Inhibitory effects of α, β-unsaturated carbonyl-based compounds and their pyrazoline derivatives on the phagocytosis of human neutrophils

Article abstract of DOI:10.1007/s00044-018-2163-3

A series of α, β-unsaturated carbonyl-based compounds (curcumin analogs and chalcone derivatives) and their pyrazoline derivatives were investigated for their in vitro inhibitory effects on the phagocytosis of human neutrophils. The effects of the compoun

Full text of DOI:10.1007/s00044-018-2163-3

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2163-3
ORIGINAL RESEARCH
Inhibitory effects of α, β-unsaturated carbonyl-based compounds
and their pyrazoline derivatives on the phagocytosis of human
neutrophils
1
Laiba Arshad1 Ibrahim Jantan
Syed Nasir Abbas Bukhari2 Shajarahtunnur Jamil3
●
●
●
Received: 25 December 2017 / Accepted: 26 February 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A series of α, β-unsaturated carbonyl-based compounds (curcumin analogs and chalcone derivatives) and their pyrazoline
derivatives were investigated for their in vitro inhibitory effects on the phagocytosis of human neutrophils. The effects of the
compounds on the chemotactic migration, CD11a/18 expression, phagocytic activity, and reactive oxygen species (ROS)
production by human whole blood cells (WBC) and isolated human polymorphonuclear neutrophils (PMNs) were assessed
by using 24-well cell migration assay kit, flow cytometer, Phagotest assay kit, and luminol/lucigenin-based
chemiluminescence assay, respectively. Compounds 4, 5, 6, 13, 23, 33, 39, and 41 showed strong inhibitory activity
against PMNs chemotaxis with IC50 values, ranging from 0.22–1.68 µM which were much lower than those of ibuprofen and
curcumin (IC50 values 11.02 and 5.0 µM, respectively). All compounds showed low or moderate inhibition of cell adhesion
molecule expression except for compound 15, while compounds 4, 5, 8, and 21 at 100, 25, and 6.25 μg/mL showed strong
inhibition of opsonized bacteria engulfment by neutrophils with the highest suppressive effects exhibited by compound 21 at
37.4%. Compounds 4, 11, 13, 14, 24, 25, 27, 33, and 34 significantly suppressed ROS generation by PMNs and WBC.
Compounds with N-methyl 4-piperidone and 4-piperidone linkers (4, 13, 14, 23, 24) and 2-pyrazoline-1-carboxamide and 2-
pyrazoline-1-carbothioamide derivatives showed strong inhibition on chemotactic and phagocytic activities, and ROS
production. The results suggest that some of the α, β-unsaturated carbonyl-based compounds strongly modulated the innate
immune responses of phagocytes at different steps, highlighting their potential as a source of new immunomodulatory
agents.
●
●
●
●
Keywords α, β-Unsaturated carbonyl-based compounds Immunosuppressive effect Chemotaxis Phagocytosis Reactive
oxygen species
Introduction
Polymorphonuclear neutrophils (PMNs) are professional
phagocytes playing important roles in our innate immune
defense against pathogens such as bacteria (Casulli et al.
Electronic supplementary material The online version of this article
2014). Normally, in a healthy person, neutrophils prevail as
(https://doi.org/10.1007/s00044-018-2163-3) contains supplementary
resting state in blood circulation, which prevents the acci-
material, which is available to authorized users.
dental release of toxic intracellular granules. However,
certain agents i.e., bacterial products, chemokines, and
cytokines can cause the activation of neutrophils which then
* Ibrahim Jantan
profibj@gmail.com
1
migrate towards the inflamed tissue and come across acti-
vating signals to elicit bacterial killing (Ahmad et al. 2014).
Upon activation, the movement of neutrophils from the
circulation to the site of infection is the first step of pha-
gocytosis (Muller 2013). The surface receptors of neu-
trophils i.e., L-selectins make loose connections with the
Drug and Herbal Research Center, Faculty of Pharmacy, Universiti
Kebangsaan Malaysia, Kuala Lumpur 50300, Malaysia
2
3
Pharmaceutical Chemistry Department, College of Pharmacy, Jouf
University, Aljouf, Sakaka 2014, Saudi Arabia
Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru
81310, Malaysia

