Effects of synthetic chalcone derivatives on oxidised palmitoyl arachidonoyl phosphorylcholine-induced proinflammatory chemokines production

Article abstract of DOI:10.1039/c5ra11073d

Oxidised 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) induces the production of proinflammatory chemokines has been widely studied for its role in vascular inflammation. There is increasing evidence on the role of chalcones as potent

Full text of DOI:10.1039/c5ra11073d

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Effects of Synthetic Chalcone Derivatives on Oxidised Palmitoyl
Arachydonoyl Phosphorylcholine-Induced Proinflammatory
Chemokines Production
Received 00th January 20xx,
Accepted 00th January 20xx
Lim Sock-Jin, Endang Kumolosasi , Norazrina Azmi , Syed Nasir Abbas Bukhari, Malina Jasamai and
Norsyahida Mohd Fauzi*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Oxidised 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) induces the production of
proinflammatory chemokines has been widely studied for its role in vascular inflammation. There is
increasing evidence on the role of chalcones as potential anti-inflammatory agents but less is known about
its effects on OxPAPC-induced chemokines production and the involvement of unfolded protein response
(UPR) signalling, particularly through XBP1 pathway. The present study sought to investigate the inhibitory
potential of synthetic chalcone derivatives on the release of interleukin-8 (IL-8) and monocyte
chemoattractant protein-1 (MCP-1), induced by OxPAPC through XBP1 signalling pathway on differentiated
U-937 macrophages. The effects of synthetic chalcone derivatives on the chemokines productions were
investigated using enzyme-linked immunosorbent assays, while the inhibitions of XBP1 signalling were
detected using western immunoblot. Results show that all the three tested synthetic chalcone derivatives
inhibited OxPAPC-induced chemokines production in a concentration-dependent manner. Compound 1.5
exhibited the strongest inhibition of IL-8 and MCP-1 at 61.4 ± 4.23% and 63.8 ± 2.16%, respectively.
Compound 1.5 also achieved the lowest IC50 values for both IL-8 (18.33 ± 1.59 µM) and MCP-1 (8.42 ± 1.05
µM) inhibitions. For XBP1 protein expression, both compound 1.4 and 1.5 exhibited significant concentration-
dependent suppression of the protein expressions. The results suggest that synthetic chalcone derivatives
may serve as potential alternatives for future development of anti-inflammatory agents, particularly in
vascular inflammation.
Introduction
as
oxidised
1-palmitoyl-2-arachidonoyl-sn-glycero-3-
phosphorylcholine (OxPAPC) can induce the release of these
Chemokines play various important roles in inflammatory chemokines. However, induction of IL-8 and MCP-1 by OxPAPC is
1
processes. Diverse factors such as oxidised phospholipids (OxPLs) , independent of the classic nuclear factor kappa b (NFκβ) pathway
2
3
ultraviolet radiation exposure and endotoxin are known to be and therefore unlike LPS, OxPAPC did not induce certain pro-
involved in the generation of chemokines. Overproduction of inflammatory cytokines such as TNF-alpha and IL-1β. Moreover,
proinflammatory chemokines such as interleukin-8 (IL-8) and stimulation with OxPAPC will generally result in more prolonged
monocyte chemoattractant protein-1 (MCP-1) has been associated upregulation of these chemokines 9.
4
with various inflammatory diseases including atherosclerosis
.
MCP-1 is widely known to play important role in the accumulation Oxidised phospholipids (OxPLs), the bioactive components of
and transmigration of mononuclear cells such as monocytes to the oxidised low density lipoprotein (OxLDL) particles, have received
5
sites of inflammation while IL-8 is responsible for monocytes considerable attentions in their effects on vascular inflammatory
6,7
10,11,12
arrestment to vascular endothelium
.
