Synthesis of Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents

Article abstract of DOI:10.1002/ardp.200300763

New series of 3,5-bis(substituted benzylidene)-4-piperidones, 2,7-bis(substituted benzylidene)cycloheptanones, 1,5-bis(substituted phenyl)-1,4-pentadien-3-ones, 1,7-bis(substituted phenyl)-1,6-heptadien-3,5-diones, 1,1-bis(substituted cinnamoyl)-cyclopentanes, and 1,1-bis(substituted cinnamoyl)cyclohexanes have been synthesized and tested for their antioxidant activity. Among the tested compounds, compounds II ₄, II₉ II₁₀, II₁₁, V₁, and V₄ exhibited higher free radical scavenger activity with % inhibition values of 90.71, 91.24, 96.91, 94.26, 99.23, and 99.85%, respectively. Moreover, compound V₁ is the safest member toward peripheral multinuclear neutrophils (PMNs) with a % viability value of 91%. Detailed synthesis, spectroscopic, and biological data are reported.

Full text of DOI:10.1002/ardp.200300763

42 Youssef et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Khairia M. Youssefa,
Magda A. El-Sherbenya,
Faiza S. El-Shafiea,
Hassan A. Faraga,
Synthesis of Curcumin Analogues as Potential
Antioxidant, Cancer Chemopreventive Agents
New series of 3,5-bis(substituted benzylidene)-4-piperidones, 2,7-bis(substituted
benzylidene)cycloheptanones, 1,5-bis(substituted phenyl)-1,4-pentadien-3-ones,
1,7-bis(substituted phenyl)-1,6-heptadien-3,5-diones, 1,1-bis(substituted cin-
namoyl)- cyclopentanes, and 1,1-bis(substituted cinnamoyl)cyclohexanes have
been synthesized and tested for their antioxidant activity. Among the tested com-
pounds, compounds II4,II9 II10,II11,V1, andV4 exhibited higher free radical scavenger
activity with % inhibition values of 90.71, 91.24, 96.91, 94.26, 99.23, and 99.85 %,
respectively. Moreover, compoundV1 is the safest member toward peripheral multi-
nuclear neutrophils (PMNs) with a % viability value of 91 %. Detailed synthesis,
spectroscopic, and biological data are reported.
Omar A. Al-Deeba,
Sit Albanat A. Awadallab
a
Department of Medicinal
Chemistry,
College of Pharmacy and
Department of Physiology,
b
College of Medicine,
King Saud University,
Riyadh, Saudi Arabia
Keywords: Curcumin derivatives; Antioxidants; Free radical scavengers; Chemilu-
minescence test
Received: January 7, 2003; Accepted: April 22, 2003 [FP763]
DOI 10.1002/ardp.200300763
levels of oxyradicals may accumulate in tissues [11]. A
number of naturally occuring compounds as
Introduction
Chemoprevention is the process of reversing, suppress-
ing, or preventing carcinogenesis.A number of naturally-
occuring and synthetic compounds of potential chemo-
preventive properties has been identified [1–4]. Some of
these compounds appear to block activation of chemical
carcinogens and prevent the initiation phase [5]. Lipid
peroxidation is a term that refers to the oxidative degen-
eration of unsaturated lipids in cell membrane and other
organized assemblies. Interest in biological lipid peroxi-
dation has been intensified in recent years due to the
growing awareness that lipid peroxidation may play an
important role in aging and in a wide variety of pathologi-
cal disorders such as arteriosclerosis, inflammatory dis-
ease, and cancer [6, 7].It is a free radical-mediated chain
reaction, in which lipid hydroperoxides are produced.Ac-
cumulation of lipid hydroperoxides in a membrane dis-
rupts membrane functions, leads to decreased fluidity,
increased leaking of ions, and damage to membrane
proteins such as receptors and enzymes [8, 9]. The hu-
man body uses antioxidative mechanisms to quench this
biochemical fire; among these are the enzymes super-
oxide dismutase (SOD), catalase, and glutathione per-
oxidase (GSH-Px) [10]. Tissue stores of small mole-
cules, such as α-tocopherol, reduced glutathione
(GSH), and ascorbate, are also used. However, when
these defenses are overwhelmed by pre-oxidants, toxic
β-carotene[12, 13], vitamin E [14], reduced glutathione
[15, 16], green tea polyphenols [17], resveratrol [18], cur-
cumin [19] and synthetic medicinals such as aspirin,
sulindac, and piroxicam [20, 21] were investigated for
their potential role in different types of animal model can-
cers as urinary bladder, lung tumorgenesis, and colon
cancer.Curcumin has recently received considerable at-
tention due to its pronounced anti-inflammatory [22], an-
tibacterial [23], antihepatotoxic [24], and antioxidative
properties [25]. The potent antioxidant activity of curcu-
min (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadi-
en-3,5-dione, Figure 1) has currently received remarka-
ble interest as it has a unique conjugated structure and
shows a typical radical trapping ability as a chain-break-
ing antioxidant.
