% at concentrations of 5, 2.5, and 1.25 mg/kg, respectively, as
compared to the non-treated group (Figure 3A). Since neutrophils are the first-line
immune cells recruited into the inflammatory site, the cell migration was evaluated. The
data show that ENIMF reduced the recruitment of neutrophils to the mice peritoneal
cavity as compared to the control group. ENIMF reduced (66%, 61%, and 65%) the
number of neutrophils at concentrations of 5, 2.5, and 1.25 mg/kg, respectively (Figure
3B). The neutrophil migration observed in the ENIMF-treated mice was similar to that
8

in the dexamethasone-treated group (used as the anti-inflammatory standard drug).
Based on this data, the ENIMF dose of 5 mg/kg was selected for subsequent
experiments.
3.3.2 Vascular signs of inflammation
The effects of ENIMF on vascular signs of inflammation were investigated by
Evans blue-labeled plasma protein extravasation assay. The results demonstrated that
ENIMF (5 mg/kg) was able to reduce the carrageenan-induced increase in vascular
permeability by 24% as compared to that in the control group. The ENIMF-treated mice
showed exudation (1.3 ± 0.05) similar to that in the control group that received vehicle
(1.1 ± 0.02) (Figure 4A). Because edema is a consequence of increased vascular
permeability during inflammation, carrageenan-induced paw edema in the mice was
evaluated. Figure 3 showed that ENIMF, at a dose of 5 mg/kg, inhibited edema at all-
time points as compared to that in the untreated group. The anti-edematogenic effect of
ENIMF after 4 h of treatment was 66% as compared to the control treatment (Figure
4B). This effect was similar to that of dexamethasone. DMSO (2%) used as the vehicle
showed similar results as that in the saline-treated group (data shown in the
supplementary material).
3.4 NO production
The anti-inflammatory activity of ENIMF was also evaluated based on NO
production by macrophages stimulated with LPS. The results demonstrated that ENIMF
caused a reduction in nitrite macrophage production at all doses in a dose-dependent
manner (Figure 5A). To confirm that ENIMF could inhibit NO production, the nitrite
concentration was assessed in the peritoneal exudate of the ENIMF-treated mice (Figure
5B). The data show that ENIMF, at concentrations of 5 and 2.5 mg/kg, reduced the
concentration of nitrite (3.2 ± 0.9 and 2.5 ± 1.5, respectively) which was similar to that
in the negative control group (0.7 ± 0.3). The data corroborate the results of in vitro NO
production.
4 Discussion
The bioactivity of synthetic compounds is closely related to the profile of the
substituents attached in their structure and may include a wide range of biological
activities (Gonzalez et al., 2009; Sondhi et al., 2006). Among the compounds with
potential pharmacological applications, nitro-derivatives are highlighted because they
have an important pharmacophoric moiety in their molecules, which facilitate their use
9