Medicinal Chemistry Research
vascular endothelium which induce the conformational
changes in integrin adhesion molecules (CD11b/CD18) that
further get tangled with intercellular adhesion molecule
(ICAM-1 and 2), vascular cell-adhesion molecule-1
(VCAM-1), eventually resulting in strong adherence
(Schmidt et al. 2013). After neutrophils have interacted with
the endothelial cells they move along a chemotactic gradient
to the site of infection. Neutrophils at the injured tissue
phagocytose the infecting pathogens and this engulfment
activity is initiated when complement surface receptors on
PMNs detect complement-opsonized microbes (Haugland
et al. 2012) ultimately resulting in oxygen burst and release
of toxic granules for the prompt killing of bacteria (Yuanda
and Husain 2017). Although this process is imperative in
protecting the body from the infectious microbes, yet
sometimes, it becomes lethal in case of inappropriate acti-
vation of neutrophils, hence incongruous release of ROS
and other toxic molecules can cause harm to the host tissue,
ultimately resulting in the pathology of various immune-
related disorders.
for their inhibitory effects on human cancer cell lines and on
the diphenolase activity of mushroom tyrosinase and it was
found that some of the compounds exhibited highly potent
anticancer and tyrosinase inhibiting activities (Qin et al.
2015). Besides, this series of compounds has also been
evaluated against the sPLA2-V, COX-1, COX-2, and
release of pro-inflammatory cytokines including IL-6 and
TNF-α (Bukhari et al. 2015a).
These findings encouraged us to evaluate the effects of
these compounds on the innate immune system and hence
explored the relationship between the inhibitory effects and
chemical structures of the compounds. Therefore, in this
study we investigated the in vitro inhibitory effects of this
series of α, β-unsaturated carbonyl-based compounds along
with their pyrazoline derivatives on the successive steps of
phagocytosis. The phagocytosis steps include chemotaxis of
PMNs, expression of adhesion molecule CD11b/CD18,
phagocytic activity and inhibition of intracellular and
extracellular reactive oxygen species (ROS) production by
PMNs and human whole blood.
The α,β-unsaturated carbonyl-based compounds are
considered to be the reactive substructures of natural pro-
ducts or synthetic molecules, hence, owing miscellaneous
pharmacological activities i.e., potent antioxidant, anti-
inflammatory, antiviral, antibacterial, antifungal, anti-
tubercular, and immunomodulatory properties. In the α,β-
unsaturated carbonyl-based compounds, the carbon-carbon
double bond and carbon-oxygen double bond are segre-
gated by only one carbon-carbon single bond i.e., double
bonds are conjugated. These conjugations not only give the
properties of the functional group attached but also give
some other properties to the compounds. The most impor-
tant and widely studied α, β-unsaturated carbonyl-based
compounds include the natural products, curcumin, chal-
cones and their analogs and derivatives (Arshad et al. 2017).
In fact one-sixth of the currently known natural products
contain these potentially reactive substructures (Rodrigues
et al. 2016). Curcumin, extracted from the rhizomes of
Curcuma longa is a hydrophobic polyphenol as well as the
active constituent of turmeric found to exhibit a wide range
of activities (Anand et al. 2008) while flavonoids, either
isolated from natural sources or chemically synthesized,
have also acquired continuous importance due to their
ample biological activities (Elias et al. 1999; Ni et al. 2004;
Bukhari et al. 2013). Previously, we synthesized different
novel series of α, β-unsaturated carbonyl-based compounds.
The first series of compounds (1–30) were investigated as
acetylcholinesterase and butyrylcholinesterase inhibitors
(Bukhari et al. 2014a) along with the inhibition of enzymes
involved in arachidonic acid metabolism pathway, and it
was documented that these compounds have been found to
be strong inhibitors of aforementioned enzymes (Bukhari
et al. 2014b). Other compounds (31–41) were investigated
Methods and materials
General method for the synthesis of α, β-
Unsaturated carbonyl-based compounds
A novel series of α, β-unsaturated carbonyl-based com-
pounds have been synthesized previously by our group
using Claisen-Schmidt condensation reaction. The first
series of compounds (twenty one compounds, 1–7, 11–17,
21–27) was prepared by condensation between different
ketones and suitable aryl aldehydes at a molar ratio 1: 2 in
the presence of NaOH in ethanol. Compounds (8–10, 18–
20, 28–30) resembling chalcone analogs were synthesized
by same Claisen Schmidt condensation using molar ratio
1:1 of ketone and aldehyde (Fig. 1) (Bukhari et al. 2014b).
Other compounds related to chalcone analogs were syn-
thesized by substituting thioacetamide and arylacetamide
groups at their N1 position in three different series i.e.,
chalcones (31–34) in which equimolar ketones and alde-
hydes were reacted in the presence of a base. Further, these
chalcones (31–34) and thiosemicarbazide were refluxed at
alkaline pH to synthesize pyrazolines (35–37). A similar
reaction was used to yield compounds (38–41) by refluxing
chalcones (31–34) with N-(4-chlorophenyl) semicarbazide
in alkaline conditions (Fig. 2) (Qin et al. 2015). Curcumin,
ibuprofen and aspirin were used as reference compounds in
this study based on their role in inflammation and that they
have been used as positive controls in similar studies
(Jantan et al. 2011; Bukhari et al. 2015b).

Medicinal Chemistry Research
Fig. 1 General synthesis of α, β-unsaturated carbonyl-based compounds. Reprinted from the European Journal of Medicinal Chemistry (Bukhari
et al. 2014a)
Chemicals and reagents
were obtained from BD Biosciences (Franklin Lakes, NJ,
USA). The flow cytometer BD FACS Canto II equipped
with 488 nm argonion laser was used. Lymphoprep™ gra-
dient (1077 mg/mL), trypan blue reagent, phosphate buffer
saline tablet (PBS), Hanks Balance Salt Solutions (HBSS),
and dimethylsulfoxide (DMSO) were purchased from
Sigma (St Louis, MO, USA).
Serum opsonized zymosan A (Saccharomyces cerevisiae
suspensions and serum), luminol (3-aminophthalhydrazide),
phorbol 12-myristate 13-acetate (PMA), lucigenin (10, 10’-
dimethyl-9, 9’-biacridinium, dinitrate), acetyl salicylic acid
(purity 99%) and ibuprofen (purity 99%) were purchased
from Nacalai Tesque, Japan. Chemiluminescence mea-
surements were carried out on a Luminoskan Ascent
luminometer (Thermo Scientific, UK). Cytoselect 24-well
cell migration assay kits, Roswell Park Memorial Institute
(RPMI)-1640, fetal bovine serum (FBS) penicillin, strep-
tomycin were obtained from Cell Bio labs, Inc. (San Diego,
CA, USA). A CO2 incubator from Shell Lab, USA, and the
inverted microscope was purchased from Olympus ck 30,
Tokyo, Japan. Phagotest kit was obtained from Glycotope
Technology, Germany and allophycocyanin (APC)-con-
jugated CD11b, FITC-conjugated CD18, and IgG-FITC
Isolation of human polymorphonuclear neutrophils
(PMNs)
The isolation of PMNs from the whole blood from healthy
human volunteers aged ≥18 years old was performed by
following the method described by Koko et al. with slight
modification (Koko et al. 2008). The volunteers fulfilled the
following inclusion criteria; fasted overnight and did not
take any supplements or medicine and non-smoker. Briefly,
10 mL of fresh blood collected from the volunteers was

Medicinal Chemistry Research
Fig. 2 Structures and synthesis of chalcone derivatives (scheme 1) and pyrazoline derivatives (scheme 2, 3). Reprinted from the Chemical Biology
and Drug Design (Bukhari et al. 2015a, 2015b)
allowed to sediment for 30 min. After the specified time
interval, the separated layer of plasma was carefully layered
into a 10 mL falcon tube containing 5 mL of lymphoprep
gradient (1077 mg/mL) without any break to the gradient.
The Falcon tube, with two-step gradients of lymphoprep
and blood was centrifuged at 400×g for 20 min. The neu-
trophils and erythrocytes sedimented through the lympho-
prep layer during centrifugation. For the lysis of RBCs,
1 mL of distilled water was added followed by the addition
of HBSS and centrifuged at 300×g for 5 min. The super-
natant was aspirated carefully without disturbing the pellet
and resuspended in HBSS. Finally, the cell suspension was
diluted with HBSS to obtain a final cell suspension of 1 ×
106/mL. The use of human blood was approved by the
Ethics Committee of the Universiti Kebangsaan Malaysia
(approval no. FF/2012/Ibrahim/23May/432-May2012-
August 2013).
with the blue dye uptake were considered as dead cells and
the percentage cell viability was calculated from the total
cell counts (Jantan et al. 2012).
Cell migration assay
The inhibitory effect of the compounds on neutrophils
migration was quantitatively measured by using modified
method of Ilangkovan et al. (2015). The neutrophils isolated
from the heparinized whole blood were adjusted to a final
concentration of 1.5 × 106 cells. Cytoselect 24-well cell
migration assay kit was used to perform the experiment.
Initially, a total of 500 μL RPMI media comprising of
chemotactic stimulant, 10% FBS was added to the lower
chambers of the 24-well plate while the cell suspension,
with 1.5 × 106 cells in 300 μL serum-free media and serial
dilutions of tested sample (10, 5, 2.5, 1.25 µg/mL) was
added to the upper chamber (consisting of a polycarbonate
membrane insert, with 3 μm pore-sized filters) of a 24-well
tissue culture plate. Afterwards, the plate was incubated for
2.5 h at 37 °C in 5% CO2 incubator for the cells to migrate
towards the chemoattractant. The migratory cells passed
through polycarbonate membrane and adhered to the bot-
tom side. After the specified time interval, the inserts were
shifted to the new wells containing cell detachment solution
Cell viability
Trypan blue exclusion assay was employed to assess the
viability of cells. The synthetic compounds at four different
doses i.e., 200, 100, 50, and 6.25 µg/mL were incubated
with PMNs (1 × 106/mL) in 5% CO2 incubator, each in
triplicate, at 37 °C for 2 h at room temperature. The cells

