Effects of diarylpentanoid analogues of curcumin on chemiluminescence and chemotactic activities of phagocytes

Article abstract of DOI:10.1111/j.2042-7158.2011.01423.x

Objectives A series of 43 curcumin diarylpentanoid analogues were synthesized and evaluated for their inhibitory effects on the chemiluminescence and chemotactic activity of phagocytes in vitro. Methods The effects of the compounds on the respiratory burst of human whole blood and isolated human polymorphonuclear leukocytes (PMNs) were evaluated using a luminol-based chemiluminescence assay and their effect on chemotactic migration of PMNs was investigated using the Boyden chamber technique. Key findings Compounds 6, 17, 25 and 30 exhibited significant inhibitory activity on the oxidative burst of PMNs. The presence of methoxy groups at positions 2 and 5, and methoxylation and fluorination at positions 4 and 2 of both phenyl rings, respectively, may contribute significantly to their reactive oxygen species inhibition activity. Compounds 7, 17, 18, 24 and 32 showed strong inhibition of the chemotaxis migration of PMNs. Chlorination at various positions of both phenyl rings of cyclohexanone diarylpentanoid resulted in compounds with potent inhibitory effects on PMN migration. Conclusions The results suggest that some of these diarylpentanoid analogues are able to modulate the innate immune response of phagocytes at different steps, emphasizing their potential as a source of new immunomodulatory agents.

Full text of DOI:10.1111/j.2042-7158.2011.01423.x

Research Paper
Journal of Pharmacy
And Pharmacology
Effects of diarylpentanoid analogues of curcumin on
chemiluminescence and chemotactic activities of phagocytes
Ibrahim Jantana, Syed Nasir Abbas Bukharia, Nordin Haji Lajisb, Faridah Abasb, Lam Kok Waia and
Malina Jasamaia
aDrug and Herbal Research Center, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur and bInstitute of Bioscience, Universiti Putra
Malaysia, Selangor, Malaysia
Keywords
chemotactic activity; chemiluminescence;
Abstract
Objectives A series of 43 curcumin diarylpentanoid analogues were synthesized
and evaluated for their inhibitory effects on the chemiluminescence and chemo-
tactic activity of phagocytes in vitro.
curcumin diarylpentanoid analogues;
immunomodulatory; polymorphonuclear
leukocytes
Methods The effects of the compounds on the respiratory burst of human whole
blood and isolated human polymorphonuclear leukocytes (PMNs) were evaluated
using a luminol-based chemiluminescence assay and their effect on chemotactic
migration of PMNs was investigated using the Boyden chamber technique.
Key findings Compounds 6, 17, 25 and 30 exhibited significant inhibitory activity
on the oxidative burst of PMNs. The presence of methoxy groups at positions 2 and
5, and methoxylation and fluorination at positions 4 and 2 of both phenyl rings,
respectively, may contribute significantly to their reactive oxygen species inhibition
activity. Compounds 7, 17, 18, 24 and 32 showed strong inhibition of the chemotaxis
migration of PMNs. Chlorination at various positions of both phenyl rings of cyclo-
hexanone diarylpentanoid resulted in compounds with potent inhibitory effects
on PMN migration.
Correspondence
Ibrahim Jantan, Drug and Herbal Research
Center, Faculty of Pharmacy, Universiti
Kebangsaan Malaysia, Jalan Raja Muda Abdul
Aziz, 50300 Kuala Lumpur, Malaysia.
E-mail: ibj@pharmacy.ukm.my
Received September 8, 2011
Accepted November 9, 2011
doi: 10.1111/j.2042-7158.2011.01423.x
Conclusions The results suggest that some of these diarylpentanoid analogues
are able to modulate the innate immune response of phagocytes at different steps,
emphasizing their potential as a source of new immunomodulatory agents.
Introduction
Professional phagocytes such as polymorphonuclear
neutrophils (PMNs), peripheral blood mononuclear and
macrophage cells play important roles in our innate immune
defence against infectious microbes. Several steps are
involved in phagocytosis: migration of phagocyte cells to the
site of infection and adherence towards vascular endothelial
cells, followed by degradation of the pathogen.[1] Chemot-
axis is the movement of phagocytes to the site of infection
and is the first step of the immune response. The ability
of these cells to be chemotactically attracted to the site of
the initial microbial invasion or to an inflammatory focus is
fundamental for the full activation of the immune response
that follows.[2] Accumulation of phagocytes at the infection
site can be induced by endogenous chemoattractants such
as interleukin 8, leukotriene B4, platelet activating factor,
or exogenous chemoattractants such as formyl methionyl-
leucyl-phenylalanine (fMLP) derived from bacterial cell
products.[3]
The PMNs adhere stably to the endothelium cells because
these express all three CD11/CD18 leucocyte integrins on
their cell surfaces.[4] Binding of a receptor-specific ligand
may lead to, besides signal-transduction events, internali-
zation of the receptor–ligand complex, which leads to a sub-
sequent down-regulation of surface receptor expression.
Phagocytosis will occur at the site of infection by means
of phagocyte–ligand interaction, leading to a sequence of
events known as oxidative burst. The oxidative burst involves
increased oxygen consumption and generation of highly
-
reactive superoxide anion radicals (O2 ) by the NADPH–
oxidase complex. O2- is further dismutated to hydrogen per-
oxide (H2O2), either spontaneously or enzymatically by
superoxide dismutase. Highly damaging hydroxyl radicals
(OH-) are formed from O2- and H2O2 via a Fenton reaction.