in medicinal chemistry and pharmacology. ENIMF is a part of a class of compounds
called imines or Schiff bases and has a nitro group (NO2) in its structure. In
nitroaromatic compounds, one or more nitro groups are directly attached to the aromatic
ring system (Patterson and Wyllie, 2014). In the aromatic ring, it has an electron
withdrawing group that can interact with proteins, amino acids, nucleic acids, and
enzymes (Nepali et al., 2018; Olender et al., 2018; Strauss, 1979). Studies have shown
that compounds containing a nitro group have a variety of biological activities including
anti-Trypanosoma cruzi (Almeida et al., 2018), antifungal (Andrade et al., 2019), and
anti-leishmania (Mendonça et al., 2019) activities.
The nitro group can induce toxicity (Nepali et al., 2018), and consequently,
limits its use in drug designing. The toxicity of nitro-group correlates with ROS
production by enzymatic reduction of the nitro groups, such as nitro radical anion,
nitroso, and hydroxylamide. In the biological systems, these intermediates may react
with the biomolecules and lead to adverse effects, such as mutagenicity and
carcinogenesis (Olender et al., 2018; Plošnik et al., 2016). The possible toxic effect of
the nitro group can be modified by modulating stereoelectronic properties and the
appropriate position of the group and its substituents (Landge et al., 2016; Purohit and
Basu, 2000).
Firstly, the toxicity of ENIMF to murine erythrocytes and macrophages was
investigated. The data demonstrated that ENIMF did not induce erythrocyte membrane
lysis and did not alter the viability of macrophages at 24 h and 48 h post exposure.
Likewise, ENIMF also did not cause polychromatic micronucleus formation, suggesting
that it does not trigger DNA damage during mitotic division. The absence of a
genotoxic effect may be related to the capacity of ENIMF to inhibit NO synthesis. Once
a high level of NO increases peroxynitrite production, it triggers undesirable effects on
the target cells, such as cytotoxicity and mutagenicity (Kim, 2017). This preliminary
data indicate that ENIMF can be used in in vivo studies.
Following this, the anti-inflammatory effects of ENIMF were evaluated. The
onset of the inflammatory process is characterized by a cellular and vascular response in
which a series of mediators trigger various processes, such as vessel dilation, increased
capillary permeability, hyperemia, leukocyte recruitment, and nociceptor sensitization
(Pober and Sessa, 2007). Though neutrophils are the first line of defense of the host
organism, a high neutrophil count is detrimental in inflammation-related diseases
(Headland and Norling, 2015). In this context, drugs capable of reducing the number of
10

neutrophils are crucial in controlling this condition. The data showed that ENIMF
inhibited the recruitment of neutrophils to the mice peritoneal cavity, suggesting the
anti-inflammatory effects of the compound on the cellular signs of inflammation.
The anti-inflammatory effects of ENIMF were also investigated on the vascular
signs of inflammation. The results elucidated that ENIMF reduced vascular
permeability and carrageenan-induced paw edema. Under normal physiological
conditions, the endothelium allows selective transport of substances and is impermeable
to macromolecules, such as albumin. In the presence of inflammation, this selective
transport is affected, resulting in vascular leakage and, consequently, the development
of edema (Radu and Chernoff, 2013; Yao et al., 2018). The results suggest that ENIMF
can effectively inhibit the inflammatory response.
The data demonstrate that ENIMF inhibits leukocyte recruitment, exudation, and
edema on carrageenan-induced models of inflammation. The mechanisms involved in
the anti-inflammatory effects of ENIMF can be related to the inhibition of NO
production. ENIMF inhibited NO production both in the peritoneal exudate and LPS-
stimulated macrophage culture. The macrophages and neutrophils present in the
peritoneal exudate are the possible sources of NO production during inflammation
(Biswas et al., 2001; Wright et al., 1989).
NO is a mediator with several functions in the biological system. It exists in
three different isoforms, of which two are constitutive forms (eNOS: responsible for the
vascular tone and leukocyte aggregation and nNOS: acts as a neuromodulator) and one
is an inducible form (iNOS: produced by macrophages and other cells and plays
important roles in inflammatory processes). NO has a dual role in inflammation and
may have pro- and anti-inflammatory characteristics. NO can activate and deactivate the
gene expression of a diverse range of proteins, thereby modulating the activation profile
of immune cells (Connelly et al., 2001). Inflammatory stimulus induces the production
of high levels of NO, which regulate inflammatory signals such as vascular
permeability, vasodilation, and leukocyte transmigration to the inflamed sites. NO
production can be stimulated by pro-inflammatory cytokines, as well as by microbial
products, such as LPS (Guzik et al., 2003; Korhonen et al., 2005; Pautz et al., 2010).
NO synthesis inhibitors, especially selective iNOS inhibitors, are candidates for anti-
inflammatory drugs (Coleman, 2001; Guzik et al., 2003; Korhonen et al., 2005).
Schiff base derivatives are reported as potential anti-inflammatory drugs (Arshad
et al., 2014; Pandey et al., 2012; Sondhi et al., 2006) and the insertion of a nitro group
11