Figure 1.
The antioxidant mechanism of curcumin has attracted
much attention. Generally, it has a dual effect in oxygen
radical reactions, thus it can act as a scavenger of hy-
droxyl radicals or it catalyses the formation of hydroxyl
radicals depending on the experimental conditions [26].
Curcumin is stable at a pH below 6.5, the instability of
Correspondence: Khairia M. Youssef, Department of
Medicinal Chemistry, College of Pharmacy, King Saud
University, P.O. Box 22452, Riyadh 11495, Saudi Arabia.
Phone: +966-1-477 5464, Fax: +966-1-477 5464; e-mail:
Khairia_m@yahoo.com
© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents 43
curcumin at a pH above 6.5 is attributed to the active
methylene group, omitting the active methylene group
leads to potent antioxidative compounds. In this work, a
series of 3,5-bis(substituted benzylidene)-4-piperi-
dones II1–16, 2,7-bis(substituted benzylidene)cyclohep-
tanones III1–5, 1,5-bis(substituted phenyl)-1,4-pentadi-
en-3-ones IV1–6, 1,7-bis(substituted phenyl)-1,6-hepta-
dien-3,5-diones V1–6, 1,1-bis-(substituted cinnamoyl)cy-
clopentanesVI1–6, and cyclohexanesVII1–6 are prepared
and evaluated for their antioxidant activity compared
with that of curcumin.
through condensation of the appropriate aldehyde with
either 1-cycloheptanone or acetone, respectively
(Scheme 1).The interaction of the appropriate aldehyde
with acetyl acetone using alcoholic NaOH afforded the
corresponding chalcone 1,7-bis(substituted phenyl)-
1,6-heptadien-3,5-diones V1–6. Upon treatment of com-
poundsV1–6 with NaOEt, the corresponding sodium salts
were obtained, which were subjected to cyclization us-
ing either 1,4-dibromobutane or 1,5-dibromopentane in
ethanol to yield the required 1,1-bis(substituted cin-
namoyl)cyclopentanesVI1–6 and 1,1-bis(substituted cin-
namoyl)cyclohexanes VII1–6, respectively (Scheme 2).
Chemistry
The designed target compounds are depicted in
Schemes 1 and 2. 3,5-bis(substituted benzylidene)-N-
alkyl-4-piperidones II1–16 were obtained through conden-
sation of the appropriate 4-piperidone with a variety of
aromatic aldehydes under acidic condition.Following the
same reaction condition 2,7-bis(substituted benzy-
lidene)-cycloheptanones III1–5 and 1,5-bis(substituted
phenyl)-1,4-pentadien-3-ones IV1–6 were produced
Biology
In vitro chromatographic determination of
free radical scavenging activity (Diphenylpic-
rylhydrazyl free radical test)
The reactivity of the prepared compounds, as free radi-
cal scavengers, were tested by their action on the 2,2-
diphenyl-1-picrylhydrazyl free radical.The test is simple
Scheme 1.
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44 Youssef et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Scheme 2.
and was carried out in acetonitrile without any need of
biological media. Diphenylpicrylhydrazyl absorbs
strongly at 515 nm. The reaction was carried out under
pseudo-first-order conditions using excess of the tested
compounds compared to the reagent. The mixture was
monitored for the decrease of absorption at 515 nm.
Form the linear regression line of the absorbancy
tivities are observed with II3, II4, II6, II9, II10, II11, II12, II13,
and II15 with Kapp min–1 × 10–2 equal 7.30, 10.25, 4.40,
12.15, 16.32, 12.41, 9.32, 6.91, and 14.80, respectively.
