Chemico-Biological Interactions 206 (2013) 63–75

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation

and microbial killing of human neutrophils
Luciana M. Kabeya a,⇑, Carolina N. Fuzissaki a, Silvia H. Taleb-Contini a,1, Ana Maria da C. Ferreira b,
Zeki Naal a, Everton O.L. Santos a, Andréa S.G. Figueiredo-Rinhel a, Ana Elisa C.S. Azzolini a,
Roberta B. Vermelho a, Alberto Malvezzi b,2, Antonia T. -do Amaral b, João Luis C. Lopes a,
Yara Maria Lucisano-Valim a
a
Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto da Universidade de São Paulo. Av. do Café s/n, 14040-903 Ribeirão Preto, SP, Brazil
b
Departamento de Química Fundamental, Instituto de Química da Universidade de São Paulo. Av. Prof. Lineu Prestes 748, 05508-900 São Paulo, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methyl-
Received 16 April 2013 coumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils
Received in revised form 14 July 2013 and display antioxidant activity. These compounds decreased the ability of neutrophils to generate super-
Accepted 17 August 2013
oxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate
Available online 28 August 2013
their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous
acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand,
Keywords:
the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimu-
7-Hydroxycoumarin
Neutrophil
lated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxyl-
Degranulation ated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical
Myeloperoxidase intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemilumines-
Candida albicans cence assay. These species also oxidized ascorbic acid and the spin traps a-(4-pyridyl-1-oxide)-N-tert-
Prooxidant butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives
investigated here were able to modulate the effector functions of human neutrophils and scavenge reac-
tive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperox-
idase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the
biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neu-
trophils to produce relevant anti-inflammatory effects.
Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction cells. These molecules help regulate the initiation of specific T

Neutrophils are professional killers of invading microorganisms
and also contribute to the regulation of inflammatory processes.
These cells secrete types of chemokines, cytokines, lipid mediators,
granule proteins, and reactive oxygen species (ROS) that serve as
signaling molecules for subsequent recruitment of inflammatory
and B cell immunity and the resolution phase of inflammation
[1–3]. Thus, modulating the effector functions of neutrophils is a
promising therapeutic strategy to manage inflammation.
Coumarin and its derivatives have arisen as promising natural
products with potent anti-inflammatory effect and low toxicity
to humans [4–8]. They constitute a large class of phenolic

Abbreviations: AAPV, methoxy-succinyl-Ala-Ala-Pro-Val-chloromethylketone; ABAH, 4-aminobenzoic acid hydrazide; Asc, ascorbic acid; CL, chemiluminescence; CL-luc,
lucigenin-enhanced chemiluminescence; CL-lum, luminol-enhanced chemiluminescence; DMPO, 5-dimethyl-1-pyrroline-N-oxide; DMPOX, 5,5-dimethyl-1-pyrrolidone-2-
oxyl-1; DMSO, dimethyl sulfoxide; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical; EPR, electron paramagnetic resonance; n-fMLP, n-formyl-methionyl-leucyl-phenylalanine;
HBSS, Hank’s balanced saline solution; HBSS-gel, Hank’s balanced saline solution supplemented with gelatin; HRP, horseradish peroxidase; IC50, concentration inhibiting a
biological response by 50%; LDH, lactate dehydrogenase; MPO, myeloperoxidase; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; NADPH, reduced
form of nicotinamide adenine dinucleotide phosphate; PI, propidium iodide; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; 4-POBN, a-(4-pyridyl-1-oxide)-N-
tert-butylnitrone; ROS, reactive oxygen species; SAAVNA, N-succinyl-Ala-Ala-Val-p-nitroanilide; SOD, superoxide dismutase; SOZ, serum-opsonized zymosan.
⇑ Corresponding author. Address: Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo. Av. do Café s/n,
Bairro Monte Alegre, 14040-903 Ribeirão Preto, SP, Brazil. Tel.: +55 16 36024212; fax: +55 16 36024880.
E-mail address: luciana_kabeya@yahoo.com.br (L.M. Kabeya).
1
Present address: Unidade de Biotecnologia, Universidade de Ribeirão Preto (UNAERP), Avenida Costábile Romano 2201, Bairro Ribeirânea, 14096-380 Ribeirão Preto, SP, Brazil.
2
Present address: Universidade Nove de Julho (UNINOVE), Avenida Dr. Adolpho Pinto 109, Barra Funda, São Paulo, SP 01156-050, Brazil.