IL-8 and MCP-1 are events
. Oxidised 1-palmitoyl-2-arachidonoyl-sn-glycero-3-
chemokines that are widely known to play a major role in vascular phosphorylcholine (OxPAPC) in particular is present at high
13
inflammations 8. These chemokines can be induced by conventional concentration in atherosclerotic plaques
and is thought to
pro-inflammatory inducers such as lipopolysaccharide (LPS) through contribute to the inflammatory processes induced by OxLDL.
activation of NF-kB pathway. Similarly, oxidised phospholipid such OxPAPC has been shown to induce the expressions of inflammatory
genes such as IL-8 and MCP-1 in human endothelial cells through
UPR pathway14, which is one of the pathways that are activated
during endoplasmic reticulum stress. The levels of OxPAPC-induced
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IL-8 and MCP-1 release were also reported in human THP-1 cell
Journal Name
15
.
Furthermore, an in vivo study carried out by Furnkranz and co- Synthesis of chalcone derivatives
workers showed that topical administration of OxPAPC on C57BL/6J Synthetic chalcone derivatives used in this study were synthesised
mice carotid arteries resulted in enhanced mRNA expression of by members of research group using Claisen-Schmidt condensation
MCP-1 and keratinocyte-derived chemokine (KC) 16, of which the reaction and reported previously (Table 1). Briefly, an amount of 10
latter is a murine chemokine with closest resemblance to IL-8 in mmol of the respective ketones was added to a solution of the
human system.
respective aldehydes (10 mmol) in ethanol (15 mL). NaOH (50%)
solution was added dropwise as catalyst, and the reaction mixture
Endoplasmic reticulum (ER) stress is characterised as imbalanced of was stirred at room temperature for 2-24 h accordingly. Reaction
cellular protein modifications. Various stimuli which initiate ER completion was monitored by TLC and appearance of precipitate
stress include accumulation of unfolded proteins, deprivation of and colour changes of the reaction mixture were an indicative of
glucose, disruption in calcium homeostasis as well as accumulation product formation. Upon completion, the reaction mixture was
of free cholesterol and oxidised phospholipids 17,18. Continual ER poured into acidified ice, extracted with ethyl acetate (50 mL),
stress causes activation of unfolded protein response (UPR) washed with water (2 x 150 mL), dried and concentrated in vacuo to
signalling, which involves inositol requiring enzyme (IRE) 1, RNA- give oils and solids. The crude products were further purified either
depending protein kinase-like endoplasmic reticulum kinase (PERK) by column chromatography or recrystallization28
and activating transcription factor (ATF) 6 19. Among the three
.
signalling arms, IRE1 is the most evolutionarily conserved pathway, All compounds were characterised as reported previously28. H and
whereby activation of IRE1 is responsible for X-box protein (XBP) 1 13C NMR spectra were recorded with a JEOL ECP spectrometer
activation. It is linked to various inflammatory disorders such as operating at 500 MHz, with Me4Si as internal standard and CDCl3 or
1
21
22
atherosclerosis 20, inflammatory bowel disease
and colitis
.
DMSO-d6 as solvent. High resolution mass spectra (HRMS) were
Besides, it was reported that UPR is responsible in effector cells of determined by the electrospray ionisation mass spectrometry (ESI-
the immune response through the enhanced release of MS) on MicroTOF-Q mass spectrometer (Bruker, Coventry, UK).
proinflammatory mediators 23,24
.
Microanalyses data were obtained from the Fison EA 1108
elemental analyser. Infrared spectra were recorded using KBr disc
Chalcones (1,3-diphenyl-2-propen-1-one) are natural polyphenolic on a Perkin Elmer 400 (FTIR) spectrometer.
compounds biosynthesized abundantly in fruits, soy beans, spices
4-(4-Diethylamino-phenyl)-1,1-diphenyl-but-3-en-2-one (1.2) 28
and vegetables. They are composed of open-chain flavanoids which
are joined by α,β-unsaturated ketone moiety 25. Chalcones have
been a considerable interest among scientists due to their broad
range of pharmacological activities and simple synthesis. The anti-
inflammatory effects of chalcones have been reported extensively.