Medicinal Chemistry Research
and again incubated at 37 °C (5% CO2) for 30 min to
remove the cells from the bottom of inserts. Finally, the
inserts were discarded and dislodged cells were stained by
adding the lysis buffer/CyQuant® GR dye solution. The
control wells contained RPMI with 10% FBS and cells but
no compound. Ibuprofen and curcumin were used as posi-
tive controls. The number of cells migrating to lower
chambers was determined by fluorescence, per manu-
facturer’s instructions, on a Victor 2 plate reader (Perkin
Elmer, Inc.).
on ice to stop the process of phagocytosis followed by the
addition of quenching solution which suppressed fluores-
cence of the bacteria to bind to the outside of the cell. The
sample tubes were vortexed well and centrifuged at 250×g
for 5 min (4 °C) after adding 3 mL of washing solution.
After washing twice, 2 mL of lysing solution was added and
incubated in dark for 20 min at 37 °C. At the end of incu-
bation, the samples were again washed and centrifuged at
the same speed. Finally, 200 μL of DNA staining solution
was added to each tube and vortexed. The phagocytic
activity was analyzed by flow cytometer and was deter-
mined as the percentage of phagocytizing neutrophils. Live
populations were gated by the software program in the
scatter diagram (FCS versus SSC).
Determination of MAC-1 expression
The Mac-1 expression assay was carried out by following
the method of Harun et al. (2015) with slight modification.
Aliquots (100 μL) of heparinized whole blood were incu-
bated with 20 μL of tested samples at three different con-
centrations (100, 25, and 6.25 µg/mL) in 5% CO2 incubator
at 37 °C for 30 min. After half an hour, cells were stimu-
lated with LPS at a concentration of 0.25 µg/mL and incu-
bated for 90 min. Thereafter the tubes were simultaneously
placed on ice in order to stop the reaction. Next, 10 µL of
APC-conjugated CD11b and FITC-conjugated CD18 were
added to the mixture. Meanwhile, 10 µL of IgG-FITC was
added as a negative control and tubes were incubated on ice
for 60 min. After that, FACS lysing solution was added to
each tube and incubated in dark for 20 min at 37 °C to lyse
the red blood cells. Subsequently, the tubes were cen-
trifuged at a speed of 250×g for 10 min and the supernatant
was discarded while retaining the pellet. Finally, the cells
within the pellet were washed twice with PBS at the same
speed. The inhibition of adhesion molecule expression was
analyzed by flow cytometer. The specific mean fluorescence
intensity for cells stained with each antibody was reported
as a percentage of CD11b and CD18 expression. For each
sample, 10,000 cells were analyzed.
Chemiluminescence assay
Luminol and lucigenin based chemiluminescence assay
were performed as previously described by Bukhari et al.
(2015a, 2015b). Luminol and lucigenin served as a probe
for the detection of intracellular and extracellular ROS
production. Briefly 25 µL of whole blood diluted with
sterile PBS (1:50; pH 7.4) and 25 µL of PMNs (with a final
concentration of 1 × 106 cells/mL) suspended in HBSS was
mixed with 25 µL of synthetic analogs dissolved in DMSO
and distilled water at a ratio of 5:95. The final concentration
of DMSO in the reaction mixture was maintained at 0.5% to
eliminate the effect of the solvent on the chemilumines-
cence while the final concentrations of the synthetic com-
pounds were 12.5, 6.25 3.125, 1.56, and 0.78 µg/mL. The
reaction mixture was incubated at room temperature for
30 min. At the end of incubation, the cells were activated by
25 µL of opsonized zymosan while for extracellular ROS
production, phorbol 12-myristate 13-acetate (PMA) was
used to stimulate PMNs, followed by the addition of 25 µL
of luminol and lucigenin, respectively. Thereafter, the final
volume of each well in 96-well plate was adjusted to 200 µL
by adding HBSS and incubated in the thermostatically
controlled chamber of the luminometer for 50 min at 37 °C.
However, the control wells contained no compound but
0.5% DMSO, HBSS, luminol, and cells. Ibuprofen and
curcumin were used as positive controls. The luminometer
results were monitored as the chemiluminescence reading
per luminometer unit (RLU) with peak and total integral
values set with repeated scans at 30 s intervals and 1 s points
measuring time. The inhibition percentage (%) for each
compound was calculated using the following formula:
Determination of phagocytic activity
The phagocytic activity assay was determined by using the
Phagotest assay kit, as previously described (Ilangkovan
et al. 2013). Briefly, a total of 100 μL heparinized blood was
mixed with 20 μL of test samples at two different con-
centrations (100, 25, and 6.25 µg/mL) and incubated in a
closed shaking water bath at 37 °C with 60 r.p.m. speeds for
30 min. The negative control tube without test sample was
kept on ice. After the incubation, the tubes were immedi-
ately transferred to the icebox and 20 μL of fluorescent-
labeled Escherichia coli (E. coli) was added into each tube
which was then incubated in shaking water bath at 37 °C for
10 min, while for negative control, the samples remained on
ice. Thereafter, the samples were immediately transferred
À
Á
RLUcontrol À RLUsample Â100%
Inhibition ð%Þ¼
RLUcontrol