0009-2797/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.cbi.2013.08.010
64 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
compounds produced by plants of different botanical families,
primarily in Angiospermae [9]. The coumarin (1,2-benzopyrone;
5,6-benzo-a-pyrone) has been clinically used to treat cancer,
lymphoedema, venous insufficiency and chronic infections [5–8].
Coumarin alone is ineffective in treating some diseases, but it
can increase the beneficial effects of other compounds in combina-
tion therapy [8]. However, it has a short half-life in humans due to
hepatic metabolism (70–90%) to 7-hydroxycoumarin (Fig. 1) and
its glucuronide. Coumarin is considered a prodrug and 7-hydroxy-
coumarin is the bioactive molecule [6,8].
7-Hydroxycoumarin, also known as umbelliferone, occurs in
plants and edible fruits and roots, such as golden apple, bitter or-
ange, carrot, coriander, garden angelica, and mouse-ear hawkweed
[10–11]. This coumarin displays a wide range of beneficial bioac-
tivities: it reduces lymphoedema in humans [6] and exerts anti-
hyperglycemic [12], bronchodilating [10], antinociceptive [13],
and antiedematogenic [4,14,15] effects in rats. The in vitro and
in vivo antioxidant, anti-inflammatory and immunomodulatory ef-
fects of 7-hydroxycoumarin have been recently reported
[4,5,11,16–22]. This compound inhibits migration of neutrophils
and eosinophils [10], degranulation of mast cells [15], release of
NO by macrophages [19–21], release of cytokines [10,13], biosyn-
thesis of prostaglandin, and activity of cycloxygenase-2 and
5-lipoxygenase [4,5,14]. In addition, 7-hydroxycoumarin activates
the protective immune responses of mice against influenza virus
[19] and Salmonella typhimurium[22].
However, there are few reports on the action of 7-hydroxy-
coumarin on neutrophils. 7-Hydroxycoumarin and 7-hydroxy
-4-methylcoumarin inhibit the generation of O 2 by zymosan-
stimulated rabbit neutrophils [23], phorbol-12-myristate-13
-acetate (PMA)-stimulated human neutrophils, and n-formyl-
methionyl-leucyl-phenylalanine (fMLP)-stimulated rat leukocytes
[5]. 7-Hydroxycoumarin also inhibits the migration of neutrophils
in mouse models of inflammation [10,13]. Therefore, the high po-
tential of 7-hydroxycoumarin for the development of an effective
anti-inflammatory drug and the fundamental contribution of
neutrophils to the regulation of inflammatory processes have moti-
vated us to evaluate the modulator effect of 7-hydroxycoumarin,
7-hydroxy-4-methylcoumarin, and their acetylated analogs
(Fig. 1) on some effector functions of human neutrophils. In partic-
ular, we investigated their effects on the oxidative metabolism,
degranulation, microbial killing ability, and myeloperoxidase and
elastase activity of neutrophils. We also evaluated their physico-
chemical properties, antioxidant activity, and HOCl scavenging po-
tential in cell-free systems.
2. Materials and methods
2.1. Chemicals
L-Ascorbic acid (Asc), cytochalasin B, 1,1-diphenyl-2
-picrylhydrazyl radical (DPPH), N,N-dimethylformamide, 5-di-
methyl-1-pyrroline-N-oxide (DMPO), a-(4-pyridyl-1-oxide)
-N-tert-butylnitrone (4-POBN), horseradish peroxidase (HRP, type
Fig. 1. Chemical structure of coumarins.
VI-A, EC.1.11.1.7), luminol (5-amine-2,3-dihydro-1,4-phtalazinedi-
one), lucigenin (N,N’-dimethyl-9,90 -biacridinium dinitrate), 3-(4,5-
dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide
(MTT), phorbol-12-myristate-13-acetate (PMA), superoxide dis-
mutase (SOD), trizma hydrochloride (Tris(hydroxymethyl)amino-
methane hydrochloride), 3,30 ,5,50 -tetramethylbenzidine, Triton
X-100, Trypan blue, and Zymosan A were purchased from Sigma–
Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), ethanol,
n-octanol, hydrogen peroxide, n-formyl-methionyl-leucyl-phenyl-
alanine (n-fMLP), N-succinyl-Ala-Ala-Val-p-nitroanilide (SAAVNA),
methoxy-succinyl-Ala-Ala-Pro-Val-chloromethylketone (AAPV),
human leukocyte myeloperoxidase (MPO, EC 1.7.1.11) and 4-ami-
nobenzoic acid hydrazide (ABAH) were obtained from Merck
(Darmstadt, Germany). We acquired the following products from
different suppliers: gelatin (microbiological grade, Difco Laborato-
ries, Detroit, MN, USA), propylene glycol (Synth, Diadema, SP,
Brazil), RPMI 1640 medium (Cultilab, Campinas, SP, Brazil),
Sabouraud’s dextrose agar (Acumedia, Michigan, USA), LDH Liqui-
form™ kit (Labtest Diagnostica, Lagoa Santa, MG, Brazil) and
APOPTEST™-FITC (propidium iodide (PI) + Annexin V stained with
fluorescein isothiocyanate) apoptosis detection kit (Dako, Glostrup,
Denmark). All the other chemicals and solvents used in this work
were of analytical grade and purchased from commercial sources.
2.2. Coumarins
7-Hydroxycoumarin (1) was purchased from Sigma–Aldrich
(St Louis, MO, USA). It was used as raw material to synthesize 7-hy-
droxy-4-methylcoumarin (2), 7-acetoxycoumarin (3), and 7-acet-
oxy-4-methylcoumarin (4), by the methods described by Vogel
[24] and Lopes et al. [25], with slight modifications. The purity of
all the compounds was 97–100%. Their chemical structures are
illustrated in Fig. 1.
2.3. Isolation of human neutrophils
Twenty adult volunteers who met the criteria described by Pau-
la et al. [26] were recruited for this study. The experimental proce-
dures were approved by the local Research Ethics Committee on
Human Experimentation (CEP/FCFRP-USP, protocol 96), according
to the rules of the 196/96 Resolution of the Brazilian National
Health Council and the Helsinki Declaration of 1975 (revised in
2008).
Blood was collected by venous puncture and diluted 1:2 in
Alsever solution (anticoagulant). Neutrophils were isolated using
sterile and lipopolysaccharide (LPS)-free solutions, as previously
described [26]. The cell pellets were suspended in Hanks balanced
saline solution (HBSS) containing 1 mg mL-1 gelatin (HBSS-gel).
The cell preparations contained 90–95% neutrophils with viability
higher than 95%, as established by exclusion with Trypan Blue.
2.4. Generation of ROS by neutrophils
The generation of ROS by neutrophils was measured as de-
scribed previously [26]. Zymosan particles were prepared and
opsonized with normal human serum as described by Kanashiro
et al. [27]. Serum-opsonized zymosan (SOZ) was suspended in
HBSS for use (10 mg mL1). A 10 mM PMA stock solution in
DMSO was diluted 1:1000 in HBSS prior to use [26].
Briefly, the reaction mixtures (0.5 mL) containing neutrophils
(1  106 cells mL1), the chemiluminescent probe (100 lM luminol
or 100 lM lucigenin) and a coumarin (0.01–100 lM), ABAH
(1–200 lM) or DMSO (0.1% v/v, control) were incubated for
3 min, at 37 °C. The reaction was initiated by adding SOZ
(1 mg mL1) or PMA (0.1 lM) and the luminol- or lucigenin-
enhanced chemiluminescence (CL-lum and CL-luc, respectively)
 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
was measured for 15 min, at 37 °C, in a luminometer (Auto Lumat
LB953, EG&G Berthold, Bad Wildbad, Germany). The light emission
was recorded in c.p.m. (photons counts per minute). Background
CL produced by non-stimulated cells was subtracted from all the
samples. The integrated areas of CL were determined and used to
calculate the percentage of CL inhibition or increase promoted by
the tested compound.
2.5. Degranulation assay
Degranulation of neutrophils was evaluated as described previ-
ously [28], using elastase as marker. Briefly, aliquots of neutrophils
(1  106 cells mL1) were incubated in 96-well microplates with a
coumarin (100 lM), DMSO (0.1% v/v, vehicle), or HBSS (control) for
20 min or 60 min, at 37 °C. Then, cytochalasin B (1 lM) was added
to the mixture and incubated for 10 min, at 37 °C, before adding n-
fMLP (1 lM) to trigger degranulation. After 30 min of incubation at
37 °C, the elastase substrate SAAVNA (330 lM) was added to the
medium and the mixture was incubated for further 30 min, at
37 °C. The absorbance was recorded at 405 nm in a microplate
reader (EIA Multi-well Reader II, Sigma Diagnostics, St. Louis,
MO, USA). The difference between the absorbance readings before
and after addition of SAAVNA was used to calculate the percentage
of degranulation elicited by each sample.
2.6. Elastase activity of human neutrophils
The elastase-rich supernatant was obtained from human neu-
trophils treated with cytochalasin B and n-fMLP, as described by
Kanashiro et al. [28]. Aliquots of the supernatant were mixed with
one of the coumarins (100 lM), DMSO (0.1% v/v, vehicle) or HBSS
(control), and incubated for 20 min or 60 min, at 37 °C. Then SAAV-
NA (330 lM) was added to all the samples, and the absorbance of
the product p-nitroaniline was recorded at 405 nm in a plate read-
er, after 30 min of incubation at 37 °C. Appropriate blanks without
the substrate were subtracted from all the samples.
2.7. Microbial killing by neutrophils
This assay was performed in 96-well microplates, according to
the method described by Mosmann [29], with modifications. Star-
ter cultures of Candida albicans (ATCC 64548) were grown for 48 h,
at 37 °C, in Sabouraud’s agar containing 1% chloramphenicol. The
cells were harvested, washed with sterile 0.15 M NaCl (3000g,
5 min, 25 °C), and suspended in RPMI medium. The suspension
containing C. albicans was kept on ice-bath until use.
Aliquots of neutrophils (2  106 cells mL1) were treated with
one of the coumarins (1–100 lM), ABAH (50 lM), DMSO (0.02%)
or HBSS (control), for 30 min, at 37 °C, before adding the suspen-
sion of C. albicans (1  106 CFU mL1) to the reaction mixture. After
2-h incubation at 37 °C in a shaker, the plates were centrifuged,
and the pellets were treated with 1% (v/v) Triton X-100 for
10 min, at 25 °C. The plates were centrifuged again, and the pellets
were washed three times with 0.15 M NaCl. MTT (0.25 mg mL1 in
RPMI medium) was added to all the wells, and the plates were
incubated in a shaker for 4 h, at 37 °C, in the dark. Next, the plates
were centrifuged, and the formazan crystals were dissolved in
DMSO. The absorbance was recorded in a plate reader at 545 nm
after 10-min incubation at 25 °C. All the centrifugation steps de-
scribed in this paragraph were carried out at 3220g, for 10 min,
at 25 °C. Controls in the absence of neutrophils represent 100% of