seems to augment the anti-inflammatory activities as compared to the other substituents
(Ali et al., 2019; Gacche et al., 2006; Geronikaki et al., 2004; Rakesh et al., 2015). The
antifungal, antimicrobial, and anti-leishmania activities of ENIMF have been reported
(Abirami and Nadaraj, 2015; Al-Kahraman et al., 2010); however, there are no reports
on its anti-inflammatory activities. The mechanism of action of anti-inflammatory
compounds containing nitro groups is still unknown. Corroborating our data, studies
with nitroaromatics have shown reduced NO production in LPS-activated macrophages
and decreased expression of iNOS, cyclooxygenase-2 (COX-2), interleukin 1-β (IL1-β),
and tumor necrosis factor-α (TNF-α) (Tumer et al., 2017). The nitro group also appears
to be crucial for the inhibition of NO production (Cowart et al., 1998). Concomitantly,
the activity of ENIMF may be related not only to the presence of the nitro group but
also to azomethine binding of the Schiff base. It is also considered that the mechanism
of action of ENIMF may involve other inflammatory pathways such as prostaglandin
biosynthesis inhibition through COX inhibition, phospholipase A2 inhibition, or 5-
lipoxygenase inhibition (Patil et al., 2019). In this regard, the present work introduces a
new starting point for Schiff bases with nitro derivatives as potential anti-inflammatory
agents.
5 Conclusion
A nitro-Schiff base was synthesized and evaluated for toxicity and anti-
inflammatory activity. Collectively, the results support that ENIMF have an effective
anti-inflammatory activity. The results encourage further studies that might help in a
better understanding of the mechanism of action of this compound.
12

Competing interests
All authors declare that they have no conflict of interest.
Acknowledgments
The authors are grateful to the Coordination for the Improvement of Higher
Level Education Personnel (CAPES) for financial support. We thank Aspesi GH and
Souza JVF for their help in drafting this manuscript.
13

Credit Author Statement
Author
Credit Author Statement
Roriz, B
PhD student, responsible for conducting
experiments in vivo (anti-inflammatory,
analgesic and antimutagenic effect of
ENIMF); data analysis, statistical analysis
and writing - original draft.
Buccini, D.F
Santos, B. F
Post-doctoral student, assisted in the
performance of in vitro tests of ENIMF
(cytotoxicity and hemolytic activity), data
analysis and writing - review & editing
draft.
PhD student, performed the synthesis and
analysis of the compound (E)-2-(((4-
nitrophenyl)imino)methyl) phenol
(ENIMF).
Silva, S. R
Master's student, helped to carry out studies
to evaluate the genotoxicity of the ENIMF.
Senior researcher responsible for the design
and supervision of the synthesis of the
ENIMF, data analysis and writing - review
draft.
Domingues, N.L.C.
Moreno, S.E
Supervisor of the PhD student Roriz, B.,
responsible for the conceptualization nd
supervision of the project, data formal
analysis; funding acquisition, writing –
review draft.
14