In the phagosome, H2O2 and myeloperoxidase enzyme
(MPO) activate a halogenating system, giving rise to
hypochlorous acid (HClO), which is a potent bactericidal
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
404

Ibrahim Jantan et al.
Immunomodulatory effects of diarylpentanoids
agent (myeloperoxidase–H2O2–Cl- system). MPO is also
involved in the production of highly toxic nitric oxides
(NO).[5] Besides the defensive roles during the infections,
when excessively or inappropriately deployed the phagocyte–
microbe interactions can damage host tissues and contribute
to the pathogeny of various immune and non-immune
chronic inflammatory diseases, including some rheumatoid
disorders. Therefore, inhibitors of phagocyte reactive oxygen
species (ROS) production can be used in the treatment of a
variety of disorders, including inflammation.[6]
A series of 43 diarylpentanoids has been synthesized based
on the chemical structure of curcumin by eliminating the
b-diketone and modifying it into conjugated double bonds,
i.e. two identical aromatic ring regions separated by five
carbon linkers. To the best of our knowledge, curcumin-
related diarylpentanoid analogues have not been investigated
for their immunomodulatory effects. We therefore evaluated
these compounds for their effects on the ROS production of
human whole blood and isolated PMNs, and chemotaxis of
PMNs induced by the bacterial peptide N-formyl-methionyl-
leucyl-phenylalanine (fMLP).
Curcumin
(1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,
6-heptadiene-3,5-dione) is the most studied curcuminoid
of the species Curcuma. Curcumin is well known for its
anti-inflammatory effects and over the past few decades
its numerous biological and pharmacological activities
have been reported.[7,8] Curcumin has been shown to
inhibit the metabolism of arachidonic acid and the activities
of cyclooxygenase-2 (COX-2), lipoxygenase, proinflamma-
tory cytokines,inducible nitric oxide (iNOS),protein kinases,
transcription factors such as nuclear factor-kB and release
of steroids.[9–11] Other activities of curcumin include inhi-
bition of low-density lipoprotein oxidation, reduction of
blood cholesterol level, inhibition of platelet aggregation,
suppression of thrombosis and myocardial infarction, treat-
ment of rheumatoid arthritis, inhibition of HIV replication,
protection from liver injury, as well as anticancer and immu-
nomodulatory activities.[12–14]
Curcumin is known to have a poor bioavailability, as
orally administered curcumin undergoes hepatic conjuga-
tion, leading to the formation of glucoronides and sulphates,
and systematic administration causes it to undergo reduc-
tion.[15] Many studies have been carried out to improve the
bioavailability of curcumin by modifying its molecular struc-
ture, i.e. eliminating the unstable b-diketone moiety and
modifying the heptadiene linker while retaining the phenolic
OH groups.[16,17] The presence of the b-diketone moiety will
result in rapid metabolism by aldo-keto reductase in the liver,
thus limiting the beneficial effects of curcumin on many
types of disease. Various curcumin analogues have been syn-
thesized and evaluated for activity against known biological
targets in order to investigate their structure–activity rela-
tionships (SAR). Recent SAR studies on curcumin analogues
have demonstrated anti-inflammatory, anticancer, antioxi-
dant, anti-tyrosinase and antiosteoporosis properties.[17–20]
The presence of phenolic OH on both phenyl ring structures
has been shown to be important for antioxidant activity.[21]
Our previous studies on synthesized diarylpentanoids and
pyrazoline derivatives have revealed that these compounds
inhibit NO production in IFN-gLPS-activated RAW 264.7
and U937 cells. More importantly, one of the compounds
suppressed both the iNOS gene and enzyme expression while
displaying a strong inhibitory effect on MCP-1 and IL-10
secretion and gene expression.[22,23]
Materials and Methods
Chemicals and equipment
Serum opsonized zymosan A (Sacromyces cerevisae sus-
pensions and serum), luminol (3-aminophthalhydrazide),
phosphate buffer saline tablet (PBS), Hanks balance salt
solutions (HBSS++), fetal calf serum (PAA Laboratories,
USA), Ficoll, Hanks balance salt solution (HBSS–), N-formyl-
methionylleucyl-phenylalanine (fMLP), acetyl salicylic acid
(purity > 99%), ibuprofen (purity > 99%), dimethylsulfox-
ide (DMSO), methanol and ammonium chloride of analyti-
cal grades were purchased from Sigma (St Louis, MO, USA).
Chemiluminescense measurements were carried out on a
Luminoskan Ascent luminometer (Thermo Scientific, UK).
fMLP was stored as a stock solution of 108 m in DMSO at
-80°C and diluted in Hanks solution prior to assay. Haema-
toxylin and xylene for staining were obtained from BDH
(UK). A Boyden 48-well chamber with a 2 mm polycarbonate
membrane filter separating the upper and lower compart-
ments was purchased from Neuro Probe (Cabin John, MD,
USA). A CO2 incubator (Shell Lab, USA) and light micro-
scope (Leitz Watzler, Germany) were used in this assay.
Synthesis of diarylpentanoid analogues
The diarylpentanoid analogues of curcumin were synthe-
sized by direct coupling of the appropriate aromatic aldehyde
with the three ketones, acetone (1–17), cyclohexanone
(18–33) or cyclopentanone (34–43) at a molar ratio of 1 : 2,
using the base-catalysed Claisen–Schmidt condensation
reaction. Figure 1 illustrates the general synthesis of diaryl-
pentanoids. Briefly, the appropriate aromatic aldehyde
(20 mmol, 2 equiv.) and the appropriate ketone (10 mmol, 1
equiv.) were mixed and dissolved in 15 ml of ethanol and
stirred at 5°C for a few minutes. Four drops of 40% NaOH
solution in ethanol was added dropwise over several minutes.