C. albicans viability (0% of killing).
65
2.8. Evaluation of the cytotoxicity
The cytotoxic effect of coumarins on neutrophils was evaluated
as previously described [30]. Briefly, aliquots of neutrophils
(1  106 cells mL1) were incubated with one of the coumarins
(200 lM), DMSO (0.1% v/v, vehicle), HBSS (non-treated cells), or
Triton X-100 (0.2% v/v, positive control) for 20 min or 60 min, at
37 °C. The cell pellets were immediately suspended in HBSS-gel
after centrifugation (755g, 10 min, 4 °C), and cellular viability
was determined using the Trypan Blue exclusion test, by counting
a total of 200 cells for each sample. The activity of lactate dehydro-
genase (LDH) released into the supernatant was measured with the
LDH Liquiform™ test kit.
Neutrophils (1  106 cells mL1) were treated with coumarin 1
(100 lM) for 60 min, at 37 °C. The cell pellets were immediately
suspended in Annexin V-FITC binding buffer after centrifugation
(440g, 10 min, 4 °C) and kept on ice for 10 min in the dark. PI
was added to the samples, and they were immediately analyzed
in the BD FACSCanto flow cytometer (Becton, Dickinson and Com-
pany; Franklin Lakes, NJ, USA). Data from 10,000 events were col-
lected and analyzed by the BD FACSDiva Software. We followed the
instructions of the manufacturer of the APOPTEST™-FITC apoptosis
detection kit to perform this assay.
2.9. HOCl scavenging assay
The HOCl scavenging potential of coumarins was assessed as
described by Andrade et al. [31]. The working solution of one of
the coumarins (0.05–50 lM) or propylene glycol (0.1% v/v, control)
was added to HOCl (50 lM) and allowed to react for 10 min. The
residual HOCl was detected by rapidly mixing the developing re-
agent (2 mM 3,30 ,5,50 -tetramethylbenzidine in 400 mM acetate
buffer pH 5.4 containing 10% N,N-dimethylformamide and
100 lM KI). The absorbance was recorded at 650 nm in a plate
reader after 5 min of incubation. All the reaction steps were con-
ducted in the dark at 25 °C.
2.10. DPPH scavenging assay
The DPPH free radical scavenging potential of coumarins was
examined by the method of Blois [32], with modifications. The
DPPH stock solution was prepared in ethanol and diluted 6:4 in
40 mM acetate buffer pH 5.5 for use. The DPPH working
solution (100 lM) was incubated with one of the coumarins
(0.1–100 lM), DMSO (0.1% v/v, vehicle), or HBSS (control) for
5 min, at 25 °C, and the absorbance was recorded at 517 nm. The
difference between absorbance readings before and after sample
addition was used to calculate the percentage of DPPH reduction
by each sample.
2.11. MPO-H2O2-luminol chemiluminescence assay
The enzymatic activity in the MPO stock solution was deter-
mined as described by Marquez et al. [33]. The assay buffers were:
PBS pH 7.4 (10 mM sodium phosphate, 140 mM NaCl, 10 mM KCl)
and 50 mM phosphate buffer pH 7.4 (chloride-free medium). The
effect of the coumarins on the MPO-H2O2-luminol reaction was
evaluated as described by Kitagawa et al. [34], with modifications.
Reaction mixtures (0.5 mL) containing the assay buffer, luminol
(20 lM), MPO (50 mU.mL1), and one of the coumarins
(0.01–10 lM) or DMSO (0.01% v/v, control), were incubated at
37 °C for 2 min. The reaction was initiated with H2O2 (50 lM),
and the CL was measured in a luminometer for 30 min, at 37 °C.
The integrated chemiluminescence area was used to calculate the
percentage of CL increase promoted by the tested compounds.
66 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
2.12. Oxidation of coumarins by MPO-H2O2
The coumarin solution (50 lM) or DMSO (0.1% v/v, control) was
mixed with MPO (0.5 U mL1). A small aliquot of H2O2 (50 lM) was
added to the reaction mixture, and the spectra (200–600 nm) were
recorded in quartz cuvettes (1-cm light path) at different times
(0.5–20 min) of the reaction at 25 °C (U3501 spectrophotometer;
Hitachi Instruments, Tokyo, Japan). The assay buffers were PBS
pH 7.4 and 50 mM phosphate buffer pH 7.4.
2.13. Oxidation of ascorbic acid by HRP-H2O2
Aliquots of HRP solution (20 U mL1) were mixed with ascorbic
acid (Asc, 0.4 mM) and one of the coumarins (0–120 lM) or DMSO
(0.8% v/v; control). The reaction mixture was immediately trans-
ferred to a 200-lL flat quartz cell after addition of H2O2
(0.4 mM). The EPR spectra were registered using modulation
amplitude of 1 G and one scan, at 22 °C.
2.14. Electron paramagnetic resonance (EPR) spin trap measurements
DMPO was purified by recommended methods; it was treated
with charcoal [35] or freshly distilled [36] and stored at 80 °C.
The coumarin stock solutions were prepared in DMSO; all the other
reactants were prepared in 0.1 M sodium phosphate buffer pH 7.4.
Aliquots of HRP solution (20 U mL1) were mixed with one of
the coumarins (0–120 lM) or DMSO (0.8% v/v), and one of the spin
traps: DMPO (80 mM) or 4-POBN (100 mM). The reaction was
started by adding H2O2 (0.5 mM); the reaction mixture was imme-
diately transferred to a 200-lL flat quartz cell.
The EPR signals were registered at different time points of the
reaction course (0.5–30 min), at 22 °C, using the Bruker EMX spec-
trometer operating at the X-band (9.5 GHz), with modulation fre-
quency of 100 kHz, microwave power of 20 mW, and modulation
amplitude of 1 G. Ten scans were accumulated for each sample,
to obtain the final spectra. The Win-EPR software package (Bruker,
USA) was used to analyze the spectra by double integration and
simulate their hyperfine coupling constants.
2.15. Measurement of redox potential by cyclic voltammetry
Aliquots of the sample – coumarin (0.1–0.5 mM), luminol
(0.5 mM), ascorbic acid (Asc, 0.1–0.5 mM) or DMSO (0.1% v/v) –
were added to PBS pH 7.4 previously bubbled with oxygen-free
argon for 10 min. Cyclic voltammograms were recorded by a
potentiostat (model MQI 12/8PCC, Microquímica, Brazil) at a
sweep rate of 100 mV s1, at 25 °C. Conventional electrochemical
cells were employed; a disk glassy carbon electrode (diame-
ter = 2 mm), a platinum wire, and sodium chloride saturated sil-
ver/silver chloride [Ag/AgCl/NaCl(sat)] were used as the working,
counter, and reference electrodes, respectively. The argon gas flow
was kept near the solution surface when registering the cyclic
voltammogram.
2.16. Physicochemical properties of coumarins
The molecular volume of the tested compounds was calcu-
lated using the SYBYL Molecular Modeling System (version 6.7,
2001, Tripos Associates Inc., St. Louis, MO, USA). The theoretical
partition coefficient value (ClogP) was determined by the ClogP
for Windows (version 1.0.0, 1995, ByoByte Corp., USA). The
apparent partition coefficient (LogPapp) was obtained by the
shake-flask method, according to the procedure described by
Amaral et al. [37]. The partition system consisted of saturated
n-octanol and trizma buffer pH 7.4 with 0.1 M ionic strength, ad-
justed with NaCl. Room temperature was controlled and set to
25 ± 1 °C. The concentration of the tested compounds in the
aqueous phase was measured spectrophotometrically at 325 nm
(coumarins 1 and 2: e = 11,475 and 11,055 M1 cm1, respectively)
or 275 nm (coumarins 3 and 4: e = 10,039 and 9335 M1 cm1,
respectively).
2.17. Data analysis
Experimental data were processed and analyzed using the
GraphPad Prism Software (version 3.00 for Windows, GraphPad
Software Inc., San Diego, CA, USA). Statistical analysis was per-
formed by Analysis of Variance (ANOVA) followed by the Tukey’s
test. A value of p < 0.05 was considered significant.
3. Results
3.1. Modulation of the ROS generation by neutrophils
We evaluated the modulator effect of the four coumarins (1–4)
on the production of O2 and total ROS by SOZ- or PMA-stimulated
human neutrophils using the lucigenin-(CL-luc) and luminol-
(CL-lum) enhanced chemiluminescence assays, respectively.
All the tested coumarins inhibited the PMA- and SOZ-stimu-
lated neutrophil CL-luc in a concentration-dependent manner
(Fig. 2A–D). They inhibited the SOZ-stimulated CL-luc twice as
effectively as they inhibited the PMA-stimulated CL-luc. The
hydroxylated coumarins 1 and 2 were more effective to inhibit
both the PMA- and SOZ-stimulated CL-luc than their acetylated
counterparts 3 and 4.
In contrast, the four coumarins increased the PMA- and SOZ-
stimulated CL-lum of neutrophils (Fig. 2F–K). The hydroxylated
coumarins 1 and 2 had similar enhancing effects, which were sig-
nificantly higher than the effect of their acetylated analogs (Fig. 2F
and I). The percentage enhancement of the PMA-stimulated
CL-lum of neutrophils and the peak height of the CL-lum response
increased as a function of the concentration of the coumarin (Fig. 2I
and K). On the other hand, coumarins 1 and 2 had a biphasic effect
on the SOZ-stimulated CL-lum: the CL-lum response increased at
concentrations up 10 lM but decreased at higher concentrations
(Fig. 2F). The kinetics of CL-lum produced by the SOZ-stimulated
neutrophils changed significantly: the peak height decreased, and
the time necessary to reach the maximum CL-lum response was
longer in the presence of increasing concentrations of the couma-
rins (Fig. 2H).
ABAH, a specific MPO inhibitor, suppressed the CL-lum of neu-
trophils in a concentration-dependent way; the maximum inhibi-
tory effect lay around 60% at 50 lM. ABAH did not inhibit the
CL-luc of neutrophils, indicating its specificity for MPO (Fig. 2E).
This compound mitigated the effect of coumarins on the
PMA- and SOZ-stimulated CL-lum of neutrophils (Fig. 2G and J).
Therefore, the fact that coumarins enhanced CL-lum depended on
the activity of MPO.
3.2. Oxidation of coumarins by MPO
We used cell-free systems to evaluate the oxidation of couma-
rins by MPO-H2O2. The hydroxylated coumarins (1, 2) increased
the oxidation of luminol in a concentration-dependent manner,
but their acetylated counterparts (3, 4) did not interfere signifi-
cantly in this reaction (Fig. 3A). In the chloride-free reaction med-
ium, the prooxidant effect of coumarins 1 and 2 was fourfold
higher than the effect observed in the presence of chloride
(Fig. 3B). However, in the absence of luminol, the hydroxylated
compounds were oxidized a little faster in the reaction medium
containing chloride (Fig. 3D).
 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75 67