Medicinal Chemistry Research
Table 1 Percentage inhibition at 10 μg/mL and IC50 values (µM) of α,
β-unsaturated carbonyl based compounds and pyrazoline derivatives
on activities of PMN cell movement
Statistical analysis
All the data were analyzed by using the Statistical
Package for Social Sciences (SPSS). Each sample was
measured in triplicate and the data are presented as a
mean standard error of mean (SEM). To determine the
IC50 values of active compounds, Graph Pad Prism 5
Software was used and the values were obtained from at
least three determinations. Data were analyzed using a
one-way analysis of variance (ANOVA) for multiple
comparisons, and P < 0.05 was considered to be statisti-
cally significant.
Compounds % inhibition of chemotactic activity IC50 values (µM)
3
61 3.4*
91 2.0**
91 2.3**
90 2.0**
73 1.4*
75 2.0*
75 3.1*
89 1.4**
65 2.0*
85 3.0**
75 1.4*
90 1.2**
87 0.8*
88 1.7*
65 1.7*
85 1.7*
59 1.4*
83 2.0**
80 2.3*
76 7.0
8.79 1.6
1.10 1.1
0.22 2.0
1.11 1.7
6.83 1.4
3.19 1.2
1.46 2.4
2.29 1.8
10.8 2.1
1.12 1.1
4.35 1.4
6.13 1.6
5.66 1.4
1.68 1.5
8.87 2.1
4.20 1.4
7.78 1.5
0.58 0.8
0.25 1.6
11.02 3.1
5.0 0.6
4
5
6
8
11
13
15
16
23
Results and discussion
24
31
All the synthetic compounds evaluated for cytotoxicity on
PMNs and human whole blood cells were found safe at
three different doses i.e., 100, 50, and 6.25 µg/mL, showing
percent cell viability ≥90% after 2 h of incubation. How-
ever, at 200 µg/mL, the cell viability was less than 50%.
Thus, the safe concentrations used in this study were below
100 µg/mL. The results of trypan blue exclusion method
indicated that the compounds were not toxic to immune
cells and could possibly modify the cellular immune
responses.
32
33
36
37
38
39
41
Ibuprofen
Curcumin
79 0.8
Values are presented as mean SD (n = 3). (–) IC50 values were not
determined. Significance of differences with respect to control: **P <
0.01; *P < 0.05
Effect of α, β unsaturated carbonyl based
compounds on PMN chemotaxis
The effects of the forty-one α, β-unsaturated carbonyl-based
compounds were studied at a single dose of 10 µg/mL on
the migration of PMNs towards the chemoattractant (10%
FBS). Chemoattractant buffer (DMSO and RPMI, 1:1 ratio)
was used as a negative control. Compounds 3–6, 8, 11, 13,
15, 16, 23, 24, 31–33, 36–39, 41 showed significant
blockade on the directional movement with percentage
inhibition ranging from 59 to 91% (Table 1). These com-
pounds were further studied at serial dilutions of 10 to 1.25
µg/mL and compared with the positive controls, ibuprofen
and curcumin. As shown in Fig. 3, all the selected samples
tested followed a concentration-dependent manner of inhi-
bition while compounds 4, 5, 6, 13, 23 33, 39, and 41 were
found to be highly effective, with IC50 values, ranging from
0.22–1.68 µM which were much lower than those of the
positive controls, ibuprofen and curcumin (IC50 values of
11.02 and 5.0 µM, respectively)(Table 1). Earlier, Bukhari
et al. suggested that compounds 4 and 23 were potent anti-
inflammatory agents (Bukhari et al. 2014a, 2014b). Other
compounds listed in Table 1 were also more active or
comparable to ibuprofen as they showed IC50 values ran-
ging from 2.29 to 10.8 µM.
Effect of α, β unsaturated carbonyl based
compounds on MAC-1 expression
The effect of α, β-unsaturated carbonyl-based compounds
on MAC-1 expression was measured by a flow cytometer.
Table 2 shows the percentage of CD 11b/18 expression
activity of neutrophils at 100.0, 25, and 6.25 μg/mL. Among
all the α, β unsaturated carbonyl based compounds, curcu-
min analogs exhibited weak to mild inhibition on CD 11b/
18 expression compared with the untreated sample as a
positive control. On the other hand, compounds synthesized
in three different schemes (chalcone and pyrazoline deri-
vatives) did not show any inhibitory effects on Mac-1
expression (Data are not shown). Of all the compounds
tested, compound 15 showed the most active inhibitory
effects on CD 11b/18 expression activity with percentage
expression of 40.27% at 100 µg/mL compared to the posi-
tive control. Compounds 8, 16, 23, and 27 also exhibited
moderate inhibition of CD 11b/18 expression at 100 µg/mL
i.e., 72.93 1.58–78.23% (Table 2). The inhibition of this
cell adhesion by the compounds might be correlated with
the blockade of various intracellular and extracellular

Medicinal Chemistry Research
Fig. 3 Inhibitory effects of α, β-unsaturated carbonyl-based compounds and pyrazoline derivatives at 10, 5, 2.5, 1.25 µg/mL on activities of PMN
cell movement. Significance of differences with respect to control: **P < 0.01, *P < 0.05
mechanisms particularly inhibition of NFκB pathway acti-
vation. Recently, Zhao et al. reported that monocarbonyl
analog with a 2-nitro substitution strongly suppressed TNF-
α which was responsible for activating NFκB pathway
(Zhang et al. 2014). Also in another study, it was reported
that compounds with thiopyrone structure and 4-piperidone
derivative strongly inhibited cytokines release and NFκB
inhibition (Zhao et al. 2015).
highest suppressive effects exhibited by compound 21 i.e.,
37.4%. Chalcones synthesized by three different schemes
also showed good inhibition of bacteria engulfment by
neutrophils ranging from 72.4 to 76.5%. Figure 4 shows
flow cytometer representation of E. coli engulfment by
neutrophils; inhibitory effects of α, β-unsaturated carbonyl-
based compounds at 100 and 6.25 μg/mL on PMN phago-
cytic activity. CR1, CR2, and CR3, complement surface
receptors
on
neutrophils
immediately
recognize
Effect of α, β-unsaturated carbonyl-based
compounds on phagocytic activity
complement-opsonized bacteria while FcαR, FcεRI, FcγRI
receptors of PMNs, specific for the Fc region of antibody,
identify antibody-coated microbes (Shokunbi and Odetola
2008). The results indicate that the enhanced inhibition of
ingestion of immunoglobulins and complement opsonized
E. coli by the tested samples might be due to the blockade
of aforementioned significant receptors.
The ability of PMNs to phagocytize opsonized bacteria was
determined by Phagotest kit and analyzed by flow cyto-
metry. The phagocytic inhibitory activity at 0 °C was used
as a negative control while at normal condition (37 oC) as a
positive control, and the phagocytic activity was calculated
by comparing the percentage of phagocytosis with the
decline in the percentage of E. coli ingestion by phagocytic
cells. The phagocytic activity of the cells when mixed with
the α, β-unsaturated carbonyl-based compounds was less
than the positive control, signifying that the compounds
reduced the percentage of FITC-labeled E. coli intake and
thus inhibiting the phagocytic cells. Notably, the phagocytic
inhibitory activity of PMNs by the compounds was dose-
dependent however; more pronounced effects were
observed at higher doses. As shown in Table 3, compounds
4, 5, 8, and 21 at 100, 25, and 6.25 μg/mL showed strong
inhibition of bacteria engulfment by neutrophils with the
Effect of α, β-unsaturated carbonyl-based
compounds on ROS production
The compounds were initially investigated on the human
whole blood for the purpose of preliminary screening and it
was seen that all compounds exhibited significant dose-
dependent inhibition of ROS generation during the meta-
bolic phase of phagocytosis i.e. increase in the concentra-
tion of the tested samples led to increase in percentage
inhibition (Table 4). Both intracellular and extracellular
ROS production in the whole blood was strongly inhibited
by curcumin analogs 3, 5, and 23 with IC50 values of 5.84,