References
Abirami, M., Nadaraj, V., 2015. Antimicrobial activity of salicylaldimine Schiff bases.
International Journal of PharmTech Research 8, 558-561.
Al-Kahraman, Y.M., Madkour, H.M., Ali, D., Yasinzai, M., 2010. Antileishmanial,
antimicrobial and antifungal activities of some new aryl azomethines. Molecules 15,
660-671.
Alessandri, A.L., Sousa, L.P., Lucas, C.D., Rossi, A.G., Pinho, V., Teixeira, M.M.,
2013. Resolution of inflammation: mechanisms and opportunity for drug development.
Pharmacology & therapeutics 139, 189-212.
Ali, S.S., Kenawy, E.-R., Sonbol, F.I., Sun, J., Al-Etewy, M., Ali, A., Huizi, L., El-
Zawawy, N.A., 2019. Pharmaceutical Potential of a Novel Chitosan Derivative Schiff
Base with Special Reference to Antibacterial, Anti-Biofilm, Antioxidant, Anti-
Inflammatory, Hemocompatibility and Cytotoxic Activities. Pharmaceutical research
36, 5.
Almeida, T.C., Ribeiro, L.H.G., dos Santos, L.B.F., da Silva, C.M., Branquinho, R.T.,
de Lana, M., Gadelha, F.R., de Fátima, Â., 2018. Synthesis, in vitro and in vivo anti-
Trypanosoma cruzi and toxicological activities of nitroaromatic Schiff bases.
Biomedicine & Pharmacotherapy 108, 1703-1711.
Andrade, J.T., Alves, S.L.G., Lima, W.G., Sousa, C.D.F., Carmo, L.F., De Sá, N.P.,
Morais, F.B., Johann, S., Villar, J.A.F.P., Ferreira, J.M.S., 2019. Pharmacologic
potential of new nitro-compounds as antimicrobial agents against nosocomial
pathogens: design, synthesis, and in vitro effectiveness. Folia microbiologica, 1-13.
Arshad, N., Ahmad, M., Ashraf, M.Z., Nadeem, H., 2014. Spectroscopic,
electrochemical DNA binding and in vivo anti-inflammatory studies on newly
synthesized Schiff bases of 4-aminophenazone. Journal of Photochemistry and
Photobiology B: Biology 138, 331-346.
Biswas, S.K., Sodhi, A., Paul, S., 2001. Regulation of Nitric Oxide Production by
Murine Peritoneal Macrophages Treated in Vitro with Chemokine Monocyte
Chemoattractant Protein 1. Nitric Oxide 5, 566-579.
Ceyhan, G., Köse, M., Tümer, M., Demirtaş, İ., 2015. Anticancer, photoluminescence
and electrochemical properties of structurally characterized two imine derivatives.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 149, 731-743.
Coleman, J.W., 2001. Nitric oxide in immunity and inflammation. International
immunopharmacology 1, 1397-1406.
Connelly, L., Palacios-Callender, M., Ameixa, C., Moncada, S., Hobbs, A., 2001.
Biphasic regulation of NF-κB activity underlies the pro-and anti-inflammatory actions
of nitric oxide. The Journal of Immunology 166, 3873-3881.
15

Cowart, M., Kowaluk, E.A., Daanen, J.F., Kohlhaas, K.L., Alexander, K.M., Wagenaar,
F.L., Kerwin, J.F., 1998. Nitroaromatic amino acids as inhibitors of neuronal nitric
oxide synthase. Journal of medicinal chemistry 41, 2636-2642.
Dwivedi, A.K., Gurjar, V., Kumar, S., Singh, N., 2015. Molecular basis for
nonspecificity of nonsteroidal anti-inflammatory drugs (NSAIDs). Drug Discov Today
20, 863-873.
Freire, M.O., Van Dyke, T.E., 2013. Natural resolution of inflammation.
Periodontology 2000 63, 149-164.
Gacche, R., Gond, D., Dhole, N., Dawane, B., 2006. Coumarin Schiff-bases: As
antioxidant and possibly anti-inflammatory agents. Journal of enzyme inhibition and
medicinal chemistry 21, 157-161.
Geronikaki, A., Vicini, P., Incerti, M., Hadjipavlou-Litina, D., 2004. Thiazolyl and
isothiazolyl azomethine derivatives with anti-inflammatory and antioxidant activities.
Arzneimittelforschung 54, 530-537.
Gonzalez, A., Gomez, E., Cortes-Lozada, A., Hernández, S., Ramírez-Apan, T., Nieto-
Camacho, A., 2009. Heptacoordinate tin (IV) compounds derived from pyridine Schiff
bases: synthesis, characterization, in vitro cytotoxicity, anti-inflammatory and
antioxidant activity. Chemical and Pharmaceutical Bulletin 57, 5-15.
Graesser, D., Solowiej, A., Bruckner, M., Osterweil, E., Juedes, A., Davis, S., Ruddle,
N.H., Engelhardt, B., Madri, J.A., 2002. Altered vascular permeability and early onset
of experimental autoimmune encephalomyelitis in PECAM-1–deficient mice. The
Journal of Clinical Investigation 109, 383-392.
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum,
S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Analytical
biochemistry 126, 131-138.
Grosser, T., Ricciotti, E., FitzGerald, G.A., 2017. The cardiovascular pharmacology of
nonsteroidal anti-inflammatory drugs. Trends in pharmacological sciences 38, 733-748.
Guzik, T., Korbut, R., Adamek-Guzik, T., 2003. Nitric oxide and superoxide in
inflammation. Journal of physiology and pharmacology 54, 469-487.
Halade, G.V., Ma, Y., 2016. Neutrophils: friend, foe, or contextual ally in myocardial
healing. Journal of molecular and cellular cardiology 97, 44-46.
Hameed, A., al-Rashida, M., Uroos, M., Abid Ali, S., Khan, K.M., 2017. Schiff bases in
medicinal chemistry: a patent review (2010-2015). Expert opinion on therapeutic
patents 27, 63-79.
Headland, S.E., Norling, L.V., 2015. The resolution of inflammation: principles and
challenges, Seminars in immunology. Elsevier, pp. 149-160.
Heddle, J.A., 1973. A rapid in vivo test for chromosomal damage. Mutation
Research/Fundamental and Molecular Mechanisms of Mutagenesis 18, 187-190.
16