A comparison of activities within compounds II shows
that the introduction of electron donating substituents at
the ortho-position of the phenolic hydroxy group produc-
es compounds with higher activity as shown by II3 and
II4, II6 II1, Kapp min-1—10-2 are 7.30, 10.25, 4.40 >
against time, the pseudo-first-order rate constant (Kapp
)
and the corresponding half-life time (t1/2) of the reaction
were calculated.This reaction is valuable in studying the
difference between the chemical and the in vitro biologi-
cal testing [27].
2.15, which is also true for II9, II10, II11, II12 > II7 and II15
>
II13. Moreover, increasing the lipophilicity of the piperi-
done ring by substitution of methyl with either an ethyl or
propyl moiety, potentiates the activity as shown by II13
and II7 > II1, Kapp min–1 × 10–2 are 6.91, 3.06 > 2.15. Re-
placement of the piperidone moiety with cyclohepta-
none affords compounds which still possesses high free
radical scavenging activity, as shown by III1 and III5 with
Chemical determination for the free radical scavenging
activity indicates that most of the tested compounds
show higher activity compared to curcumin with Kapp
min–1 × 10–2 equal 3.30.In case of compounds II, high ac-
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents 45
Table 1. Reaction of Curcumin analogues with free radical.
Compound
Structure
Kmin–1 × 10–2
2.15 0.116
r
T 1/2
25.3
II1
– 0.998
II3
7.30 0.11
– 0.999
–0.996
19.5
17.5
II4
10.25 0.103
II6
4.40 0.023
3.06 0.057
– 0.997
– 0.995
20.5
12.9
II7
II9
12.15 0.120
16.32 0.035
12.41 0.032
– 0.991
–0.993
–0.992
10.5
15.3
12.7
II10
II11
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46 Youssef et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Table 1. (continued)
Compound
Structure
Kmin–1 × 10–2
9.32 0.114
r
T 1/2
16.8
II12
– 0.991
II13
6.91 0.325
– 0.998
– 0.991
18.3
11.3
II15
14.80 113
III1
III5
5.91 0.130
11.92 111
– 0.992
–0.995
18.3
13.3
IV1
IV5
1.41 0.013
3.41 0.113
– 0.985
– 0.993
19.7
20.3
IV6
1.82 0.124
2.83 0.328
– 0.996
– 0.994
23.6
18.2
V1
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents 47
Table 1. (continued)
Compound
Structure
Kmin–1 × 10–2
1.81 0.052
r
T 1/2
16.6
V4
– 0.996
VI1
1.90 0.111
1.33 0.230
– 0.991
– 0.998
19.3
21.6
VII1
Curcumin
3.30 0.11
–0.998
19.5
Kapp min–1 × 10–2 5.91 and 11.92, respectively.Upon sub-
stitution of the cyclic structure either piperidone or cy-
cloheptanone with acetone leads to a remarkable de-
crease in the free radical scavenging activity; com-
using medium containing serum and agarose-coated
microplates.Both solid tumors and hematological malig-
nancies have been tested with the TCA-100. The high
sensitivity allows to replicate the testing of six single
agents or drug combinations at seven concentrations
with less than two million cells. The system provides
dose-response results and is able to detect heterogenity
of drug sensitivity between tumors and drug combina-
tion effects consistent with clinical experience, to estab-
lish the relationship between energy (ATP) use and oxy-
gen metabolite production in human peripheral blood
phagocytes. The system is also able to determine the
ability of alterations in these parameters to distinguish
toxic from metabolic effects on phagocyte function [28,
29]. In this work, normal human phagocytes obtained
from healthy male and female volunteers were used and
separated by the use of neutrophil isolation medium
(NIM) (Cardinal Associates, Santa Fe, NM, USA).
pounds IV1, IV5 < II10, II11, II15, and III1 with Kapp min–1
×
10–2 1.41, 3.41 < 16.32, 12.41, 14.80, and 5.91, respec-
tively. Upon increasing the length of the unsaturated
chain using acetyl acetone instead of acetone, leads to
an increase in the activity as shown by compoundV1 with
Kapp min–1 × 10–2 2.83.Upon ommitting the active methyl-
ene group in compounds V1–6 using either cyclopen-
tanone or cyclohexanone, yielding compoundsVI1–6 and
VII1–6, leads to a decrease in the free radical scavenging
activity (Table 1).