The mixture was stirred at room temperature for 10 h.
The reaction was neutralized with dilute HCl to form a pre-
cipitate, which was subjected to further purification by
recrystallization from appropriate solvents or column chro-
matography. The structures of the synthesized compounds
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
405

Immunomodulatory effects of diarylpentanoids
Ibrahim Jantan et al.
O
O
1,5-bis(2,5-dimethoxyphenyl)-1,4-
O
R
pentadiene-3-one (compound 6)
R
R
EtOH
H
+
1H NMR (500 MHz, acetone-d6) d in ppm: 7.98 (d, 2H,
J = 15.5 Hz), 6.88 (d, 2H, J = 15.5 Hz), 6.84 (d, 4H,
J = 8.0 Hz), 6.82 (s, 2H), 3.96 (s, 6H, OCH3), 3.85 (s, 6H,
OCH3); mp 105–106°C; MS (EI): [M]+ at m/z 354. UV
(MeOH): lmax 396 (e 20 000), 316 (21 000), 207 (38 300) nm;
IR (KBr, cm-1): 1493, 1578, 1611.
NaOH
1
2. R = 3-OMe, 4-OH
3. R = 2-OH
4. R = 2-OMe, 3-OMe
5. R = 2-OMe, 4-OMe
6. R = 2-OMe, 5-OMe
7. R = 2-OMe, 6-OMe
10. R = 4-OH
11. R = 3-OH
12. R = 4-Cl
13. R = 3-Cl
14. R = 2-Cl
15. R = 4-F
1,5-bis(2,6-dimethoxyphenyl)-1,4-
pentadiene-3-one (compound 7)
8. R = 2-OMe, 4-OMe, 6-OMe 16. R = 3-F
9. R = 3-OMe, 4-OMe, 5-OMe 17. R = 2-F, 4-OMe
1H NMR (500 MHz, acetone-d6) d in ppm: 8.17 (d,
2H, J = 16.3 Hz), 7.59 (d, 2H, J = 16.3 Hz), 7.36 (t, 2H,
J = 8.5 Hz), 6.75 (d, 4H, J = 8.5 Hz), 3.90 (s, 12H); mp 153–
155°C; MS (EI): [M]+ at m/z 354. UV (MeOH): lmax 366 (e
28 100), 213 (42 900) nm; IR (KBr, cm-1): 1568, 1604, 1637.
O
O
O
R
R
R
EtOH
H
+
NaOH
27
1,5-bis(2-fluoro, 4-methoxyphenyl)-1,4-
pentadiene-3-one (compound 17)
26. R = 2-F
18. R = 2-Cl
19. R = 3-F
20. R = 2-OH
21. R = 3-OMe, 4-OH
22. R = 4-OH
23. R = 4-Cl
28. R = 2-OMe, 3-OMe
29. R = 2-OMe, 4-OMe
30. R = 2-OMe, 5-OMe
1H NMR (500 MHz, acetone-d6) d in ppm: 7.84 (d, 2H,
J = 15.5 Hz), 7.81 (m, 2H), 7.24 (d, 2H, J = 15.5 Hz), 6.90 (m,
4H), 3.91 (s, 6H); mp 115–117°C; EIMS: [M]+ at m/z 330. UV
(MeOH): lmax 357 (e 33 900), 325 (17 600), 200 (37 800) nm;
IR (KBr, cm-1): 1105, 1508, 1612.
31. R = 2-OMe, 3-OMe, 4-OMe
32. R = 3-OMe, 4-OMe, 5-OMe
33. R = 2-F, 4-OMe
24. R = 3-Cl
25. R = 4-F
O
O
O
R
R
2,6-bis(2-chlorobenzylidene)-cyclohexanone
(compound 18)
R
EtOH
H
+
NaOH
1H NMR (500 MHz, acetone-d6) d in ppm: 7.85 (s, 2H), 7.56
(m, 4H), 7.45 (m, 4H), 2.87 (t, 4H, J = 6.5 Hz), 1.78 (q, 2H,
J = 6.5 Hz); mp 90–92°C; (EI): [M]+ at m/z 343. UV (MeOH):
lmax 313 (e 21 700), 203 (46 000) nm; IR (KBr, cm-1): 1051,
1576, 1603.
39. R = 4-F
40. R = 2-OMe, 3-OMe
41. R = 2-OMe, 5-OMe
34. R = 2-Cl
35. R = 3-Cl
36. R = 4-Cl
37. R = 2F
42. R = 2-OMe, 3-OMe, 4-OMe
43. R = 3-OMe, 4-OMe, 5-OMe
38. R = 3-F
1,5-bis(3-chlorobenzylidene)-cyclohexanone
(compound 24)
Figure 1 General synthesis of diarylpentanoid analogues.