For example, hydroxychalcones were shown to be effective in
inhibition of lipopolysaccharide (LPS)-induced tumour necrosis
This compound was obtained by reacting 1,1-diphenylacetone (2.10
g, 10 mmol) with 4-diethylamino benzaldehyde (1.76 g, 10 mmol) to
give light red solids (1.25 g, 34%). RF 0.55 (EtOAc-PE 1:3 v/v); mp:
1
106–107 °C; H NMR (500 MHz, CDCl3): d = 7.84 (d, J = 7.5 Hz, 2H),
7.74 (d, J = 8.50, 1H), 7.61 (d, J = 7.0 Hz, 1H), 7.35 (m, 10H), 6.70 (d J
= 7.0 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 5.41 (s, 1H), 3.45 (q, J = 7.0 Hz
4H), 1.23 (t, J = 10.0 Hz, 6H). 13C NMR (500 MHz, CDCl3): d=190.10,
149.68, 144.40, 141.32, 130.74, 129.30, 128.57, 128.49, 128.09,
126.93, 110.60, 62.91, 44.50, 12.57; HRMS (ESI) m/z: 370.21 [M + H]
+; Anal. calcd for C26H27NO: C 84.51, H 7.37, N 3.79, found C 84.53, H
7.67, N 3.81.
26
factor-α (TNF-α) and LPS-induced inducible nitric oxide synthases
(iNOS) 27 on RAW 264.7 macrophages.
Nevertheless, synthetic chalcone derivatives have not been
extensively studied in OxPAPC-induced proinflammatory
chemokines release. Therefore, this study was aimed to investigate
the inhibitory effects of synthetic chalcone derivatives on the
release of IL-8 and MCP-1 induced by OxPAPC on differentiated U-
937 macrophages and the mechanism of action. Results obtained
demonstrated that the synthetic chalcones derivatives tested were
able to reduce the release of IL-8 and MCP-1 through inhibition of
XBP1 signalling.
1,1,5-Triphenyl-hex-3-en-2-one (1.4) 28
This compound was obtained by reacting 1,1-diphenylacetone (2.10
g, 10 mmol) with 2-phenylpropionaldehyde (1.34 mL, 10 mmol) to
give white powder (2.8 g, 86%). RF 0.72 (EtOAc-PE 2:3 v/v); mp:
156–157 °C; 1H NMR (500 MHz, CDCl3): d = 7.66 (d, J = 8.0 Hz, 1H),
7.30 (m, 10H), 7.11 (m, 5H), 6.88 (d, J = 8.0 Hz, 1H), 4.95 (s, 1H),
3.65 (m, 1H), 1.61 (d, J = 6.0 Hz, 3H); 13C NMR (500 MHz, CDCl3):
d=207.71, 153.41, 143.06, 140.14, 128.65, 128.60, 127.26, 126.94,
126.21, 125.92, 122.98, 63.00, 41.82, 16.97; Anal. calcd for C24H22O:
C 88.31, H 6.79, found C 88.15, H 6.97.
Materials and methods
3-(5-methyl-furan-2-yl)-1-naphthalen-1-yl-propenone (1.5) 28
Chemicals and reagents
This compound was obtained by reacting 1-acetonaphthone (1.52
mL, 10 mmol) with 5-methyl furfural (1 mL, 10 mmol) to give brown
crystals (2.2 g, 84%). RF 0.38 (EtOAc-PE 1:3 v/v); mp: 138–139 °C;
1H NMR (500 MHz, CDCl3): d = 8.39 (d, J = 7.5 Hz, 1H), 8.00 (d, J = 7.5
Hz, 1H), 7.91 (d, J = 17.5 Hz, 1H), 7.77 (d, J = 18.5 Hz, 1H), 7.55 (m,
3H), 7.31(d, J = 7.5 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H), 6.65 (s, 1H), 6.14
(s, 1H), 2.34 (s, 3H); 13C NMR (500 MHz, CDCl3): d = 195.20, 156.29,
All cell culture reagents were purchased from Invitrogen
(Burlington, ON, Canada). 1-palmitoyl-2-arachidonoyl-sn-glycerol-3-
phosphocoline (PAPC) was purchased from Avanti Polar Lipids
(Alabaster, AL, USA) and ELISA kits were purchased from
eBioscience (San Diego, CA, USA). Other chemicals were purchased
from Sigma (St. Louis, MO, USA) unless otherwise stated.