Medicinal Chemistry Research
Table 2 Percentage of Cd11b/Cd18 expression activity of neutrophils
at 100, 25, and 6.25 μg/mL of α, β-unsaturated carbonyl based
compounds
Table 3 Percentage of PMN phagocytic activity at 100, 25, and 6.25
μg/mL of α, β-unsaturated carbonyl based compounds and pyrazoline
derivatives
Compounds
PMN Mac-1 expression activity SEM (%)
Compounds
PMN Phagocytic activity SEM (%)
100 µg/mL
25 µg/mL
6.25 µg/mL
100 µg/mL
25 µg/mL
6.25 µg/mL
1
97.7 0.53
97.94 0.28
95.04 1.15
93.38 1.58
89.28 1.50*
91.28 1.66
94.04 1.49
90.61 1.12
85.28 1.04*
86.94 0.94*
94.38 1.18
88.28 0.51*
94.04 0.93
90.94 0.81*
86.61 0.70
66.90 1.98*
79.55 2.09*
94.71 0.42
93.71 1.25
93.04 0.96
90.94 1.78
94.38 0.61
86.40 1.29*
83.40 0.56*
88.28 0.51*
91.61 1.12
95.38 0.48
83.41 0.57*
89.79 0.28*
90.61 1.12
94.71 0.84
99.00 0.60
98.27 0.44
96.47 0.69
96.57 1.18
95.50 1.80
93.23 2.29
97.07 0.26
91.53 0.52
96.00 0.50
90.20 1.15
96.47 0.72
94.37 1.66
96.60 0.67
94.87 1.55
94.7 1.50
1
62.20 1.01**
73.56 1.73**
62.40 1.44*
49.37 0.93**
43.73 1.23**
58.43 1.07**
70.41 1.62*
49.73 1.34**
53.00 1.46**
63.10 0.81*
62.13 1.22*
66.57 1.56**
57.73 2.28*
53.70 1.63*
57.00 1.85*
51.00 2.02**
70.67 0.74*
69.80 0.74*
67.73 0.38**
70.53 1.70**
37.47 1.05**
62.17 1.16*
67.8 1.18**
68.37 2.47*
64.77 1.03**
64.63 1.15*
58.67 0.95**
60.03 0.98*
70.37 0.61*
79.23 1.65*
73.5 0.93*
69.27 2.25*
74.13 1.05*
68.13 2.01*
53.67 4.67*
55.37 4.12*
62.57 3.24*
72.47 2.48*
55.37 4.12*
62.91 2.92*
68.50 2.21*
64.11 2.53*
68.39 2.11*
61.45 4.28*
57.38 3.40**
63.17 3.11*
58.52 1.74*
74.13 1.05*
71.80 1.93*
70.27 1.88*
72.13 1.62*
57.73 1.86**
65.11 1.71*
70.93 1.34*
77.13 0.75
69.39 1.18*
68.06 0.90*
70.27 1.30*
64.78 1.39*
73.13 0.98*
86.28 2.09
78.13 1.14*
77.47 0.48*
78.47 0.75*
76.47 0.55*
77.94 1.89*
75.80 0.72
76.13 1.05*
79.81 1.77*
74.47 0.48*
76.47 0.55*
75.47 1.43*
94.97 0.99
74.83 1.41*
75.50 1.43*
73.13 2.24*
58.03 1.16*
60.87 0.66*
66.13 0.96*
78.59 1.59*
70.60 1.42*
69.93 0.27*
71.80 0.90*
65.30 1.44*
72.93 0.26*
63.63 1.46*
60.30 0.87*
67.80 1.91*
67.67 1.54*
76.10 0.67*
74.83 0.67*
74.73 0.74*
75.62 1.70*
63.63 1.49*
68.10 1.46*
74.10 1.81*
79.17 1.08*
76.43 1.81*
71.73 0.66*
76.50 1.75*
74.37 1.47*
77.43 0.93*
91.2 0.82
2
94.33 0.50
2
3
90.07 1.75*
84.13 2.47*
87.17 4.35*
92.33 0.90
3
4
4
5
5
6
6
7
88.77 0.75**
77.10 1.27**
81.50 1.29*
91.50 0.64*
81.50 0.64*
91.13 0.66
7
8
8
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
87.73 1.42*
81.30 0.76
40.27 1.39**
72.93 1.58**
92.13 0.64*
91.43 1.21*
92.50 0.44*
87.40 0.69*
89.53 0.41*
81.93 1.92*
78.23 0.41**
82.50 1.21*
86.60 1.10*
92.57 0.67*
76.57 2.32**
89.20 0.52*
85.67 1.86*
94.57 0.66
96.10 0.29
95.57 0.20
95.27 0.52
95.17 1.39
94.37 0.69
92.03 1.01
96.33 1.26
95.00 1.93
92.00 1.08
93.97 0.81
93.50 0.76
97.23 0.45
90.67 1.76*
90.73 0.52*
93.60 1.04
96.03 0.93
Positive control
81.6 1.16
76.4 1.23*
78.7 0.66*
81.1 0.96
Values are presented as mean SEM (n = 3). Significance of
differences with respect to control: **P < 0.01; *P < 0.05
76 1.07*
74.4 1.62*
79.7 1.59*
82.7 1.42
73 1.34*
72.7 1.46**
73.6 0.81*
81.4 0.27
3.13, and 5.92 µM, respectively, suggesting that they were
highly potent. Compounds 4, 11, 13, 14, 18, 24, and 25 also
displayed high inhibitory effects with IC50 values ranging
from 3.97–5.14 µM for luminol-enhanced ROS production
while 7.24–20.42 µM for lucigenin amplified ROS genera-
tion as compared to aspirin (15.24 and 43.6 µM, respec-
tively) and curcumin (7.4 and 14.1 µM, respectively). On
the other hand, compounds 8, 9, 14, 16, 17, 18, 21, 22 and
29 with IC50 values ranging from 3.84–7.33 µM and
7.24–33.92 µM exhibited the good inhibitory activity of
both intracellular and extracellular reactive oxygen species
80.8 0.90
75.5 1.22*
85 1.44
72.4 1.56**
75.6 2.28*
80.7 0.26
78.3 1.46*
78.3 0.87*
74.0 1.63*
Positive control
Values are presented as mean SEM (n = 3). Significance of
differences with respect to control: **P < 0.01; *P < 0.05