1H NMR (500 MHz, acetone-d6) d in ppm: 7.65 (s, 2H), 7.56
(s, 2H), 7.51 (t, 4H, J = 5.5 Hz), 7.44 (m, 2H), 2.99 (m, 4H),
1.86 (quintet, 2H, J = 6.0 Hz); mp 136–138°C; EIMS: [M]+ at
m/z 343. UV (MeOH): lmax 323 (e 38 100), 234 (21 400), 207
(58 400) nm; IR (KBr, cm-1): 1092, 1577, 1606.
were elucidated by spectroscopic techniques and compari-
son of their spectroscopic data made with those of related
compounds. All the chemicals used for synthesis of 2,6-
bisbenzylidene derivatives were of research grade, purchased
from Sigma-Aldrich, Merck and Acros Organics. Analytical
TLC plates (silica gel F254 precoated, 0.2 mm thickness) were
obtained from Merck. Melting points were determined using
a hot-stage melting point apparatus equipped with a micro-
scope, XSP-12 model 500X, and were uncorrected. IR spectra
were recorded on a Perkin Elmer RX1 FT-IR spectrometer as
KBr disk or thin film. Electron impact mass spectra (EIMS)
were performed on a Thermo Finnigan POLARISQ spec-
trometer at 70 ev. The 1H and 13C spectra were carried out on
a Varian 500 MHz NMR spectrometer.
1,5-bis(4-fluorobenzylidene)-cyclohexanone
(compound 25)
1H NMR (500 MHz, acetone-d6) d in ppm: 7.68 (s, 2H), 7.56
(s, 2H), 7.49 (d, 4H, J = 8.3 Hz), 7.42 (d, 4H, J = 8.3 Hz), 2.99
(t, 2H, J = 5.5 Hz), 1.83 (quintet, 2H, J = 6.0 Hz); mp
86–88°C; EIMS: [M]+ at m/z 310. UV (MeOH): lmax 328
(e 35 400), 230 (21 000), 201 (36 600) nm; IR (KBr, cm-1):
1158, 1507, 1608.
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
406

Ibrahim Jantan et al.
Immunomodulatory effects of diarylpentanoids
to detect the level of ROS affected by the synthetic com-
pound. The molecular weight of luminol is relatively small
and it can enter the cells and then react with intracellular
ROS.[26] For the assay, 25 ml diluted whole blood (1 : 50 dilu-
tion in sterile PBS, pH 7.4) or 25 ml PMN (1 ¥ 106/ml) sus-
pended in HBSS++ were incubated with 25-ml serial dilutions
of synthetic compounds in DMSO and H2O (5 : 95 ratio)
(6.25–100.00 mg/ml). The cells were stimulated with 25 ml of
opsonized zymosan followed by 25 ml of luminol (7 ¥ 105 m)
and then HBSS++ was added to adjust the final volume to
200 ml. The final concentrations of the samples in the mixture
were 12.5, 6.25, 3.13, 1.56 and 0.78 mg/ml. Tests were per-
formed in white 96-well microplates, which were incubated
at 37°C for 50 min in the thermostatically controlled
chamber of the luminometer. The control wells contained
0.6% DMSO, HBSS++, luminol and cells but no compound.
Curcumin and acetylsalicylic acid, a non-steroidal anti-
inflammatory drug (NSAID), were used as positive controls.
The final concentration of DMSO in the mixture was 0.6%, to
eliminate the effect of the solvent on the chemiluminescence.
The luminometer results were monitored as the chemilumi-
nescence 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:
1,5-bis(2,5-dimethoxybenzylidene)-
cyclohexanone (compound 30)
1H NMR (500 MHz, acetone-d6) d in ppm: 7.89 (s, 2H), 7.02
(d, 2H, J = 8.5 Hz), 6.97 (d, 4H, J = 8.5 Hz), 3.84 (s, 6H), 3.80
(s, 6H), 2.92 (t, 4H, J = 6.0 Hz), 1.78 (quintet, 2H, J = 6.0 Hz);
mp 161–163°C; EIMS: [M]+ at m/z 394.UV (MeOH): lmax 376
(e 8700), 313 (11 000), 279 (9400), 202 (30 100) nm; IR (KBr,
cm-1): 1494, 1582, 1603.
1,5-bis(3,4,5-trimethoxybenzylidene)-
cyclohexanone (compound 32)
1H NMR (500 MHz, acetone-d6) d in ppm: 7.64 (s, 2H), 6.86
(s, 4H), 3.90 (s, 12H), 3.79 (s, 6H), 2.82 (t, 4H, J = 6.5 Hz),
1.77 (quintet, 2H, J = 6.5 Hz); mp 147–149°C; EIMS: [M]+ at
m/z 454. UV (MeOH): lmax 357 (e 18 300), 547 (10 400), 200
(42 900) nm; IR (KBr, cm-1): 1505, 1604, 1579.
Isolation of human polymorphonuclear
leukocytes
For isolation of PMNs, venous blood was obtained in
heparin-containing tubes by aseptic vein puncture from
healthy human volunteers aged Ն18 years old who fulfilled
the following inclusion criteria: non-smoker,fasted overnight
and did not take any medicine or supplements.The blood was
centrifuged initially at 170g to remove the platelet-rich
plasma and then at 1000g to eliminate platelet-poor plasma.
The buffy coat of white cells was diluted with PBS (pH 7.2).
Dextran was added and the mixture was left for 45 min at
room temperature (26°C) for sedimentation. The superna-
tant was centrifuged by Ficoll-gradient separation and then
washed twice with distilled water to remove red blood cells.
A pellet of PMNs was collected from the tube base. The
cells were suspended in HBSS (pH 7.4) with Ca2+ and Mg2+
(HBSS++). Cell suspensions were counted using a haemocy-
tometer and light microscope and diluted with HBSS to
obtain a final cell suspension of 1 ¥ 106/ml.[24] The use of
human blood was approved by the Human Ethical Commit-
tee of UKM (approval no. FF-220–2008).