Fig. 2. Modulation of the SOZ- and PMA-stimulated oxidative metabolism of human neutrophils by 7-hydroxycoumarin derivatives. (A–D): Concentration-dependent
2 by neutrophils stimulated with SOZ (A, B) and PMA (C, D), assessed by the lucigenin-amplified chemiluminescence (CL-luc) assay [(A) and
inhibition of the generation of O
(C): effect of the four coumarins; (B) and (D): time-course of the CL-luc reaction in the presence of coumarin 1]. (E): Concentration-dependent inhibition of the PMA-
stimulated neutrophil CL-lum and CL-luc by ABAH. (F–K): Concentration-dependent increase of the generation of total ROS by neutrophils stimulated with SOZ (F–H) and
PMA (I–K), assessed by the luminol-enhanced chemiluminescence (CL-lum) assay [(F) and (I): effect of the four coumarins; (G, H, J and K): CL-lum produced by the
neutrophils treated with coumarins 1 and 2 (50 lM) in combination with ABAH (50 lM); (G) and (J): integrated CL-lum area; (H) and (K): time-course of the CL-lum reaction].
Results are expressed as mean ± standard deviation of at least four independent experiments, assayed in duplicate. Statistics: values not sharing the same letter (a–f) are
significantly different from each other (p < 0.05; ANOVA and Tukey’s post-hoc test).