2 | J. Name., 2012, 00, 1-3
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149.93, 137.45, 133.85, 131.85, 131.36, 130.53, 128.40, 127.32,
126.89, 126.39, 125.81, 124.58, 122.73, 118.40, 109.47, 14.01;
HRMS (ESI) m/z: 285.06 [M + Na]+; Anal. calcd for C18H14O2: C 82.42,
H 5.38, found C 82.76, H 5.45.
laboratory showed that significant level of IL-8 and MCP-1 were
started to be observed from the respective time points forward
(supplementary fig. I & II). A negative control group received
OxPAPC (30 µg/mL) whilst a positive control group received
simvastatin and vehicle control group received 0.1% DMSO.
Supernatants were collected and enzyme-linked immunosorbent
assay (ELISA) was carried out according to the manufacturer’s
instructions. The percentage of inhibition (%) at highest treatment
concentrations (50 µM) and IC50 values for respective compounds
were determined. Percentages of inhibition (%) were calculated
using formula as follows:
Autoxidation of PAPC
Autoxidation of PAPC was carried out using the method previously
described 13. PAPC (1 mg) was dissolved in high performance liquid
chromatography (HPLC)-grade methanol and evaporated under a
nitrogen stream. The dried PAPC was left to autoxidise at room
temperature for 7 days. Both the purity of PAPC and the
autoxidation status of OxPAPC were analysed and monitored using
the positive ion-electrospray ionization-mass spectrometry (ESI-
MS).
% inhibition = ꢀꢁꢂꢀꢃ x 100%
ꢀꢁ
Where Cn is the concentration of chemokines (pg/mL) at 0 µM
treatment (negative control), while Cx is the concentration of
chemokines (pg/mL) at corresponding concentrations (µM).
Cell culture and cell differentiation
Western immunoblot
U-937 (ATCC® CRL-1593.2 TM) cell line was purchased from American
Type Culture Collection (ATCC) (Manassas, VA, United States). U-
937 mononuclear cell line was grown in the Rosewell Park
Memorial Institute-1640 (RPMI-1640) culture media, enriched with
10% (v/v) foetal bovine serum (FBS) and 1% (v/v) penicillin
G/streptomycin in a 75 cm2 cell culture flask. The cultured cells
were maintained at 37oC humidified atmosphere with 5% CO2. Prior
to treatment with the tested compounds, U-937 cells (5 x 105
cells/mL) were incubated with phorbol myristate acetate (PMA) at
concentration 200 nM/mL for 24 hours to allow differentiation from
monocytes to macrophage-like phenotypes. Subsequently, cells
were incubated with serum free media overnight for recovery
XBP1 protein expressions was determined using western
immunoblot. Cells were pre-treated with test compounds with
concentrations ranging from 6.25 to 50 µM, followed by stimulation
of OxPAPC for 2 hours. Cells treated with complete media and
OxPAPC only were used as negative controls while cells treated with
4-phenylnutyric acid (4-PBA) 32 were used as positive controls. Total
protein lysates of 1 mg/mL were loaded and separated by sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
and transfer onto polyvinylidene difluoride (PVDF) membranes. The
western immunoblot techniques used were as previously described
33
.
Rabbit polyclonal anti-XBP1 antibody (1: 5000, abcam,
phase 29
.
Cambridge, MA) was used to detect XBP1 protein expressions (42
kDa) while mouse monoclonal anti-beta actin antibody (1: 10,000,
abcam, Cambridge, MA) was used as loading control. The
Measurement of cell viability
established
bands
were
visualised
using
enhanced
The cytotoxic effect of the synthetic chalcone derivatives was
chemiluminescence substrate (ECL) (Perkin Elmer, USA) and the
relative intensity of each band was quantified using Fusion-Capt
Advance Software (Vilber Loumat, Germany).
determined
using
the
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay 30. Differentiated U-937
macrophages were treated with different concentrations of
synthetic chalcone derivatives and simvastatin (6.25, 12.5, 25, 50
and 100 µM) in the presence and absence of OxPAPC for 24 hours.