RLUcontrol − RLUsample ×100%
(
( )
% =
)
inhibition
RLUcontrol
Chemotaxis assay
The assay was performed using a modified 48-well Boyden
chamber with formyl-methionyl-leucyl-phenylalanine
(fMLP) as a chemotaxin,as previously described by Sacerdote
et al.[27] Briefly, aliquots of 25 ml of fMLP (10-8 m) were added
to the lower chamber. Serial dilutions (5 ml) of each com-
pound in DMSO and H2O (5 : 95 ratio) (6.25–100 mg/ml)
were added six times each (n = 6) to the upper chamber con-
taining 45 ml PMNs (1 ¥ 106 cells per ml) suspended in HBSS-
and incubated for 1 h at 37°C in a CO2 incubator. The final
concentrations of the samples in the mixture were 10, 5, 2.5,
1.25 and 0.625 mg/ml. The final concentration of DMSO in
the reaction mixture was fixed at 0.5% to avoid interference
with the chemotactic study. Migrated cells that had adhered
to the distal part of the filters were fixed and stained by
haematoxylin and xylene. The cell migration distance was
measured by using a light microscope. The chemoattractant
buffer (DMSO and HBSS, 1 : 1 ratio) was added as a control
and curcumin and ibuprofen were used as positive controls.
The chemotactic effect of each compound was measured at
different concentrations. The percentage inhibition (%) was
calculated using the following formula:
Cell viability
Cell viability was determined by the standard trypan blue
exclusion method. The PMNs and macrophage cells (1 ¥ 106/
ml) were incubated with 6.25 or 100 mg/ml of synthetic com-
pounds, each in triplicate at room temperature for 2 h. The
blue dye uptake was an indication of cell death. The percent-
age viability was calculated from the total cell counts.[24]
Chemiluminescence assay
Luminol-based chemiluminescence assay was carried out as
described by Jantan et al.[25] The luminol was used as a probe
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
407

Immunomodulatory effects of diarylpentanoids
Ibrahim Jantan et al.
Table 1 Percentage inhibition at 12.5 mg/ml and IC50 values (mmol/l) of
ROS inhibitory activity of synthetic diarylpentanoid analogues on human
whole blood and PMNs assayed by luminol-amplified chemiluminescence
distance travelled by control
(
− distance travelled by sample ×100%
)
distance travelled by control
Mean Ϯ SEM
IC50 values
Compound Whole blood PMNs
Whole blood PMNs
Statistical analysis
1
2
36.1 Ϯ 1.4
42.9 Ϯ 0.5
71.6 Ϯ 0.8
56.8 Ϯ 5.0
14.3 Ϯ 1.4
76.5 Ϯ 1.0
53.0 Ϯ 0.5
42.7 Ϯ 2.0
42.2 Ϯ 1.7
57.6 Ϯ 1.2
54.0 Ϯ 1.6
37.8 Ϯ 1.3
37.7 Ϯ 1.4
34.7 Ϯ 5.3
61.5 Ϯ 1.4
58.4 Ϯ 2.8
88.8 Ϯ 0.3
41.7 Ϯ 1.1
84.2 Ϯ 0.9
50.8 Ϯ 3.0
58.9 Ϯ 0.5
54.3 Ϯ 0.1
35.2 Ϯ 5.6
31.7 Ϯ 3.8
87.4 Ϯ 2.8
52.7 Ϯ 0.9
46.2 Ϯ 1.7
73.3 Ϯ 0.5
57.2 Ϯ 0.4
76.8 Ϯ 2.1
50.7 Ϯ 2.0
54.3 Ϯ 1.8
59.2 Ϯ 1.1
38.2 Ϯ 2.5
40.4 Ϯ 1.1
38.3 Ϯ 0.6
50.6 Ϯ 2.7
52.7 Ϯ 1.3
45.6 Ϯ 1.8
51.5 Ϯ 3.0
50.0 Ϯ 1.4
45.7 Ϯ 1.6
42.1 Ϯ 2.8
80.5 Ϯ 2.4
71.9 Ϯ 0.4
46.6 Ϯ 0.4
54.3 Ϯ 0.1
89.6 Ϯ 0.3
–
–
–
All the data were analysed using Statistical Package for Social
Sciences (SPSS) version 15.0. Each sample was measured in
triplicate and the data presented as mean Ϯ standard error of
mean (SEM). The IC50 values were calculated using GraphPad
Prism 5 software. The values were obtained from at least three
determinations. Data were analysed using a one-way analysis
of variance (ANOVA) for multiple comparisons. P < 0.05 was
considered to be statistically significant.
30.8 Ϯ 0.5
3
7.5 Ϯ 0.6
12.4 Ϯ 0.2
4
56.5 Ϯ 1.4 19.0 Ϯ 6.4
16.5 Ϯ 0.1
31.2 Ϯ 0.7
5
–
–
6
91.0 Ϯ 0.3 10.5 Ϯ 0.5
62.9 Ϯ 0.1 34.3 Ϯ 0.3
5.2 Ϯ 0.7
7
24.5 Ϯ 0.3
8
54.2 Ϯ 0.6
54.0 Ϯ 0.5
–
–
27.2 Ϯ 0.3
9
27.2 Ϯ 0.4
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
42
43
Curcumin
Aspirin
67.1 Ϯ 0.4 43.2 Ϯ 2.4
60.3 Ϯ 0.5 43.9 Ϯ 3.1
32.2 Ϯ 0.4
41.9 Ϯ 0.7
Results and Discussion
49.9 Ϯ 0.4
49.9 Ϯ 0.4
49.5 Ϯ 1.6
–
–
–
–
–
Inhibition of reactive oxygen
species generation
–
43.2 Ϯ 0.3 26.2 Ϯ 1.0
50.7 Ϯ 0.8 27.8 Ϯ 3.6
–
37.4 Ϯ 1.1
The cell viability test carried out to evaluate the cytotoxicity
of the 43 curcumin-like diarylpentanoid analogues on PMNs
at 6.25 and 100 mg/ml indicated that the cells were viable
(>90%) after 2 h of incubation. Preliminary screening of
the compounds on the whole blood showed that compounds
3, 6, 17, 19, 25, 28 and 30 exhibited high inhibitory activity
for luminol-enhanced chemiluminescence with IC50 values
ranging from 5.7 to 10.5 mm (Table 1). Phagocyte cells, upon
activation by serum opsonized zymosan (SOZ), release ROS.