The absorption spectra of coumarins 1 and 2 changed in the
presence of MPO-H2O2: the intensity of the characteristic absorp-
tion of coumarins at 324 nm decreased (Fig. 3E) in the first 5 min
of reaction (Fig. 3D), whereas the typical absorption band of the
catalytic intermediates of MPO, compounds I and II, appeared in
the range of 410–430 nm. In addition, the spectra of these
hydroxylated coumarins did not change in the absence of either
MPO or H2O2, suggesting that MPO metabolized these compounds
after this enzyme was activated by H2O2. The absorption spectra of
coumarins 3 or 4 did not change significantly (Fig. 3C); therefore,
MPO-H2O2 did not oxidize these two coumarins under the assessed
experimental conditions.
68 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75

Fig. 3. Oxidation of the 7-hydroxycoumarin derivatives by myeloperoxidase (MPO). (A) Concentration-dependent enhancement of the MPO-H2O2-luminol (0.5 U mL1/
50 lM/20 lM) CL by the four coumarins, assayed in a reaction medium with 100 mM Cl-. (B) MPO-H2O2-luminol CL enhancement by coumarin 1, assayed in the presence of
100 mM Cl- or not. (C) Typical time-course of oxidation of the coumarins tested by MPO-H2O2; the absorption changes were recorded at 324 nm (coumarins 1 and 2) or
310 nm (coumarins 3 and 4) up to 20 min. (D) Oxidation of coumarin 1 (50 lM) by MPO-H2O2 (0.5 U mL1/50 lM) assayed in the presence of 100 mM Cl- or not; the
absorption changes were recorded at 324 nm. (E) Superimposed absorption spectra recorded during oxidation of coumarin 1 (50 lM) by MPO-H2O2 (0.5 U mL1/50 lM); the
dotted line is the spectra recorded before addition of MPO-H2O2. Data are expressed as mean ± standard deviation of at least three independent experiments, assayed in
duplicate.

In the cell-free systems, ABAH completely inhibited the oxida-
tion of coumarin by MPO in PBS and in the chloride-free buffer,
both in the presence and absence of luminol (data not shown).
3.3. DPPH and HOCl scavenging effect
DPPH displayed reduced ability to inhibit the four coumarins
(2.5–5.5%), and inhibition was not significantly different from the
control (3.68 ± 0.89%, at the highest concentration tested–
100 lM). In contrast, coumarins 1–4 effectively scavenged HOCl:
their IC50 values were 4.15 ± 0.65, 4.34 ± 0.45, 7.05 ± 0.18, and
11.56 ± 1.29 lM, respectively. The hydroxylated coumarins scav-
enged HOCl at least twice as effectively as their acetylated
counterparts.
3.4. Interaction between coumarins and ascorbic acid
A reaction mixture of Asc, HRP, H2O2 and one of the hydroxyl-
ated coumarins (1 or 2) gave a strong two-line EPR signal that
was characteristic of the ascorbyl free radical (Fig. 4A.d and e).
The hyperfine coupling constant aH = 1.79 G of this signal agreed
with the values reported in the literature [38]. The EPR signal in-
creased with rising concentration of coumarins 1 and 2 up to
90 lM, but decreased at higher concentrations (Fig. 4A.g); the
intensity of this signal decreased along the reaction (data not
shown). Omission of HRP, H2O2, or the coumarins from the reaction
mixture produced a low EPR signal relative to ascorbyl radical
(Fig. 4A.a). HRP-H2O2 did not oxidize Asc at significant levels
(Fig. 4A.b) nor did the acetoxylated coumarins (3 or 4) or DMSO
modify the EPR signal detected in this reaction significantly
(Fig. 4A.c,e and f).
The EPR signal of the ascorbyl radical and the absorption spec-
tra of the coumarins and Asc did not change significantly when
these compounds were mixed in the absence of a peroxidase (data
not shown), indicating that the tested coumarins did not oxidize
Asc directly. The H2O2 did not oxidize the coumarins or Asc di-
rectly under the assessed conditions.
Cyclic voltammetry revealed that Asc oxidized at
+0.208 ± 0.019 V. Addition of equimolar concentrations of Asc
and coumarin 1 or 2 to the buffer delayed the oxidation peak of
 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75 69

Fig. 4. Analysis of the interaction between ascorbic acid (Asc) and the tested coumarins (1–4). Data show representative results of two independent experiments.
Instrumental parameters are described in the Section 2. (A) EPR spectra of the ascorbyl radical detected in the reaction mixture containing HRP (20 UI/ml), H2O2 (0.5 mM), Asc
(0.4 mM), and coumarins 1–4 (90 lM) [(a): Asc alone; (b): Control (Asc + H2O2 + HRP); (c–f): Reaction (b) with DMSO and coumarins 1–4 (90 lM); (g): Integrated areas of the
ascorbyl EPR signals detected in the reaction mixture containing HRP-H2O2-Asc and the coumarins 1–4 at different concentrations]. (B) Cyclic voltammogram of Asc(0.5 mM)
and coumarin 1 alone or in combination [(a): Asc; (b): coumarin 1; (c): Asc + coumarin 1]. (C) Total charge of the coumarin peak calculated from cyclic voltammograms of
coumarin 1 alone (0.1–0.5 mM) or combined with 0.5 mM Asc. (D) Total charge of the Asc peak calculated from cyclic voltammograms of Asc alone (0.1–0.5 mM) or 0.5 mM
Asc combined with coumarin 1 (0.1–0.5 mM). Cyclic voltammetry (B–D) was performed in 0.1 M phosphate buffer pH 7.4, with glassy carbon working electrode at
100 mV s1 scan rate.