Medicinal Chemistry Research
Fig. 4 Flow cytometer representation of E. coli engulfment by neu-
trophils. Inhibitory effects of α, β-unsaturated carbonyl-based com-
pounds and pyrazoline derivatives at 100, 25, and 6.25 µg/mL on PMN
phagocytic activity. (a) negative control, (b) positive control, (c) comp
4(100 µg/mL), (d) comp 4(25 µug/mL), (e) comp 4(6.25 µg/mL), (f)
comp 5(100 µg/mL), (g) comp 5(25 µg/mL), (h) comp 5(6.25 µg/mL),
(i) comp 8(100 µg/mL), (j) comp 8(25 µg/mL), (k) comp 8(6.25 µg/
mL), (l) comp 21(100 µg/mL), (m) comp 21(25 µg/mL), (n) comp 21
(6.25 µg/mL)
production. For the synthetic chalcones, the inhibitory
effects of the compounds were less except for compounds
32 (15.8 µM) and 33 (16.73 µM) which showed comparable
activity to aspirin i.e., 15.24 µM. for intracellular ROS
production. However, all the synthetic chalcones exhibited
strong extracellular ROS production in the whole blood
with IC50 values ranging from 5.49 to 17.19 μM. Evident by
previous studies, acetylsalicylic acid effectively inhibited
oxidative burst stimulated by both zymosan and PMA.
The compounds were further and specifically investi-
gated for their inhibitory effects on the oxidative burst by
PMNs. All the compounds particularly compounds 4, 11,
13, 22, 25, and 27 (IC50 values ranging from 3.12–4.77 µM)
presented strong inhibition as curcumin (4.0 µM) in a
concentration-dependent manner for luminol-enhanced
chemiluminescence assay whereas for extracellular oxida-
tive burst (lucigenin-induced), compounds 4, 14, and 27
(5.26–6.10 µM) demonstrated high inhibitory activity,

Medicinal Chemistry Research
Table 4 IC50 values (µM) of ROS inhibitory activity of α, β-
unsaturated carbonyl-based compounds and pyrazoline derivatives on
human whole blood and PMNs assayed by luminol and lucigenin
amplified chemiluminescence
signifying that these compounds were more efficacious than
the positive control, aspirin and curcumin which exhibited
IC50 values of 8.5 and 10.8 µM, respectively, for lucigenin-
amplified chemiluminescence. Among the chalcones ana-
logs, compound 34 showed the strongest inhibition of ROS
production with an IC50 value of 4.51 µM, stimulated by
lucigenin. The inhibitory effects of compound 33 with IC50
values of 11.67 and 11.73 μM for intracellular and extra-
cellular ROS production, respectively, was also comparable
to aspirin and curcumin.
The professional phagocytic cells after being activated
by serum-opsonized zymosan tend to accumulate at the site
of infection and phagocytose the attacking microorganisms
followed by the release of superoxide radicals and other
secondarily derived ROS, such as H2O2, hypochlorous acid,
and chloramines formed by NADPH oxidase enzyme.
NADPH oxidase is present in both phagosome and plasma
membrane and produces ROS both extracellularly and
intracellularly. In addition, H2O2 diffuses freely through
membranes and causes the release of ROS from the cell.
Extracellularly released ROS could cause tissue damage and
worsen inflammation while intracellular ROS kill phago-
cytosed pathogens or help the cell to kill them by chlor-
ination of the protein content of neutrophil phagosome or by
some other way. Therefore, it is important to differentiate
between the intracellular and extracellular production of
ROS (Rajecky et al. 2012). Owing to its small molecular
weight, luminol was used as a probe to quantify intracellular
ROS production. Therefore, the remarkable inhibition of the
reactive species by the novel synthetic compounds could be
attributed to their ability to block the interaction of zymosan
with complement receptors, ultimately inhibiting NADPH
oxidase (Rathakrishnan and Tiku 1993).
Compounds Intracellular IC50 values
(µM)
Extracellular IC50 values
(µM)
Whole blood PMNs
Whole blood PMNs
1
–
47.93 1.5
52.8 1.2
–
–
–
2
–
–
3
–
7.19 1.0 5.84 0.5
4.53 0.8 14.4 0.6
12.2 0.2
6.10 0.1
12.8 0.3
–
4
–
5
3.13 0.4
5.0 0.2
14.5 1.1
34.5 1.3
55.8 1.4
6
–
–
–
7
–
–
8
–
9.17 0.2 33.92 0.6
33.68 0.8
37.24 0.8
–
9
–
–
–
18.58 0.2
45.7 0.9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
Aspirin
Curcumin
–
3.97 1.1
4.77 0.2 12.36 0.3
58.11 1.1
–
–
–
–
4.02 0.8
4.59 0.6 11.56 0.2
6.28 0.1 20.42 0.6
42.8 0.6
4.58 1.1
9.27 1.1
70.09 1.5
36.22 1.2
12.15 0.9
–
3.84 0.6
–
9.06 0.8
4.93 0.2
–
–
6.37 0.8
–
–
32.5 0.7
6.66 0.4
10.88 0.8 7.24 0.3
–
–
61.0 1.5
25.3 1.3
18.85 0.8
–
22.05 0.7
8.19 0.5
8.51 0.3
25.0 1.0
16.27 0.8
–
–
11.12 0.2 31.14 1.1
3.12 0.1 60.17 1.5
18.34 0.8 5.92 0.8
14.82 1.2 12.5 1.0
4.39 0.4 12.34 1.0
80.32 1.1
7.33 0.8
–
5.14 1.0
Structure activity analysis of α, β-unsaturated
carbonyl-based compounds
–
3.15 0.9
–
–
5.26 0.4
60.57 1.2
25.74 0.2
–
31.00 0.4
43.20 0.9
–
6.08 0.5 31.6 1.0
α, β-Unsaturated carbonyl-based compounds possess
remarkable reactivity and have been known for their diverse
pharmacological activities conspicuously on immune sys-
tem and are considered as valuable immunomodulators.
Interestingly, these synthetic compounds exhibited variable
activity trends for different steps of phagocytosis. All the
compounds showed moderate to strong inhibition for the
migration of PMNs, phagocytic activity and ROS produc-
tion, nevertheless, very less activity was observed for
CD11b/CD18 expression of leukocytes. In perspective of
structure-activity relationship, compound 15, with 1-benzyl-
4-piperidone linker, showed good suppression on CD11b/
CD18 integrin expression, though the remaining com-
pounds did not display much activity.
–
84.26 1.2
–
–
–
14.6 0.2
58.3 1.4
–
15.80 0.3
16.3 0.6 15.5 0.7
11.67 0.1 14.8 0.4
16.73 0.4
11.73 0.8
4.51 0.2
18.6 0.6
41.5 0.9
51 1.4
–
–
–
–
–
–
–
–
–
8.74 0.1
17.19 0.8
6.38 0.2
7.54 0.3
15.16 1.0
5.98 0.5
15.9 1.2
–
–
28.60 0.2
–
19.16 0.1
53 1.2
–
–
–
48.5 1.1 8.91 0.9
10.74 0.4 43.6 1.5
26.3 0.8
8.57 0.3
10.8 0.1
15.24 0.2
7.4 0.1
4.0 0.1
14.1 0.2
The structural components found to be responsible for
the increased chemotactic inhibition by the compounds
(–) IC50 values were not determined