These were then quantified by the luminol-enhanced chemi-
luminescence assay.Fgc receptors on the surface of the phago-
cyte cells recognize SOZ and the interaction that occurs
initiates the process of respiratory burst. The luminol was
used as a probe to detect the specific ROS effected by the com-
pounds. The molecular weight of luminol is relatively small
and it can enter the cells and then react with intracellular and
extracellular ROS such as H2O2, OH- and HClO, which are
primarily produced at a later phase of oxidative burst by
phagocytic MPO.[5] The inhibitory activity of the compounds
may be due to their ability to block the complement receptors
and to inhibit NADPH oxidase.
The IC50 values of these compounds on the oxidative burst
of human whole blood were comparable to that of curcumin
(7.6 mm) and lower than that of acetylsalicylic acid (18.6 mm),
both used as positive controls (Table 1). Acetylsalicylic acid
was used as a positive control in a previous study by Parij
et al.,[28] who showed that acetylsalicylic acid inhibits
luminol-amplified chemiluminescence of human neutro-
phils. The compounds were further evaluated for their effects
on the oxidative burst of PMNs. Of the seven compounds,
compounds 6, 17, 25 and 30 were the most potent against
PMNs, with IC50 values ranging from 5.2 to 8.3 mm, respec-
tively. Their IC50 values were higher than that of curcumin
(3.8 mm) but lower than that of acetylsalicylic acid (9.5 mm)
82.3 Ϯ 0.1
32.3 Ϯ 0.3
80.9 Ϯ 0.3
5.7 Ϯ 0.4
8.3 Ϯ 0.2
–
–
9.0 Ϯ 1.0
12.4 Ϯ 0.3
34.3 Ϯ 0.9 36.7 Ϯ 3.9
47.3 Ϯ 0.1 23.6 Ϯ 1.1
43.8 Ϯ 0.1 38.2 Ϯ 0.8
–
–
53.5 Ϯ 1.0
28.3 Ϯ 1.6
28.1 Ϯ 1.5
84.7 Ϯ 0.8
–
–
51.0 Ϯ 6.7
–
6.3 Ϯ 0.3
5.7 Ϯ 0.1
43.6 Ϯ 0.3 35.1 Ϯ 1.2
41.4 Ϯ 0.5
44.7 Ϯ 0.2
–
–
71.2 Ϯ 0.2 10.4 Ϯ 0.3
45.2 Ϯ 0.1 23.5 Ϯ 0.5
10.5 Ϯ 0.1
–
86.8 Ϯ 0.6
8.9 Ϯ 0.7
6.7 Ϯ 0.4
43.3 Ϯ 0.6 24.7 Ϯ 1.3
44.4 Ϯ 0.5 23.1 Ϯ 1.2
45.8 Ϯ 0.3 25.8 Ϯ 0.5
–
–
–
39.7 Ϯ 0.7
40.3 Ϯ 0.3
39.7 Ϯ 0.2
–
–
–
–
–
–
57.8 Ϯ 0.8 38.9 Ϯ 3.1
43.9 Ϯ 0.4 36.0 Ϯ 1.3
29.9 Ϯ 0.3
–
44.7 Ϯ 0.5
–
–
43.6 Ϯ 0.9 29.3 Ϯ 1.7
43.1 Ϯ 0.4 30.0 Ϯ 1.2
–
–
47.5 Ϯ 0.5
46.5 Ϯ 0.8 33.0 Ϯ 2.8
93.5 Ϯ 0.3 7.8 Ϯ 0.3
92.5 Ϯ 0.3 18.6 Ϯ 0.2
–
–
–
3.8 Ϯ 0.2
9.5 Ϯ 0.3
(Table 1). The results showed that the compounds inhibit
ROS generation during the metabolic phase of phagocytosis
in a dose-dependent manner, i.e. as the concentration of the
samples increased the percentage inhibition increased.
Figure 2 shows the dose-dependent ROS-inhibitory effect of
compounds 6, 17, 25 and 30.
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
408

Ibrahim Jantan et al.
Immunomodulatory effects of diarylpentanoids
12.5 μg/ml
6.25 μg/ml
3.13 μg/ml
1.56 μg/ml
0.78 μg/ml
100
**
**
*
*
*
*
*
*
80
*
60
40
20
0
*
*
*
*
*
*
*
Various sample concentrations (μg/ml)
Figure 2 Effects of curcumin-related diarylpentanoid analogues on phagocytic ROS production by luminol-amplified chemiluminescence. Percentage
inhibition in oxidative burst in response to various concentrations of compounds, as compared to curcumin and aspirin with isolated PMNs. Significance
of differences with respect to control: *P < 0.05, **P < 0.01.
electron-withdrawing fluoride at position 4 (compound 25)
on both phenyl ring structures strongly increases the inhi-
bitory effect of the diarylpentanoid analogues with a linker
containing a six-member ring. The presence of a fluoride
group at position 3 (compound 19) also showed strong
inhibitory activity but to a lesser extent than compound 25.