ATP chemiluminescence assay
ATP luminescence is a sensitive and precise method for
measuring cell viability [28, 29].Since 1984, a number of
groups have used ATP luminescence to test the chemo-
sensitivity of both cell lines and tumors. The TCA-100
method described in this report uses ATP luminescence
to measure chemotherapeutic drug sensitivity and re-
sistance of dissociated tumor cells cultured for 6 d in se-
rum-free medium and polypropylene microplates to in-
hibit the survival of non-neoplastic cells. This method
was developed from a previously published technique
All of the synthesized compounds were tested for their
ability to scavenge oxygen free radical produced by pe-
ripheral multinuclear neutrophil cells (PMNs) collected
from apparently healthy blood donors. Phorpol-12-myr-
istate-13-acetate (PMA), in a final concentration of
2 ng/mL was added to PMNs (5 × 105 cells/mL) to stimu-
late respiratory burst which will be magnified by luminol
(10–4 M) for measuring in the LKB luminometer. 100 µg
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48 Youssef et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
of the prepared drugs were added to detect its effect on
the amount of oxygen radical liberated and the percent-
age of inhibition was calculated.
Effect of drugs on the viability of PMNs
To examine whether these substances have a direct cy-
totoxic effect on PMNs viability, the percentage of viable
cells was estimated by the trypan blue exclusion test
(0.2 %) of ten incubation periods 30 min each at 37 °C.In
this study, the toxicity of the tested compounds was de-
termined by adding equal parts of these compounds and
PMNs.Then the cells were incubated under normal cul-
ture conditions (37 °C, 5 % CO2 atmosphere) for 30 min-
utes. After incubation, a drop of trypan blue stain was
added to the mixture and then examined under the mi-
croscope, counting about 100 PMNs, taking the percent-
age of the viable cells (those, which did not take up the
stain) versus the dead (stained) cells. Among the tested
compounds, compound V1 showed slight toxicity with a
% viability of 91 %, while compounds II4, II5, II6, II7, II10,
II11, II12, III1, III6, IV1, IV3, V1, V2, V4, VI3, and VII1 showed
moderate toxicity with % viability range from 89–81 %.
The rest of the tested compounds showed a % viability
that range from 79–64 % (Table 2, Figure 2).
In this study, the free radical scavenger determination in-
dicates that compounds II4, II9, II10, II11, V1, and V4 show
the highest free radical scavenger activity with % inhibi-
tion values of 90.71, 91.24, 96.91, 94.26, 99.23, and
99.85 %, respectively compared to that of curcumin
with a % inhibition value of 99.55.Moreover, compounds
II1, II3, II6, II1, II12, II13 and III5 are also proved to be moder-
ately effective with % inhibition range of 88.19–70.76 %.
In addition, a % inhibition range of 65.44–60.23 % are al-
so obtained by compounds II5, III1, and V2 (Table 2, Fig-
ure 2).
Table 2. Effect of the tested compounds on the chemilu-
minescence response and cell viability of isolated hu-
man peripheral multinuclear neutrophils (PMNs).
Drugs CL Response
(mV)
% Inhibition % Viability
Conclusion
Upon determining the antioxidant activity for the synthe-
sized compounds, it was found that, most of the these
compounds showed a promising activity. Among them,
compounds II4, II9, II10, II11, V1, and V4 showed high free
radical scavenger activity with % inhibition values of
90.71, 91.24, 96.91, 94.26, 99.23, and 99.85 %, respec-
tively.
II1 110.88 18.01 70.76 3.09
64
70
81
88
85
86
68
82
88
81
75
85
79
88
81
81
77
91
82
89
75
84
85
76
92
II3
II4
II5
II6
79.61 15.09 79.01 3.88
35.24 5.33 90.71 1.22
150.83 3.23 60.23 4.48
57.18 6.36 84.92 2.64
II7 102.36 13.16 73.01 3.87
II9 33.22 4.66 91.24 2.35
II10 117.72 1.86 96.91 0.88
Depending on the data of the oxygen free radical scav-
enger activity of the tested compounds, the following
structural requirements for high antioxidative activity are
obtained:
II11
II12
II13
21.76 3.60 94.26 1.25
44.80 5.99 88.19 5.99
82.41 8.82 78.27 2.32
III1 138.70 47.81 63.42 3.52
a) p-Hydroxy phenolic structure is the most essential
component in the antioxidative structure.