Hu, G., Wang, G., Duan, N., Wen, X., Cao, T., Xie, S., Huang, W., 2012. Design,
synthesis and antitumor activities of fluoroquinolone C-3 heterocycles (IV): s-triazole
Schiff–Mannich bases derived from ofloxacin. Acta Pharmaceutica Sinica B 2, 312-317.
Kajal, A., Bala, S., Kamboj, S., Sharma, N., Saini, V., 2013. Schiff bases: A versatile
pharmacophore. Journal of catalysts 2013.
Kim, M.Y., 2017. Intracellular and extracellular factors influencing the genotoxicity of
nitric oxide and reactive oxygen species. Oncology letters 13, 1417-1424.
Korhonen, R., Lahti, A., Kankaanranta, H., Moilanen, E., 2005. Nitric oxide production
and signaling in inflammation. Current Drug Targets-Inflammation & Allergy 4, 471-
479.
Landge, S., Ramachandran, V., Kumar, A., Neres, J., Murugan, K., Sadler, C., Fellows,
M.D., Humnabadkar, V., Vachaspati, P., Raichurkar, A., 2016. Nitroarenes as
antitubercular agents: stereoelectronic modulation to mitigate mutagenicity.
ChemMedChem 11, 331-339.
Mayadas, T.N., Cullere, X., Lowell, C.A., 2014. The multifaceted functions of
neutrophils. Annual Review of Pathology: Mechanisms of Disease 9, 181-218.
Medzhitov, R., 2010. Inflammation 2010: new adventures of an old flame. Cell 140,
771-776.
Mendonça, D.B.D., Silva, R.E.C., Palace-Berl, F., Takakura, C.F., Soares, S.R.C., Braz,
L.M.A., Tavares, L.C., Lindoso, J.A.L., 2019. Nitro-Heterocyclic compounds induce
apoptosis-like effects in Leishmania (L). amazonensis promastigotes. Journal of
Venomous Animals and Toxins including Tropical Diseases 25.
Moreno, S.E., Alves-Filho, J.C., Alfaya, T.M., da Silva, J.S., Ferreira, S.H., Liew, F.Y.,
2006. IL-12, but Not IL-18, Is Critical to Neutrophil Activation and Resistance to
Polymicrobial Sepsis Induced by Cecal Ligation and Puncture. The Journal of
Immunology 177, 3218-3224.
Nagatoshi, F., Kazuo, K., 2005. Macrophages in Inflammation. Current Drug Targets -
Inflammation & Allergy 4, 281-286.
Nathan, C., Ding, A., 2010. Nonresolving inflammation. Cell 140, 871-882.
Nepali, K., Lee, H.-Y., Liou, J.-P., 2018. Nitro-group-containing drugs. Journal of
medicinal chemistry 62, 2851-2893.
Olender, D., Żwawiak, J., Zaprutko, L., 2018. Multidirectional efficacy of biologically
active nitro compounds included in medicines. Pharmaceuticals 11, 54.
Pandey, A., Rajavel, R., Chandraker, S., Dash, D., 2012. Synthesis of Schiff bases of 2-
amino-5-aryl-1, 3, 4-thiadiazole and its analgesic, anti-inflammatory and anti-bacterial
activity. Journal of Chemistry 9, 2524-2531.
Park, Y., Kim, H.N., Park, S.N., Jang, S.-H., Choi, C.-H., Lim, H.-T., Hahm, K.-S.,
2004. Design of novel analogues with potent antibiotic activity based on the
antimicrobial peptide, HP (2-9)-ME (1-12). Biotechnology letters 26, 493-498.
17