The results suggest that cyclohexanone containing linker
may play an important role in the ROS inhibitory activity
of the analogues. However, cyclopentanone containing
analogues exhibited lower ROS inhibitory activity than the
cyclohexanone and the acetone analogues.
The present study shows that the bioactive compounds
were able to inhibit the luminol chemiluminescence pro-
duced by a myeloperoxidase–H2O2–Cl- system. Interestingly,
the diarylpentanoid analogues, which showed strong ROS-
inhibitory activity, have been shown in a recent study to be
able to strongly suppress NO production, i.e. compounds
3, 6, 17, 19, 25, 28 and 30 strongly inhibit NO production in
IFN-g/LPS-activated macrophage cells.[20] Other studies – by
Ko et al.[29] and Rojas et al.[30] – showed that chalcone deriva-
tives with methoxy groups at positions 2 and 5 of their phenyl
ring structures potently inhibit NO production in activated
RAW 264.7 cells. Liang et al.[17] showed that in the presence of
a 3-methoxy group, cyclohexanone-containing analogues
of diarylpentanoids exhibit strong anti-inflammatory pro-
perties through inhibiting the LPS-induced TNF-a and IL-6
release in macrophages. It was therefore suggested that the
substitution patterns of the phenyl rings of diarylpentanoid
analogues for increased inhibitory effects on ROS, NO,
TNF-a and IL-6 release in macrophages have high degree of
similarity.
Structure–activity analysis of
diarylpentanoid analogues as
inhibitors of ROS production
Structure–activity analysis of the acetone diarylpentanoid
analogues indicated that compounds bearing hydroxyl
groups at position 2 on both the phenyl ring structures (e.g.
compound 3) possess high inhibitory activity against ROS
production in human whole blood. However, those with
hydroxyl groups present at other positions of the ring do
not show such strong inhibition. The analogue with
methoxy groups at positions 2 and 5 of the phenyl ring
structures (compound 6) demonstrated stronger inhibitory
activity than the positive controls, in fact almost a twofold
improvement in ROS inhibition of PMNs relative to acetyl-
salicylic acid. Methoxylation at position 4 and fluorination
at position 2 of both phenyl rings (compound 17) produce
compounds with strong inhibition on the oxidative burst of
whole blood and PMNs. The introduction of the cyclopen-
tanone ring as a rigid linker chain in the structures reduces
the inhibitory effect of the compound with hydroxyl groups
at position 2 on both their phenyl ring structures (com-
pound 20). However, the presence of a cyclohexanone ring
has little effect on the compound with methoxy groups at
positions 2 and 5 of their phenyl ring structure (compound
30). These result suggest that the presence of two methoxyl
groups at these positions (compounds 6 and 30) are impor-
tant structural features for ROS-inhibitory activity. Com-
pounds with methoxy groups at positions 2 and 3 on both
their phenyl ring structures (compound 28) also show
strong ROS-inhibitory activity in PMNs. The presence of
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
409

Immunomodulatory effects of diarylpentanoids
Ibrahim Jantan et al.
Table 2 Percentage inhibition at 10 mg/ml and IC50 values (mmol/l) of
synthetic diarylpentanoid analogues on PMN chemotaxis
Inhibition of PMN chemotaxis
The effects of the diarylpentanoid analogues at 10 mg/ml
on the migration of PMNs towards the chemoattractant
N-formyl-methionyl-leucyl-phenylalanine (fMLP) were
determined and their percentage inhibitions (%) are shown
in Table 2. Compounds 2, 7, 16, 17, 18, 23, 24, 32, 34 and 42
strongly inhibited migration of PMNs with percentage inhi-
bitions ranging from 64.2 to 81.7%. Chemoattractant buffer
(DMSO and HBSS, 1 : 1 ratio) was used as a control and
curcumin and ibuprofen were used as positive controls. In a
study to determine the effect of selected NSAIDs in blocking
the migration of PMNs, ibuprofen was found to be the most
effective drug.[31] The IC50 values of the compounds on
PMN chemotaxis are shown in Table 2. Compounds 7, 17,
18, 24 and 32 strongly inhibit migration of PMNs, with IC50
values of 2.0–6.4 mm, which is lower or comparable
to the values for curcumin (5.1 mm) and ibuprofen
(6.7 mm) (Table 2). All the active compounds showed a
dose-dependent effect as exemplified by compounds 7, 17,
18, 24 and 32 (Figure 3).
Compound.