III5
III6
86.99 12.88 77.06 12.88
220.43 7.17 41.87 7.78
b) Electron-donating substituents on the benzene ring
at the ortho-position of the phenolic hydroxy group
are also required.
IV1 288.92 73.32 23.81 5.82
IV3 245.07 32.91 35.37 9.34
IV6 221.87 25.66 41.49 13.04
V1
V2 131.04 37.61 65.44 1.53
V4 2.93 0.91 99.85 0.02
0.58 0.01 99.23 0.10
c) Omitting the active methylene group in 1,7-bis(sub-
stituted phenyl)-1,6-heptadien-3,5-dione V1–6 to af-
ford the corresponding 1,1-bis(substituted cin-
namoyl)-cyclopentanes VI1–6 and 1,1-bis(substituted
cinnamoyl)cyclohexanes VII1–6, leads to a decrease
in the antioxidative activities.
VI1 334.88 78.35 11.69 6.67
VI3 336.20 75.65 11.34 6.24
VII1 292.14 22.94
VII3 367.50 78.04
9.53 4.92
3.09 6.14
Curcumin
48.80 0.01 99.55 0.01
Acknowledgment
Values are expressed as mean SEM.
PMA = 2 ng/mL; PMNs = 5 × 106 cells/mL; Luminol =
The financial support of King Abdulaziz City for Science
andTechnology, Project No.AR 19–39 is greatly appreci-
ated.
10–4 M;
Curcumin derivatives = 100 µg/mL
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Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
Curcumin Analogues as Potential Antioxidant, Cancer Chemopreventive Agents 49
Figure 2. Chemiluminescence response with corresponding % inhibition and % viability of a group of water-soluble
curcumin derivatives on isolated human polymorphonuclear leukocytes (PMNs) when stimulated with PMA.
1,1-bis(substituted cinnamoyl)cyclopentane (VI1–6) and 1,1-
bis(substituted cinnamoyl)cyclohexane (VII1–6)
Experimental
Synthesis
The disodium salts ofVI1–6 (0.01 mol) were refluxed while strir-
ring with either 1,4-dibromobutane or 1,5-dibromopentane
(0.01 mol) in EtOH for 6 h. The precipitated NaBr was filtered,
and the filterate was concentrated.The separated compounds
were filtered then crystallized from the selected solvent (Table
3).
Melting points were determined on Mettler FP80 melting point
apparatus (England) and are uncorrected. Microanalysis was
performed on a Perkin-Elmer 2400 elemental analyzer (Perkin-
Elmer, Norwalk, CT, USA) at the Central Research Laboratory,
College of Pharmacy, King Saud University.Thin layer chroma-
tography was performed on pre-coated (0.25 mm) silica gel
1
plates, compounds were detected with 254 nm UV lamp, H
Biological Testing
NMR spectra were recorded on a Varian EM 360 (90 MHz) in-
strument (Varian Inc., Palo Alto, CA, USA) using TMS as inter-
nal standard (Chemical shift in δppm).Compounds II2, IV2, and
IV3 had been synthesized according to a reported procedure
[30, 31] (Scheme 2, Table 3).
Diphenylpicrylhydrazyl free radical test
To a solution of the synthesized compounds (10 mg) in ace-
tonitrile (5 mL), 2,2-diphenyl-1-picrylhydrazyl free radical
(3 mL) in acetonitrile (1 mL) was added.The mixture was kept
at 37 °C, 0.2 mL were pipetted from the reaction mixture at dif-
ferent time intervals, 5, 10, 15, 25, 30, and 35 min. The de-
crease of absorption at 515 nm was measured. Form the linear
regression line of absorbancy against time, the pseudo-first or-
der rate (Kapp) and the corresponding half-life time (t1/2) of the
reaction were calculated (Table 1).
3,5-bis(substituted benzylidene)-N-alkyl-4-piperidones (II1–16),
2,7-bis(substituted benzylidene)cycloheptanones (III1–5), and
1,5-bis(substituted phenyl)-1,4-pentandien-3-ones (IV1–6
)
A mixture of the appropriate aldehyde (0.02 mol) and the ke-
tone (0.01 mol) was heated in a water bath at 25–30 °C until a
clear solution was obtained, then 2 mL concentrated HCl was
added while stirring for 5 min. The reaction mixture was then
stirred at room temperature for 2 h. After standing for 2 d, the
mixture was treated with cold AcOH/water (1:1) and filtered.