Asc to approximately +0.300 V, but did not alter the oxidation po-
tential of coumarins 1 and 2: +0.699 ± 0.013 and +0.709 ± 0.008 V,
respectively (Fig. 4B). In the mixture, the total charge of coumarins
increased in the presence of Asc (Fig. 4C), whilst the total charge of
Asc decreased as a function of rising concentrations of the couma-
rins (Fig. 4D). Thus, these compounds act as antioxidants in this
cell-free system.
The anodic current peak height depended on the concentration
of coumarins added to the electrochemical cell (data not shown)
and its amplitude decreased after subsequent scanning. We did
not detect any significant oxidation or reduction peaks for com-
pounds 3 and 4, where the free hydroxyl group is acetylated (data
not shown). Luminol showed two oxidation peaks, at
+0.485 ± 0.011 and +0.618 ± 0.015 V.
3.5. Physicochemical properties of the coumarins
The physicochemical properties of coumarins 1–4 did not vary
significantly. Their experimental partition coefficients were
1.46 ± 0.02, 1.79 ± 0.01, 1.07 ± 0.01, and 1.37 ± 0.02, respectively,
whereas the calculated partition coefficients were 1.61, 2.11,
1.17, and 1.67, respectively. The molecular volumes of coumarins
1–4 were 119.7, 134.2, 151.5, and 167.4 Å3, respectively.
3.6. EPR spin trapping
We used DMPO and 4-POBN as spin traps to detect the couma-
rin free radical intermediates produced by MPO. The peroxidase-
catalyzed oxidation of the hydroxylated coumarins (1 or 2) in the
presence of DMPO gave the typical nine-line spectrum of 5,5-di-
methyl-1-pyrrolidone-2-oxyl-1 (DMPOX), whose characteristic
hyperfine coupling constants aN = 7.2 G and aH = 4.1 G, appeared
at g = 2.0085, agreeing with literature values [39–41] and the cor-
responding simulated spectra (Fig. 5A). The acetoxylated couma-
rins (3 or 4) did not produce a significant EPR signal in the
presence of HRP-H2O2 and DMPO under the assessed conditions.
In the absence of coumarins or HRP, neither the EPR signal nor
the absorption spectra (200–600 nm) modified significantly.
The intensity of the signal relative to DMPOX increased as a
function of the concentration of coumarin (1 and 2) from 10 to
120 lM, with the maximum intensity lying at 50 lM (Fig. 5A.i).
At this concentration, the EPR signal intensified with reaction time
until 5 min and decreased thereafter, suggesting that DMPOX
decomposed (Fig. 5A.j). Addition of superoxide dismutase to the
reaction mixture had negligible effect on the intensity of the EPR
signal (Fig. 5A.g), indicating that O
2 did not participate in the reac-
tions that produced DMPOX. Furthermore, oxidation of coumarin 1
by HRP-H2O2 did not generate significant amounts of O 2 , as as-
70 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75

Fig. 5. EPR spectra obtained from the oxidation of coumarins by HRP-H2O2 (20 U mL1/0.5 mM) using DMPO and 4-POBN as spin traps. Representative results of two
independent experiments. Instrumental parameters are described in the Section 2. (A) EPR spectra obtained with DMPO (80 mM) as spin trap after 5 min of reaction [(a):
Control (DMPO + H2O2 + HRP); (b–f): Reaction (a) with DMSO (0.8% v/v) and coumarins 1–4 (90 lM); (g): DMPO + H2O2 + HRP + coumarin 1 (90 lM) + superoxide dismutase
(SOD; 5000 U mL-1); (h): Computer simulation of the spectrum of DMPOX, where aN = 7.2 G, aH = 4.1 G, g = 2.0085, line width = 0.3 G; (i): Concentration-dependent increase
in the EPR signal area of DMPOX produced by DMPO + H2O2 + HRP + coumarin 1; (h): Time-course of the EPR signal area of DMPOX observed in the
DMPO + H2O2 + HRP + coumarin 1 (90 lM) reaction (c)]. (B) EPR spectra obtained with 4-POBN (100 mM) as spin trap after 15 min of reaction [(a): 4-POBN alone; (b) 4-
POBN + H2O2; (c): Control (4-POBN + H2O2 + HRP); (d–h): Reaction (c) with DMSO and coumarins 1–4 (90 lM); (i): Computer simulation of the spectrum of the 4-POBN/4-
POBN adduct, where aN = 14.9 G, a1H ¼ 1:8 G, and a2H ¼ 1:9 G; (j): Computer simulation of the spectrum of the POBN/coumarin adduct, where aN = 14.9 G, aH = 1.8 G; (k):
Time-course of EPR signal area observed in the 4-POBN + H2O2 + HRP + coumarin 1 (90 lM) reaction (e)].