Patil, K.R., Mahajan, U.B., Unger, B.S., Goyal, S.N., Belemkar, S., Surana, S.J., Ojha,
S., Patil, C.R., 2019. Animal Models of Inflammation for Screening of Anti-
inflammatory Drugs: Implications for the Discovery and Development of
Phytopharmaceuticals. International journal of molecular sciences 20, 4367.
Patterson, S., Wyllie, S., 2014. Nitro drugs for the treatment of trypanosomatid diseases:
past, present, and future prospects. Trends in Parasitology 30, 289-298.
Pautz, A., Art, J., Hahn, S., Nowag, S., Voss, C., Kleinert, H., 2010. Regulation of the
expression of inducible nitric oxide synthase. Nitric oxide 23, 75-93.
Plošnik, A., Vračko, M., Sollner Dolenc, M., 2016. Mutagenic and carcinogenic
structural alerts and their mechanisms of action. Arhiv za higijenu rada i toksikologiju
67, 169-182.
Pober, J.S., Sessa, W.C., 2007. Evolving functions of endothelial cells in inflammation.
Nature Reviews Immunology 7, 803.
Purohit, V., Basu, A.K., 2000. Mutagenicity of Nitroaromatic Compounds. Chemical
Research in Toxicology 13, 673-692.
Qin, W., Long, S., Panunzio, M., Biondi, S., 2013. Schiff bases: A short survey on an
evergreen chemistry tool. Molecules 18, 12264-12289.
Radu, M., Chernoff, J., 2013. An in vivo assay to test blood vessel permeability. JoVE
(Journal of Visualized Experiments), e50062.
Rakesh, K., Manukumar, H., Gowda, D.C., 2015. Schiff’s bases of quinazolinone
derivatives: synthesis and SAR studies of a novel series of potential anti-inflammatory
and antioxidants. Bioorganic & medicinal chemistry letters 25, 1072-1077.
Schmid, W., 1975. The micronucleus test. Mutat. Res. 31, 9-15.
Serhan, C.N., 2017. Treating inflammation and infection in the 21st century: new hints
from decoding resolution mediators and mechanisms. FASEB journal : official
publication of the Federation of American Societies for Experimental Biology 31, 1273-
1288.
Shahnaz, N., Puzari, A., Paul, B., Das, P., 2016. Activation of aryl chlorides in water for
Suzuki coupling with a hydrophilic salen-Pd (II) catalyst. Catalysis Communications
86, 55-58.
Sieuwerts, A.M., Klijn, J.G., Peters, H.A., Foekens, J.A., 1995. The MTT tetrazolium
salt assay scrutinized: how to use this assay reliably to measure metabolie activity of
cell cultures in vitro for the assessment of growth characteristics, IC50-values and cell
survival. Clinical Chemistry and Laboratory Medicine 33, 813-824.
Sondhi, S.M., Singh, N., Kumar, A., Lozach, O., Meijer, L., 2006. Synthesis, anti-
inflammatory, analgesic and kinase (CDK-1, CDK-5 and GSK-3) inhibition activity
18