Subsequently, MTT salt reagent was added and cells were further
incubated for 4 hours. The formazan crystals were dissolved in
DMSO and analysed at 565 nm using Tecan’s Infinite® 200PRO
NanoQuant microplate reader (Tecan Trading AG, Mannedorf,
Switzerland). The concentrations of test compounds at viability
more than 90% 31 were chosen and used for subsequent assays.
Statistical analysis
Data were obtained from at least three independent experiments
and analysed using the GraphPad Prism5 software (GraphPad
Software Inc., San Diego, CA, USA). Values were presented as mean
± standard error of mean (SEM). Statistical test analysis of variance
(ANOVA) and Bonferroni post hoc test were used for multiple
comparisons and data analysis with p-values < 0.05 was regarded as
statistically significant.
Determination of chemokines release
The chalcone derivatives were dissolved in dimethylsulphoxide
(DMSO) and further diluted in serum-free RPMI media prior to use
at the desirable concentrations. The highest concentration of DMSO
used in cell culture was at 0.1%. Differentiated U-937 macrophages
were pre-treated with synthetic chalcone derivatives at
concentration 6.25, 12.5, 25, and 50 µM (2 hours), followed by
stimulation with 30 µg/mL of OxPAPC for 6 hours and 9 hours for IL-
8 and MCP-1 quantifications, respectively. Preliminary work in our
Results
Cytotoxic effect of synthetic chalcone derivatives on PMA-
differentiated U-937 cells
All the 6 synthetic chalcone derivatives were tested towards
differentiated U-937 macrophages at concentrations ranging from 0
to 100 µM. Compounds 1.2, 1.4 and 1.5 showed 90 % or higher
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percentage of cell viability and were used in the subsequent assays concentration 30 µg/ml of OxPAPC, which is within the
(Fig. 1).
pathophysiological concentration, was able to induce significant
increase in IL-8 and MCP-1 production. This concentration was
Inhibitory effect of synthetic chalcone derivatives on OxPAPC- selected to investigate the effect of synthetic chalcones on oxidised
induced IL-8 secretion
phospholipids-induced chemokine production. In the present study,
compounds 1.2, 1.4 and 1.5 were shown to inhibit the production
All tested compounds inhibited IL-8 release induced by OxPAPC in a of IL-8 and MCP-1 as well as the expression of XBP1 induced by
concentration-dependent manner. Compound 1.5 at the maximum OxPAPC.
concentration of 50 µM showed the strongest inhibition (61.39 ±
4.23%) of OxPAPC-induced IL-8 release and achieved the lowest IC50 Chalcone derivatives have been shown to inhibit proinflammatory
value of 18.33 ± 1.59 µM. Compound 1.2 and 1.4 similarly inhibited chemokines production induced by LPS and TNF-α. Naringenin, a
OxPAPC-induced IL-8 with percentage of inhibition of 51.66 ± 0.40% chalcone derivative, was shown to suppress the LPS-induced MCP-1
and 45.46 ± 1.78%, respectively. Moreover, the percentage of in RAW 264 macrophages 35. Moreover, 4’-hydroxychalcone was
inhibition for compound 1.5 at 50 µM showed comparable effects to reported to inhibit IL-8 production induced by TNF-α in K562 cell
the inhibition of simvastatin at concentration 25 µM
percentage of inhibition and IC50 values for each tested compound synthetic chalcone derivatives used in this study were shown for
.
The line 36. To the best of the authors’ knowledge, the three tested
are presented in Table 2.