Medicinal Chemistry Research
shown in Fig. 1 (compounds 1–30) was N-methyl-4-
piperidone linker and 4-piperidone moiety which was in
accordance with our previous study. The strong suppressive
activity seen by compound 6 can be attributed to the pre-
sence of diethoxymethyl group at position 4 on the benzene
ring. Synthetic chalcones 31, 32, 37, 38, 39, and 41 bear
2,3-dimethoxy substitution pattern on naphthalene ring and
compounds 38, 39, and 37 also possess benzofuran ring
attached to an aromatic ring. These structural features may
be responsible for the inhibition of the chemotactic activity.
Compound 41 bearing 2-pyrazoline-1-carboxamide as a
linker, contains benzodioxane ring which has been reported
to be an important feature for the anti-inflammatory activity
of the synthesized compounds. Previously Bukhari et al.
(2015a, 2015b) suggested that compounds belonging to 2-
pyrazoline-1-carboxamide derivatives (38–41) and 2-
pyrazoline-1-carbothioamide derivatives (35–37) were
potent anti-inflammatory agents.
Compound 5 with the 1-benzyl-4-piperidone linker along
with compound 4 (N-methyl-4-piperidone linker) and com-
pound 8, substituted with diethoxymethyl group at position
4 on the benzene ring have indicated potential phagocytic
inhibitory effects. In addition, the results also showed that
compound 21 was found highly active towards the inhibition
of phagocytosis by PMNs. These results suggested that
substitution of the phenyl ring at position 4 is important for
good inhibitory activity. 2-Pyrazoline-1-carbothioamide
derivatives and 2-pyrazoline-1-carboxamide derivatives
also displayed good inhibition of phagocytosis by PMNs
and have been documented as strong anti-inflammatory
agents (Bukhari et al. 2015a, 2015b).
Therefore, as an overall trend, N-methyl-4-piperidone, 4-
piperidone and 1-benzyl-4-piperidone linkers remained
important in showing highest inhibitory effects along with
diethoxymethyl benzene on position 4 and phenyl 2-nitro 4-
dimethylamine substitution pattern. For compounds 33 and
37 it was observed that substitution of methoxy group at
position 2 and 3 of naphthalene ring showed strong anti-
inflammatory properties compared to when the methoxy
group was present at position 4 and 7. Further analysis of
SAR indicated that 4-dimethylamino is also responsible for
good inhibitory effects as shown by compound 34 and 38.
Conclusion
Compounds with N-methyl 4-piperidone and 4-piperidone
linkers (4, 13, 14, 23,and 24) revealed high inhibition on
ROS production stimulated by both serum opsonized
zymosan and PMA. In addition to N-methyl 4 piperidone
linker and 4-piperidone linker, compounds with acetone
linkers (5, 15) and 2-pyrazoline-1-carboxamide derivatives
(2b, 4b) also showed high inhibitory effects towards the
chemotactic and phagocytic activity. The presence of
diethoxymethyl-benzene at position 4 (8, 6, 27) and phenyl-
2-nitro-4-dimethylamine (11, 13, 14, and 16) were also
found to be responsible for the immunosuppressive activity.
Further mechanistic studies should be carried out to develop
these compounds into potent immunosuppressive agents in
treating different immune-related diseases particularly
related to inflammatory disorders.
Acknowledgements The authors thanked Universiti Kebangsaan
Malaysia for providing the grant under the Dana Impak Perdana and
Md. Areeful Haque for his technical assistance.
An insight into the structural features with respect to the
potential activity revealed that presence of 2-nitro and 4
diethylamine substitution pattern (compound 11) impart a
role in the activity of compounds. Likewise, SAR studies of
chemiluminescence assay outcomes also witnessed similar
findings. Compound 14 having N-methyl-4-piperidone linker
with substituent NCH3 and compounds 3 and 23 having 4-
piperidone linker with substituent NH were found most active
in luminol- and lucigenin-enhanced chemiluminescence
assay in both human whole blood and PMNs, respectively.
Though compounds with the aforesaid linkers but with dif-
ferent substituents (4, 24, and 13) also showed higher inhi-
bition than aspirin. Further analysis of results revealed that
ROS production by PMNs stimulated by zymosan was also
actively inhibited by compounds 11, 22, 25, 27, and 29.
Moreover, compounds 8, 9, 15, 16, and 21 also exhibited
good suppression of oxidative burst which may be due to the
effect of substitution (phenyl-4-pyrrolidine) on aromatic rings
of compounds. In the present study, the findings suggested
that in addition to specific linkers imperative for activity, the
substitution patterns on the linked aromatic rings also
imparted more or less role in determining good activity.
Funding This work was supported by Universiti Kebangsaan Malay-
sia under the Dana Impak Perdana (grant number DIP-2015-013).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Ahmad W, Kumolosasi E, Jantan I, Bukhari SNA, Jasamai M (2014)
Effects of novel diarylpentanoid analogues of curcumin on
secretory phospholipase A2, cyclooxygenases, lipo‐oxygenase,
and microsomal prostaglandin E synthase‐1. Chem Biol Drug
Des 83(6):670–681
Anand P, Thomas SG, Kunnumakkara A, Sundaram C, Harikumar
KB, Sung B, Tharakan ST, Misra K, Priyadarsini IK, Rajase-
kharan KN, Aggarwal BB (2008) Biological activities of curcu-
min and its analogues (Congeners) made by man and mother
nature. Biochem Pharmacol 76(11):1590–1611
Arshad L, Jantan I, Bukhari SNA, Haque MA (2017) Immunosup-
pressive effects of natural α, β-unsaturated carbonyl-based