evaluation of benzimidazole/benzoxazole derivatives and some Schiff’s bases.
Bioorganic & medicinal chemistry 14, 3758-3765.
Sostres, C., Gargallo, C.J., Arroyo, M.T., Lanas, A., 2010. Adverse effects of non-
steroidal anti-inflammatory drugs (NSAIDs, aspirin and coxibs) on upper
gastrointestinal tract. Best practice & research Clinical gastroenterology 24, 121-132.
Stone, W.L., Yang, H., Qui, M., 2005. Assays for Nitric Oxide Expression, in:
Krishnaswamy, G., Chi, D.S. (Eds.), Mast Cells: Methods and Protocols. Humana Press,
Totowa, NJ, pp. 245-256.
Strauss, M.J., 1979. The nitroaromatic group in drug design. Pharmacology and
toxicology (for nonpharmacologists). Industrial & Engineering Chemistry Product
Research and Development 18, 158-166.
Strober, W., 1997. Trypan blue exclusion test of cell viability. Current protocols in
immunology 21, A. 3B. 1-A. 3B. 2.
Takeuchi, M., Kobata, A., 1991. Structures and functional roles of the sugar chains of
human erythropoietins. Glycobiology 1, 337-346.
Tanaka, A., Araki, H., Komoike, Y., Hase, S., Takeuchi, K., 2001. Inhibition of both
COX-1 and COX-2 is required for development of gastric damage in response to
nonsteroidal antiinflammatory drugs. Journal of Physiology-Paris 95, 21-27.
Tumer, T.B., Onder, F.C., Ipek, H., Gungor, T., Savranoglu, S., Tok, T.T., Celik, A.,
Ay, M., 2017. Biological evaluation and molecular docking studies of nitro benzamide
derivatives with respect to in vitro anti-inflammatory activity. International
immunopharmacology 43, 129-139.
Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenin-induced edema in hind paw
of the rat as an assay for antiiflammatory drugs. Proceedings of the Society for
Experimental Biology and Medicine. Society for Experimental Biology and Medicine
(New York, N.Y.) 111, 544-547.
Wright, C.D., Mu¨lsch, A., Busse, R., Osswald, H., 1989. Generation of nitric oxide by
human neutrophils. Biochemical and Biophysical Research Communications 160, 813-
819.
Wright, H.L., Moots, R.J., Bucknall, R.C., Edwards, S.W., 2010. Neutrophil function in
inflammation and inflammatory diseases. Rheumatology (Oxford, England) 49, 1618-
1631.
Yao, L., Xue, X., Yu, P., Ni, Y., Chen, F., 2018. Evans blue dye: a revisit of its
applications in biomedicine. Contrast media & molecular imaging 2018.
Zhang, X., Goncalves, R., Mosser, D.M., 2008. The isolation and characterization of
murine macrophages. Current protocols in immunology 83, 14.11. 11-14.11. 14.
19

Figure 1. Synthesis of (E)-2-(((4-nitrophenyl)imino)methyl)phenol (ENIMF)
20

Figure 2. ENIMF did not induce micronucleus formation in the erythrocytes of mice
peripheral blood. The mice received vehicle, colchicine (5 mg/kg), or ENIMF (5
mg/kg). After 24 h, the micronucleus frequency in the erythrocytes was evaluated. The
results are expressed as the number of micronuclei in 2000 cells (mean ± SEM). One-
way ANOVA followed by Bonferroni test was performed. ***P <0.001 as compared to
the control group treated with colchicine.
21