the first time to inhibit the release of IL-8 and MCP-1 induced by
pathophysiological concentration of OxPAPC. It was observed that
Inhibitory effect of synthetic chalcone derivatives on OxPAPC- compound 1.5 with naphthalene functional group has the highest
induced MCP-1 secretion
inhibitory activity in OxPAPC-induced IL-8 and MCP-1 as compared
to compound 1.2 and compound 1.4 with diphenyl functional
All the tested compounds inhibited MCP-1 release induced by groups. It is possible that naphthalene functional group plays a
OxPAPC in a concentration-dependent manner. Among the three more important role in the inhibitory activity of synthetic chalcone
synthetic chalcone derivatives tested, compound 1.5 showed the derivatives on proinflammatory chemokines production. Moreover,
highest percentage of inhibition (63.79 ± 2.16%) and also achieved in our previous studies28 it was observed that methyl-furan group is
the lowest IC50 value, recorded at 13.05 ± 1.37 µM. On the other very important and all the compounds bearing methyl-furan group
hand, compound 1.2 and 1.4 demonstrated relatively moderate were found strong inhibitor of phagocytic activity of
inhibition at 48.23 ± 4.16% and 54.79 ± 1.81%, respectively.The polymorphonuclear neutrophils (PMNs) so strong activity of
percentage of inhibition and IC50 values for each tested compound compound 1.5 in present study can also be result of the presence of
are presented in Table 3.
methyl-furan group. On other hand, comparison of two compounds
(1.2 and 1.4) bearing same diphenyl moiety shows that substitution
pattern on aldehyde ring is also very important and effective.
Suppression of XBP1 protein expression
Differentiated U-937 macrophages incubated with media only Simvastatin was shown to be able to reduce chemokines level and
without stimulation of OxPAPC showed no induction of XBP1 therefore were used as a reference drug for inhibition of IL-8 and
37
protein expression, while 4-PBA (0.4 µM) used as positive controls MCP-1. Based on a research carried out by Rezaie-Majd et al.
,
showed significant reduction in XBP1 protein expression. Data treatment with simvastatin was able to reduce the serum levels of
obtained from western immunoblot analysis were as shown in fig. IL-8 and MCP-1 tested on patients with hypercholesterolemia.
2. The intensity of each bands were normalised with respect to beta Moreover, simvastatin was demonstrated to inhibit the production
actin protein expressions while statistical analysis was carried out of MCP-1 induced by LPS on human peripheral blood mononuclear
with respect to negative control. From the results obtained, both cells and human endothelial cells in a dose-dependent manner 38. It
compounds 1.4 and 1.5 showed significant reduction of XBP1 also effectively inhibits IL-1-induced IL-8 release on epithelial cells
protein expressions in a concentration dependent manner. Cells 39. A preliminary work carried out in our laboratory showed that
treated with these 2 compounds showed significant reduction of simvastatin was able to inhibit chemokines production induced by
XBP1 protein expressions at concentration 25 and 50 µM
.
OxPAPC (supplementary fig. III), which led to its use as a positive
Interestingly, compound 1.4 at 50 µM while compound 1.5 at control in the present study. Simvastatin at concentration of 25 µM
concentrations 25 and 50 µM showed no significant difference in was used as it is the highest non-toxic concentration towards the
XBP1 protein expression compared with positive control, suggesting treated differentiated U-937 macrophagess based on MTT assay.
a comparable effect of these compounds with 4-PBA in the
suppression of XBP1 protein expressions.
Activations of IRE1 signalling cause activation of XBP1, forming
isoform of spliced XBP1, the functionally active transcription factor
in the UPR pathway. XBP1 signalling was chosen in the present
study due to the fact that the expression of XBP1 is ubiquitous,
Discussion
14
40
Previous findings reported that concentrations of individual specifically within endothelial cells
and macrophages
. The
component of OxPAPC ranging from 1 to 50 µM are available in activated XBP1 plays dual roles, either promoting or inhibiting
human and rabbit atherosclerotic plaques 34. A preliminary study inflammation, depending on the cell type and context. Previous
carried out in our lab (data not shown) showed that OxPAPC at research has shown that expression of XBP1 is correlated with
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development of vascular inflammations. Based on a study carried
out by Zeng et al. 41, it was reported that the enhanced expression
of activated XBP1 leads to increased of inflammatory
atherosclerotic lesions. To the best of the authors’ knowledge, the
synthetic chalcones derivatives tested have shown for the first time
to cause inhibition on XBP1 protein expression tested on
differentiated U-937 macrophages.
9
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The present study demonstrated that the synthetic chalcone
derivatives possessed potential anti-inflammatory effects by
inhibiting OxPAPC-induced proinflammatory chemokines release
through inhibition of XBP-1 pathway.