sessed by the cell-free lucigenin-enhanced chemiluminescence
(data not shown).
The oxidation of coumarins 1 or 2 by HRP-H2O2 in the presence
of 4-POBN as spin trap generated a characteristic twelve-line EPR
spectrum (Fig. 5B.e,f). The signal intensified along time, reaching
a maximum after 15 min (Fig. 5B.k). The corresponding hyperfine
coupling constants were a1N ¼ 14:90 G, a2N ¼ 1:85 G and
a1H ¼ 1:80 G. On the other hand, the HRP-H2O2-4-POBN reaction
produced a very weak EPR signal that did not change after addition
of DMSO or the coumarins to the medium, up to 20 min (Fig. 5B.
a–d and g–h). This signal can be attributed to the adduct of 4-POBN
with its own radical 4-POBN, as previously described by McCor-
mick et al. [42]. Computer simulations of the spectrum of such ad-
duct using the determined parameters fit satisfactorily with the
experimental data (Fig. 5B.i).
Coumarin radicals can also originate during the reaction, giving
rise to analogous POBN/coumarin adducts. However, these ad-
ducts would have a different pattern, showing a six-line spectrum,
considering that the coupling constants aN = 14.9 G and aH = 1.8 G
are similar (Fig. 5B.j). These results suggest that the presence of
 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
the hydroxylated coumarins increased the oxidation of 4-POBN by
HRP-H2O2.
3.7. Inhibition of elastase activity, degranulation and microbial killing
We evaluated the ability of the four coumarins to inhibit the
elastase activity and suppress two important effector mechanisms
of neutrophils: degranulation and microbial killing.
None of the tested coumarins inhibited the elastase activity of
human neutrophils, at 100 lM, at least under the assessed condi-
tions (Fig. 6A). Longer incubation of elastase with the samples
(from 20 to 60 min) did not significantly contribute to inhibiting
Fig. 6. Modulation of the effector functions of human neutrophils by the 7-
hydroxycoumarin derivatives. (A) Inhibition of the elastase activity of neutrophils
after 20 and 60 min of treatment with the coumarins. AAPV: methoxy-succinyl-Ala-
Ala-Pro-Val chloromethylketone, specific inhibitor of elastase activity: (B) Inhibi-
tion of the degranulation of neutrophils after 20 and 60 min of treatment with the
coumarins. (C) Inhibitory effect of coumarin 1 on the Candida albicans killing by
neutrophils. HOCl was tested at 200 lM and ABAH (4-aminobenzoic acid hydra-
zide) at 50 lM. Data are expressed as mean ± standard deviation of at least three
independent experiments assayed in duplicate. Statistics: values not sharing the
same letter (a–e) are significantly different from each other (p < 0.05; ANOVA and
Tukey’s post-hoc test).
71
enzymatic activity: their inhibitory effect increased from
2.6 ± 2.5% to 5.7 ± 0.8%.
The hydroxylated coumarins inhibited the degranulation of
neutrophils three times more effectively than their acetylated ana-
logs (Fig. 6B). The inhibitory effect of coumarin 1 occurred at con-
centrations as low as 10 lM and was similar to the effect found at
100 lM. Cell treatment time did not increase this effect
significantly.
Coumarin 1 decreased the killing of C. albicans by neutrophils in
a concentration-dependent way (Fig. 6C). Untreated neutrophils
killed around 70% C. albicans added to the medium. At 10 lM,
the mean neutrophil killing ability decreased to 60%; at 100 lM,
this effector function of the neutrophil reduced to the same level
of MPO inhibition by ABAH (around 45%). In the absence of neutro-
phils, coumarin 1 and ABAH did not kill C. albicans, but HOCl at
200 lM killed 100% of the yeast added to the medium.
3.8. Cytotoxicity
Compared with the control, the four coumarins did not induce
significant LDH release or decrease the viability of neutrophils un-
der the assessed conditions (Table 1). After 60 min of treatment,
coumarin 1 did not induce apoptosis or necrosis; it did not reduce
cellular viability either. Thus, cytotoxicity does not underlie the
mechanism through which the tested coumarins inhibit the effec-
tor functions of neutrophils.
4. Discussion
We assessed whether 7-hydroxycoumarin (1) and its deriva-
tives (2–4) modulate the oxidative metabolism, elastase and mye-
loperoxidase activity, degranulation, and microbial killing ability of
human neutrophils. We also evaluated the physicochemical prop-
erties and antioxidant effect of the coumarins, as well as their po-
tential to scavenge HOCl in cell-free systems.
We evaluated the modulator effect of the tested coumarins on
the oxidative metabolism of SOZ- or PMA-stimulated human neu-
trophils by CL assays. We measured the production of O 2 and total
ROS by the lucigenin- and luminol-enhanced chemiluminescence
assays (CL-luc and CL-lum, respectively). SOZ is a model phagocytic
stimulus that binds to complement, IgG, and mannose membrane
receptors of neutrophils; it also triggers complex intracellular sig-
naling pathways [43]. Translocation and activation of protein ki-
nase C (PKC) is an important early event of the NADPH oxidase
assembly triggered by SOZ [43]. PMA is a soluble compound that
activates PKC directly [44].
All the tested coumarins inhibit the SOZ-stimulated CL-luc of
neutrophils more strongly than they inhibited the PMA-stimulated
CL-luc of neutrophils. Thus, inhibition of the activity of PKC by
these compounds mediates, in part, the decreased production of
2 by the neutrophils. The O2 scavenging by the hydroxylated
O 
coumarins [4,16] can also contribute to inhibiting CL-luc. Couma-
2 generation by human, rat, and rabbit
rins 1 and 2 inhibit the O
neutrophils [5,23] but not by murine RAW 264.7 macrophage cells
[45].
On the other hand, the four coumarins enhance the PMA- and
SOZ-stimulated CL-lum of neutrophils. They exert a concentra-
tion-dependent effect on PMA-stimulated cells, but influence the
SOZ-stimulated cells in a different way: as the concentration of
the hydroxylated coumarins increases, the time to reach the max-
imum CL-lum response and the peak height decrease. As a result,
the percentage of SOZ-stimulated CL-lum increases up 10 lM
and diminishes at higher concentrations of these coumarins. Stim-
ulation of the neutrophils by SOZ releases much more MPO and
less H2O2 than PMA [46]; hence, the amount of MPO released by
72 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
Table 1
Viability of neutrophils after treatment with 7-hydroxycoumarin derivatives.
Compounda 20 min
b c
Viable cells (%) LDH release (%)
HBSS 98.00 ± 1.73 4.61 ± 1.62
DMSO 97.20 ± 0.84 4.19 ± 1.26
1 (10 lM) n.d. n.d.
1 (100 lM) n.d. n.d.
1 (200 lM) 98.13 ± 1.13 5.52 ± 1.73
2 (200 lM) 97.88 ± 0.99 5.47 ± 1.24
3 (200 lM) 98.50 ± 0.84 4.78 ± 3.46
4 (200 lM) 97.50 ± 1.51 4.21 ± 2.01
n.d.: Not determined.
a
HBSS: Hank’s Balanced Salt Solution (untreated cells); DMSO: dimethyl sulfoxide (0.1% v/v; vehicle control).
b
Determined by the Trypan Blue exclusion test by counting a total of 200 cells for each sample.
c
LDH release by the samples was compared with neutrophils completely lysed by Triton X-100.
d
Determined by flow cytometry analysis of 10,000 events.
the stimuli seems to influence the pattern of prooxidant effect of
the coumarins.
The CL-lum produced by the PMA- and SOZ-stimulated neutro-
phils depends on the fusion of different types of intracellular
granules, which in turn is guided by the PKC and phosphatidylino-
sitol-3-kinase (PI3-K) signaling [43,44,47]. Together, our results
suggest that the tested coumarins interfere in these pathways.
The fact that the coumarins significantly inhibit the degranulation
of neutrophils at 10 lM supports our findings. These mechanisms
reduced the CL-lum produced by SOZ-stimulated neutrophils
occurring at coumarin concentrations higher than 10 lM. Phagocy-
tosis of SOZ particles by neutrophils mobilizes the intracellular
granules more efficiently than PMA; thus, the interference of cou-
marins in this process affects more strongly the SOZ- than the
PMA-stimulated effector functions of neutrophils. 7-Hydroxy-
coumarin decreases the degranulation of mast cells [15] and inter-
feres in early signaling mechanisms of J774 macrophages [21].
Some monohydroxycoumarins inhibit the phosphorylation of
Akt/PKB in mouse RAW 264.7 macrophages [4].
Acetylation of the free hydroxyl group of coumarins 1 and 2 re-
duce their ability to modulate the PMA- and SOZ-stimulated neu-
trophil CL-luc and CL-lum, as well as their capacity to inhibit the
degranulation of neutrophils. This structural modification remark-
ably decreases the prooxidant effect on the PMA-stimulated CL-
lum: the maximum CL-lum response of the acetylated compounds
at 100 lM is around 80% lower than that of the hydroxylated ana-
logs. In addition, the acetylated coumarins are twofold more effec-
tive to enhance the SOZ- than the PMA-stimulated CL-lum of
neutrophils. Together, these results suggest that the hydroxylated
coumarin is the pharmacologically active form, and the rate of
hydrolysis of the acetylated drugs by intracellular esterases is
higher in SOZ- than in PMA-stimulated neutrophils.
The stimulation of neutrophils with PMA and SOZ triggers the
generation of ROS in intracellular compartments much more effec-
tively than in the extracellular milieu [47,48]. Therefore, the con-
centrations of the four coumarins in the intracellular
environment are sufficient for them to exert their pharmacological
effects. Acetylation of the free hydroxyl does not seem to affect the
intracellular bioavailability of the tested coumarins, because they
have similar partition coefficient and molecular volume. We previ-
ously reported that this structural modification can increase or de-
crease the inhibitory effect of coumarin derivatives on the
neutrophil oxidative metabolism, depending on the number and
position of the substituents [23,31,49]. In addition, esterification
of hydroxyl groups has improved the pharmacological properties
of catechin and quercetin derivatives in cell-mediated oxidative
and inflammatory processes by favoring their interaction with
intracellular targets [50,51].
60 min
Viable cells (%)b LDH release (%)c Annexin-V+ cells (%)d IP+ cells (%)d
77.10 ± 8.68 13.03 ± 4.33 0.13 ± 0.05 10.30 ± 2.45
76.03 ± 4.79 14.62 ± 2.04 0.13 ± 0.05 9.89 ± 3.56
78.15 ± 3.15 15.98 ± 3.67 0.13 ± 0.05 10.21 ± 2.24
75.90 ± 5.51 13.70 ± 1.80 0.12 ± 0.04 8.47 ± 3.14
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
n.d. n.d. n.d. n.d.
Addition of the 4-methyl group to 7-hydroxycoumarin does not
change its physicochemical properties – redox potential, partition
coefficient, and molecular volume – or its ability to modulate the
effector functions of neutrophils investigated here significantly.
Recently, Witaicenis et al. [52] reported that 4-methyl-esculetin
suppressed the oxidative damage and inflammation in the intes-
tine of rats with acute colitis more effectively than esculetin
(6,7-dihydroxycoumarin). Liver P-450 monoxygenases do not
metabolize the 4-methylcoumarins to toxic 3,4-epoxide intermedi-
ates, like many other coumarins [6,53]. This suggests that using
7-hydroxy-4-methylcoumarin instead of 7-hydroxycoumarin can
be advantageous.
The reason for the increase in CL-lum can be the reaction be-
tween the hydroxylated coumarins with O 2 , yielding a free radical
product that oxidizes luminol. The other hypothesis is that MPO
oxidizes the coumarins, which we investigated in the present
study. ABAH, a specific inhibitor of MPO, suppresses up to 60% of
the PMA- and SOZ-stimulated CL-lum of neutrophils. This com-
pound significantly reduces the prooxidant effect of the coumarins,
indicating that MPO oxidizes the coumarins to free radical prod-
ucts which, in turn, oxidize luminol and increase the CL-lum. ABAH
completely inhibits the oxidative and peroxidative activities of
MPO in cell-free systems, but suppresses only 80% of the HOCl gen-
eration by human neutrophils, without interfering in the processes
of degranulation and O 2 generation in these cells [46]. Moreover,
intracellular CL-lum originates via other reactions, such as luminol
oxidation by O 2 , HOCl, and HO [54].