Figure 3. Inhibitory effects of ENIMF on the neutrophil migration to the inflammatory
sites. The mice were pretreated with vehicle, ENIMF (1.25, 2.5, and 5 mg/kg) or
dexamethasone (0.4 mg/kg) before injecting 1.5mg carrageenan (inflammatory
stimulus). After 6 h, the total and differential cell counts were performed. (A) The
number of total leukocytes and (B) the number of neutrophils immigrated into the mice
peritoneal cavity are shown. The results are expressed as the cell number × 105/mL
(mean ± SEM). One-way ANOVA followed by Bonferroni test was performed.
***P<0.001 as compared to the control group.
22

Figure 4. Inhibitory effects of ENIMF on exudation and edema. (A) Evaluation of the
effects of ENIMF on the vascular permeability by Evans blue dye assay. The mice were
pretreated with vehicle or ENIMF (5 mg/kg) before injecting 1.5% carrageenan
(inflammatory stimulus). Evans blue dye was administered. After 4 h, quantification of
the extracted Evans blue dye was performed. The results are expressed as Evans blue
dye (µM/mL) (mean ± SEM). One-way ANOVA followed by Bonferroni test was
*
performed. ***P <0.001, and P <0.05 as compared to the control group. (B) Evaluation
of the effects of ENIMF on carrageenan-induced paw edema. The mice received
vehicle, ENIMF (5 mg/kg), or dexamethasone (0.4 mg/kg). Carrageenan 1.5%
(inflammatory stimulus) was injected into the left paw of all the mice, except the
vehicle group that received the saline solution. The readings were recorded at 0, 5, 10,
15, 30, 60, 120, and 240 min after carrageenan injection. The results are expressed as
the variation (Δ) in paw volume (mean ± SEM). Two-way ANOVA followed by
**
Bonferroni test was performed. ***P <0.001, P <0.01, and *P <0.05 as compared to the
control group.
23

Figure 5. Effects of ENIMF on NO production. (A) NO production in the macrophages
stimulated by LPS (200 ng) for 24 h. (B) NO production in the exudate of mice
pretreated with vehicle, dexamethasone (0.4 mg/kg), or ENIMF (1.25, 2.5, and 5 mg/kg)
and then subjected to an inflammatory stimulus (carrageenan 1.5 mg). Nitrite was
measured by the Griess reaction method. The results are expressed as nitrite (µM/mL)
(mean ± SEM). One-way ANOVA followed by Bonferroni test was performed. ***P
<0.001, **P <0.01, and *P <0.05 as compared to the control group.
24

Table 1. Evaluation of the in vitro cytotoxicity of ENIMF.
Treatment
Hemolytic assaya Cell viability at 24 hb Cell viability at 48 hc
Negative control 12.2 ± 0.9***
100 ± 8.6
100 ± 11.1
54.1 ± 5.8*
----------------
Vehicle
15.5 ± 0.6***
100 ± 1.4
50.5 ± 4.1*
--------------
Positive control
ENIMF (µg/mL)
200
100
50
38.7 ± 8.1***
27.5 ± 5.8***
16.6 ± 0.5***
15.4 ± 4.0***
15.5 ± 1.1***
10.2 ± 1.2***
11.0 ± 0.6***
75.0 ± 4.8
90.4 ± 4.4
117.1 ± 2.5
86.7 ± 1.9
135.3 ± 0.0
93.8 ± 4.6
141.5 ± 14.6
79.6 ± 8.5
68.7 ± 10.4
104.9 ± 12.2
80.5 ± 10.9
120.4 ± 9.0
106.0 ± 3.3
134.6 ± 3.8
25
12.5
6.25
3.12
(a) Hemolytic activity in the murine erythrocytes. ***P <0.001 as compared to Triton X-
100 (positive control). (b) and (c) Cell viability in ENIMF-treated murine macrophages at
*
(b) 24 h and (c) 48 h. P <0.05 as compared to the control (untreated). The results are
expressed as percentages (mean ± SEM). One-way ANOVA followed by Bonferroni test
was performed.
25

Graphical Abstract
26