Mean Ϯ SEM
IC50 values
1
2
60.8 Ϯ 7.3
70.0 Ϯ 2.5
73.3 Ϯ 0.8
53.3 Ϯ 1.7
47.5 Ϯ 2.9
78.3 Ϯ 0.8
80.8 Ϯ 0.8
75.0 Ϯ 2.5
71.7 Ϯ 2.2
80.8 Ϯ 2.2
55.8 Ϯ 3.0
65.0 Ϯ 1.4
30.8 Ϯ 1.7
13.3 Ϯ 5.1
64.2 Ϯ 0.8
76.7 Ϯ 2.2
73.3 Ϯ 1.7
79.2 Ϯ 0.8
77.5 Ϯ 1.4
59.2 Ϯ 0.8
40.8 Ϯ 0.8
58.3 Ϯ 0.8
70.0 Ϯ 3.8
81.7 Ϯ 2.2
56.7 Ϯ 4.6
60.0 Ϯ 2.9
60.8 Ϯ 1.7
69.2 Ϯ 0.8
25.8 Ϯ 0.8
77.5 Ϯ 1.4
62.5 Ϯ 1.4
77.5 Ϯ 2.9
48.3 Ϯ 0.8
57.5 Ϯ 2.5
63.3 Ϯ 1.7
53.3 Ϯ 3.6
67.5 Ϯ 1.4
78.3 Ϯ 1.7
60.8 Ϯ 0.8
48.3 Ϯ 0.8
65.0 Ϯ 1.4
64.2 Ϯ 2.2
54.2 Ϯ 0.8
80.5 Ϯ 0.6
65.8 Ϯ 2.2
16.4 Ϯ 4.4
7.5 Ϯ 1.6
18.2 Ϯ 0.4
21.6 Ϯ 1.3
–
3
4
5
6
10.9 Ϯ 0.3
6.4 Ϯ 0.7
11.0 Ϯ 0.3
10.8 Ϯ 1.6
12.7 Ϯ 2.1
37.1 Ϯ 3.1
17.7 Ϯ 0.3
–
7
8
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
42
43
Curcumin
Ibuprofen
–
9.3 Ϯ 1.5
7.0 Ϯ 0.5
5.7 Ϯ 0.6
3.8 Ϯ 0.3
14.7 Ϯ 0.1
13.9 Ϯ 0.1
–
Structure–activity analysis of
diarylpentanoid analogues as inhibitors of
PMN chemotaxis
10.1 Ϯ 2.3
7.4 Ϯ 3.8
2.0 Ϯ 0.1
22.8 Ϯ 6.9
12.1 Ϯ 1.6
15.3 Ϯ 0.6
10.9 Ϯ 0.5
–
Compound 17, which showed strong inhibition on the
oxidative burst of whole blood and PMNs, was the most
effective acetone diarylpentanoid analogue in inhibiting
PMN migration. The presence of methoxy groups at posi-
tions 2 and 6 of the phenyl rings of the acetone diarylpen-
tanoid analogue (compound 7) may contribute to the high
inhibitory effect on PMN migration. Fluorination at posi-
tion 3 of both phenyl rings (compound 16) produced a
compound with strong inhibition of PMN chemotaxis.
Compound 2, which has a similar substitution pattern
to curcumin on its phenyl ring structures but contains a
five-carbon linker, showed weaker activity than the latter.
Chlorination at various positions of both phenyl rings of
cyclohexanone diarylpentanoid analogues resulted in com-
pounds with potent inhibitory effects on PMN migration
(compounds 18, 23 and 24). In fact, the 3-chlorinated ana-
logue (compound 24) was the most potent compound
amongst the 43 diarylpentanoid analogues tested, exhibiting
more than fourfold and threefold increases in PMN chemo-
taxis relative to curcumin and ibuprofen, respectively.
Trimethoxylation of the phenyl rings of the cyclohexanone
diarylpentanoid analogues strongly contributed to increased
inhibitory activity on PMN chemotaxis, as shown by com-
pound 32, which contains methoxy groups at positions 3, 4
and 5 of both phenyl ring structures. Trimethoxylation of
both phenyl rings of the cyclopentanone-containing linker
(compound 42) could also significantly increase the inhibi-
tory effect of the diarylpentanoid analogues. The presence
10.6 Ϯ 0.5
12.0 Ϯ 0.4
5.5 Ϯ 0.2
–
7.4 Ϯ 0.9
15.2 Ϯ 1.7
21.6 Ϯ 3.1
12.5 Ϯ 1.3
11.1 Ϯ 2.9
11.5 Ϯ 0.7
–
12.9 Ϯ 0.7
8.1 Ϯ 1.0
17.4 Ϯ 0.4
5.1 Ϯ 0.4
6.70 Ϯ 2.2
All values were significant at P Յ 0.05 when compared with the respec-
tive control.
of an electron-withdrawing chloride at position 2 of both
phenyl ring structures of the cyclopentanone diarylpen-
tanoid analogue resulted in a compound (compound 34)
with strong inhibitory activity on chemotactic migration
of PMNs.
© 2011 The Authors. JPP © 2011
Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 404–412
410

Ibrahim Jantan et al.
Immunomodulatory effects of diarylpentanoids
10 μg/ml
100
5
μg/ml
2.5 μg/ml
1.25 μg/ml
0.63 μg/ml
80
60
40
20
0
*
*
*
*
*
**
*
*
**
*
*
*
**
*
Various sample concentrations (μg/ml
)
Figure 3 Effects of curcumin-related diarylpentanoid analogues on migration of PMNs towards the chemoattractant fMLP. Percentage inhibition by
most active compounds along with curcumin and ibuprofen on migration of PMNs. Significance of differences with respect to control: *P < 0.05,
**P < 0.01.
Conclusion
Declarations
Some of these derivatives were able to modulate the
innate immune response of phagocytes at different
levels of ROS production, emphasizing their potential
for use as chemical leads for the development of new
immunomodulatory agents. Further studies are required
to elucidate the types of receptor involved in the inhibi-
tion of ROS production by the diarylpentanoid analogues,
by using other activators such as phorbal 12-myristate
13-acetate.
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Funding
This work was supported by the Ministry of Higher
Education, Malaysia, under the Fundamental Research Grant
Scheme (FRGS) (grant number 03-FRGS0029-2010).
5. Arnhold J.Free radicals-friends or foes?
Properties, functions and secretion
interaction studies. Nat Prod Rep 2011;
DOI: 10.1039/c1np00051a.
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