The solid obtained was then dried and crystallized from the ap-
propriate solvents (Table 3).
Measurement of oxygen free radicals by chemiluminescence
Isolation of PMNs
Heparinized blood (5–7 mL) was layered over 4 mL of neu-
trophil isolation medium (NIM) in a round bottom tube and then
centrifuged at 400 g for 30 min at room temperature. After cen-
trifugation, top layers of plasma and lymphocyte/monocyte
were carefully aspirated and the neutrophil layer was removed
with a pasteur pipette and transfered to a 15 mL conical centri-
fuge tube, the tube was filled with HBSS (Hank’s balanced salt
solution) and mixed to obtain a homogenous suspension which
was then centrifuged for l0 min at 350 g. After centrifugation,
the supernatant was discarded and 2 mL lysation buffer was
added and vortexed to resuspend the pellet, the tube was left
for 5–10 minutes in ice and then centrifuged for 5 min at 250 g
and then the supernatant was discarded. The pellets were
washed and resuspended with l0 mL HBSS and centrifuged at
1,7-bis(substituted phenyl)-1,6-heptadien-3,5-diones (V1–6
)
Acetyl acetone (0.01 mol) and the appropriate aldehyde
(0.02 mol) in alcoholic NaOH (50 mL, 10 %) were stirred at
room temperature for 10 min. The separated solid was filtered
and recrystallized from the suitable solvents (Table 3).
1,7-bis(substituted phenyl)-1,6-heptadien-3,5-diones sodium
salts
CompoundsV1–9 (0.01 mol) and NaOCH3 (0.04 mol) was stirred
under reflux in EtOH for 1 h.The precipitated solid was filtered
and used for the following step without further crystallization.
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54 Youssef et al.
Arch. Pharm. Pharm. Med. Chem. 2004, 337, 42–54
[11] T. E. Gram, Pharmacol. Rev., 1997, 49, 297–341.
(250 g for 5 min twice.After washing, the pellet was resuspend-
ed in 1 mL of 5 % FCS (fetal calf serum) and the neutrophils
were counted and adjusted to a final concentration of 5 × 103
cells per mL [29, 30].
[12] R.C.Moon, R.G.Mehta, K.V.N.Rao, in Retinoids, Biology,
Chemistry and Medicine, 2nd ed. (Eds: M. B. Spom, A. B.
Roberts, D. C. Goodman), Raven Press, New York, 1994,
pp. 573–595.
Separation of PMNs
[13] A. M. Smith, D. R. Parkinson, B. D. Cheson, J. Clin. Oncol.,
1992, 10, 839–864.
Fresh blood was collected by venipuncture from apparently
healthy subjects or blood donors in sterile containers with
heparin (10 IU/mL, Fischer Scientific Co., Springfield, NJ,
USA).The whole blood was then carefully layered over 4 mL of
NIM (Neutrophil Isolation Medium, Cardinal Associates) in a
labeled 15 mL round bottom tube. Then it was centrifuged at
400 g for 30 min at room temperature.The neutrophil layer was
carefully removed with a Pasteuripette and transferred to a
15 mL conical centrifuge tube, which was then filled with phos-
phate buffered saline (PBS, Sigma Chemical Co., St Louis MO,
USA) and inverted several times to obtain a homogenous sus-
pension. The tube was centrifuged again at 350 g for 10 min-
utes, then the supernatant was discarded carefully so as not to
disturb the pellet. Lysation buffer (2 mL) was added and the
tube was vortexed to resuspend the pellet and the suspension
was left to stand for 2 minutes at room temperature, after which
it was centrifuged again at 250 × g for 5 minutes.The superna-
tant was discarded and the pellet resuspended with 10 mL of
PBS, twice with centrifugation in between washing. After the fi-
nal washing step, the pellet was resuspended with 1 mL PBS
and the neutrophils were counted and adjusted to a final con-
centration of 5 × 106 cells per mL. Viability was determined by
trypan blue (0.2 %) exclusion test, only a PMN viability above
90 % was used in the experiment [29, 30].
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