13 A. D. Watson, N. Leitinger, M. Navab, K. F. Faull, S.
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Acknowledgements
This study was supported by Universiti Kebangsaan Malaysia (UKM)
through Geran Galakan Penyelidik Muda (Grant no: GGPM-2013-
059) and Ministry of Higher Education (MOHE) through
14 P. S. Gargalovic, N. M. Gharavi, M. J. Clark, J. Pagnon, W.
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Fundamental
Research
Grant
Scheme
(Grant
No:
FRGS/2/2013/SKK01/UKM/03/7). The authors would like to extend
appreciation to the Molecular Structure Characterisation
Laboratory, Centre for Research and Instrumentation Management
(CRIM), UKM for providing the ESI-MS services.
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Fig. 1
Effects of synthetic chalcone derivatives on viability of PMA-differentiated U-
937 macrophages. Data represent mean ± SEM of at least 3 independent
experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus negative control

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Compound 1.2 (A), compound 1.4 (B) and compound 1.5 (C) inhibit OxPAPC-induced expression of XBP-1 in
PMA-differentiated U937 macrophages. Data represent mean ± SEM of at least 3 independent experiments.
Data *p < 0.05, ***p < 0.001 versus negative control, while NS represent no significant different versus 4-
PBA (positive control).
93x227mm (300 x 300 DPI)

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Compound
1.1
Molecular structures
Molecular
weight
318.37
3ꢀ(2,3ꢀDimethoxyꢀphenyl)ꢀ1ꢀnaphthalenꢀ1ꢀylꢀpropenone
1.2
369.21
4ꢀ(4ꢀDiethylaminoꢀphenyl)ꢀ1,1ꢀdiphenylꢀbutꢀ3ꢀenꢀ2ꢀone
1.3
358.16
4ꢀ(2,3ꢀDimethoxyꢀphenyl)ꢀ1,1ꢀdiphenylꢀbutꢀ3ꢀenꢀ2ꢀone
1.4
1.5
326.17
1,1,5ꢀTriphenylꢀhexꢀ3ꢀenꢀ2ꢀone
262.30
3ꢀ(5ꢀmethylꢀfuranꢀ2ꢀyl)ꢀ1ꢀnaphthalenꢀ1ꢀylꢀpropenone
314.42
1.6
3ꢀ(4ꢀtertꢀbutylꢀphenyl)ꢀ1ꢀ(naphthalenꢀ1ꢀyl)ꢀ2ꢀpropenꢀ1ꢀone
Table 1: Structures of synthetic chalcone derivatives

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Table 2: Percentage of inhibition (%) and IC50 (µM) values of test compounds on
ILꢀ8 secretion.
Compound
Percentage of inhibition (%)
IC50 (µM)
51.66 ± 0.40
53.19 ± 4.28
61.39 ± 4.23 NS
77.64 ± 1.51
0.00
45.46 ± 1.78
27.32 ± 4.84
18.33 ± 1.59
5.97 ± 0.93
ꢀ
1.2
1.4
1.5
Simvastatin (25 µM
)
Negative control
(OxPAPC 30µg/ml)
Values are expressed in mean ± SEM; n=3. Percentage inhibition>10% was significant at p<0.05
NS
compared with negative control.
simvastatin.
p>0.05 was considered not significant compared with
Table 3: Percentage of inhibition (%) and IC50 (µM) values of test compounds on
MCPꢀ1 secretion.
Compound
Percentage of inhibition (%)
IC50 (µM)
48.23 ± 4.16
54.79 ± 1.81
63.79 ± 2.16 NS
75.46 ± 1.75
0.00
ND
1.2
1.4
23.45 ± 0.47
13.05 ± 1.37
8.42 ± 1.05
ꢀ
1.5
Simvastatin (25 µM)
Negative control
Values are expressed in mean ± SEM; n=3. Percentage inhibition>10% was significant at p<0.05
NS
compared with negative control.
p>0.05 was considered not significant compared with
simvastatin. ND represents IC50 value not determined.