Medicinal Chemistry Research
compounds, and their analogs and derivatives, on immune cells: a
review. Front Pharmacol 8:1–22
Bukhari SNA, Jantan I, Jasamai M (2013) Anti-inflammatory trends of
1, 3-diphenyl-2-propen-1-one derivatives. Mini Rev Med Chem
13(1):87–94
Bukhari SNA, Jantan I, Masand VH, Mahajan DT, Sher M, Naeem-ul-
Hassan M, Amjad MW (2014a) Synthesis of α, β-unsaturated
carbonyl based compounds as acetylcholinesterase and butyr-
ylcholinesterase inhibitors: characterization, molecular modeling,
QSAR studies and effect against amyloid β-induced cytotoxicity.
Eur J Med Chem 83:355–365
Jantan I, Bukhari SNA, Lajis NH, Abas F, Wai LK, Jasamai M (2012)
Effects of diarylpentanoid analogues of curcumin on chemilu-
minescence and chemotactic activities of phagocytes. J Pharm
Pharmacol 64(3):404–412
Jantan I, Harun NH, Septama AW, Murad S, Mesaik MA (2011)
Inhibition of chemiluminescence and chemotactic activity of
phagocytes in vitro by the extracts of selected medicinal plants. J
Nat Med 65(2):400–405
Koko WS, Mesaik MA, Yousaf S, Galal M, Choudhary MI (2008) In
vitro immunomodulating properties of selected Sudanese med-
icinal plants. J Ethnopharmacol 118(1):26–34
Bukhari SNA, Lauro G, Jantan I, Bifulco G, Amjad MW (2014b)
Pharmacological evaluation and docking studies of α, β-unsatu-
rated carbonyl based synthetic compounds as inhibitors of
secretory phospholipase A 2, cyclooxygenases, lipoxygenase and
proinflammatory cytokines. Bioorg Med Chem 22
(15):4151–4161
Bukhari SNA, Zhang X, Jantan I, Zhu HL, Amjad MW, Masand VH
(2015a) Synthesis, molecular modeling, and biological evaluation
of novel 1, 3‐diphenyl‐2‐propen‐1‐one based pyrazolines as anti‐
inflammatory agents. Chem Biol Drug Des 85(6):729–742
Bukhari SNA, Jantan I, Kumolosasi E, Jasamai M (2015b) Immuno-
modulatory effects of diarylpentanoid analogues of curcumin.
Med Chem Res 24(9):3405–3411
Casulli S, Coignard Biehler H, Amazzough K, Shoai Tehrani M,
Bayry J, Mahlaoui N, Elbim C, Kaveri SV (2014) Defective
functions of polymorphonuclear neutrophils in patients with
common variable immunodeficiency. Immunol Res 60(1):69–76
Elias DW, Beazely MA, Kandepu NM (1999) Bioactivities of chal-
cones. Curr Med Chem 6(12):1125
Muller WA (2013) Getting leukocytes to the site of inflammation. Vet
Pathol 50(1):7–22
Ni L, Meng CQ, Sikorski JA (2004) Recent advances in therapeutic
chalcones. Expert Opin Ther Pat 14(12):1669–1691
Qin HL, Shang ZP, Jantan I, Tan OU, Hussain MA, Sher M, Bukhari
SNA (2015) Molecular docking studies and biological evaluation
of chalcone based pyrazolines as tyrosinase inhibitors and
potential anticancer agents. RSC Adv 5(57):46330–46338
Rajecky M, Lojek A, CIZ M (2012) Differentiating between intra‐and
extracellular chemiluminescence in diluted whole‐blood samples.
Int J Lab Hematol 34(2):136–142
Rathakrishnan C, Tiku ML (1993) Lucigenin-dependent chemilumi-
nescence in articular chondrocytes. Free Radic Biol Med 15
(2):143–149
Rodrigues T, Reker D, Schneider P, Schneider G (2016) Counting on
natural products for drug design. Nat Chem 8(6):531
Schmidt S, Moser M, Sperandio M (2013) The molecular basis of
leukocyte recruitment and its deficiencies. Mol Immunol 55
(1):49–58
Harun NH, Septama AW, Jantan I (2015) Immunomodulatory effects
of selected Malaysian plants on the CD18/11a expression and
phagocytosis activities of leukocytes. Asian Pac J Trop Biomed 5
(1):48–53
Haugland GT, Jakobsen RA, Vestvik N, Ulven K, Stokka L, Werge-
land HI (2012) Phagocytosis and respiratory burst activity in
lumpsucker (Cyclopterus lumpus L.) leucocytes analysed by flow
cytometry. PloS One 7(10):47909
Shokunbi OS, Odetola AA (2008) Gastroprotective and antioxidant
activities of Phyllanthus amarus extracts on absolute ethanol-
induced ulcer in albino rats. J Med Plant Res 2(10):261–267
Yuandani, Jantan I, Husain K (2017) 4, 5, 4′-Trihydroxychalcone, 8,
8′-(ethene-1, 2-diyl)-dinaphtalene-1, 4, 5-triol and rutin from
Gynura segetum inhibit phagocytosis, lymphocyte proliferation,
cytokine release and nitric oxide production from phagocytic
cells. BMC Complement Altern Med 17(1):211
Ilangkovan M, Jantan I, Mohamad HF, Husain K, Abdul Razak AF
(2013) Inhibitory effects of standardized extracts of Phyllanthus
amarus and Phyllanthus urinaria and their marker compounds on
phagocytic activity of human neutrophils. Evid Based Comple-
ment Alternat Med 2013:603634
Ilangkovan M, Jantan I, Mesaik MA, Bukhari SNA (2015) Immuno-
suppressive effects of the standardized extract of Phyllanthus
amarus on cellular immune responses in Wistar-Kyoto rats. Drug
Des Devel Ther 9:4917–4930
Zhang Y, Jiang X, Peng K, Chen C, Fu L, Wang Z, Feng J, Liu Z,
Zhang H, Liang G, Pan Z (2014) Discovery and evaluation of
novel anti-inflammatory derivatives of natural bioactive curcu-
min. Drug Des Devel Ther 8:2161–2171
Zhao C, Zhang Y, Zou P, Wang J, He W, Shi D, Li H, Liang G, Yang
S (2015) Synthesis and biological evaluation of a novel class of
curcumin analogs as anti-inflammatory agents for prevention and
treatment of sepsis in mouse model. Drug Des Devel Ther
9:1663–1678