In cell-free systems, MPO oxidizes the hydroxylated coumarins,
but not their acetylated counterparts. Thus, the free hydroxyl
group is essential to the prooxidant effect of these compounds.
The chloride ion probably competes with the coumarin and lumi-
nol in the active site of MPO, because the presence of this ion in
the assay buffer suppresses the CL-lum elicited by the coumarin.
In the experimental system without luminol, MPO-H2O2 oxidizes
the coumarin more efficiently in the presence of Cl-. The HOCl
formed in this reaction also contributes to the oxidation of couma-
rin, because the four tested coumarins (hydroxylated and acety-
lated) are efficient HOCl scavengers. The HOCl scavenging ability
of acetylated compounds was reported previously [4,31,55].
The antioxidant activity of coumarins depends on the type of
cell-free assay system. Hoult and Payá [5] reported that coumarins
2 , HOCl, alkylperoxyl radicals,
1 and 2 do not efficiently scavenge O
or inhibit lipid peroxidation. On the other hand, Kanimozhi et al.
[16] reported that coumarin 1 effectively scavenge O 2 , HO , DPPH,

and ABTS+ (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid
radical cation) radicals. Our experiments showed that compound
1 does not reduce DPPH significantly, in agreement with Lin
et al. [56]. Detecting this effect requires a longer reaction time
 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
[16], due to the low reactivity of this coumarin. In addition, this
compound protects Asc from electrochemical oxidation but in-
creases the oxidation of Asc in the presence of a peroxidase. There-
fore, the metabolization of the hydroxylated coumarins to
oxidizing species by peroxidases mediates the prooxidant effect
of these coumarins toward Asc. Glutathione and Asc are the two
major water-soluble intracellular antioxidants that act as redox
buffers by interacting with ROS, which is an important mechanism
of their protective function against oxidative stress [57,58].
Mammalian and plant peroxidases have been reported to
produce ROS and aromatic oxyl radicals from several phenolic sub-
strates as part of their defense mechanism against pathogens [59].
These enzymes also oxidize phenolphthalein, quercetin and other
phenolic compounds of plants [60–62]. Therefore, the presence of
hydroxyl groups makes the coumarins 1 and 2 likely candidates
as substrates of endogenous peroxidases, such as MPO, lactoperox-
idase, and eosinophil peroxidase. Miller et al. [63] reported that
HRP oxidizes 6-methoxy-7-hydroxycoumarin to transient free rad-
ical intermediates.
Resonance can stabilize the free radical intermediates, or these
radicals catalyze oxidative chain reactions. We found that the oxi-
dation of coumarin 1 by MPO generates highly reactive free radi-
cals that DMPO and 4-POBN may not be able to trap. This may
happen because these free radicals oxidize the traps and prevent
the formation of the corresponding coumarin-spin trap adduct.
These radicals also oxidize Asc and luminol, so MPO oxidizes cou-
marin 1 faster than it oxidizes the other co-substrates added to the
reaction medium, even if the concentration of this coumarin is
much lower than those of the other compounds. Interestingly,
the redox potential of Asc is lower than that of coumarin 1, which
could favor its oxidation by MPO or HRP; however, Asc is poorly
oxidized by HRP in the absence of the coumarin.
Results obtained from EPR spin trapping experiments indicated
that HRP-H2O2 predominantly forms the 4-POBN/4-POBN adduct
in the presence of hydroxylated coumarins. Other studies [42] have
also observed this species, with the following spectroscopic param-
eters: aH = 1.8 G, a1N ¼ 19:9 G, and a2N ¼ 1:85 G. Under our experi-
mental conditions, we did not find that HRP-H2O2 significantly
oxidized 4-POBN, suggesting that the presence of hydroxylated
coumarins is necessary to produce significant amounts of the

4-POBN radical. HRP-H2O2 likely oxidizes the hydroxylated
coumarins, and the resulting coumarin radical reduces 4-POBN to
the 4-POBN radical, regenerating the parent phenolic compound
and establishing a redox cycling. A recycling mechanism may
explain the intensification of the EPR signal relative to the
4-POBN/4-POBN adduct, promoted by concentrations of coumarin
1,000 times lower than the concentration of 4-POBN. Furthermore,
the oxidation of DMPO to DMPOX seems to involve an analogous
mechanism, because no EPR signal corresponding to the oxidized
spin trap appears in the absence of the hydroxylated coumarins.
Therefore, since the oxidation of the spin trap DMPO only occurs
in very strong oxidizing conditions, the peroxidase system
efficiently oxidizes the hydroxylated coumarins, yielding strong
oxidants [64].
We also investigated how the pro- and anti-oxidant potential of
7-hydroxycoumarin (1) impacts the ability of the human neutro-
phil to kill microbes. We found that this coumarin impairs the kill-
ing of C. albicans, a phenomenon that is mediated by the decreased
degranulation capacity of these cells and the reduced availability of
O2 and other oxidizing species. Both the interference of this cou-
marin in the activity of MPO and its HOCl scavenging ability reduce
the availability of HOCl. Production of HOCl and other ROS is
important to kill Candida species, because some MPO-deficient
and chronic granulomatous disease patients suffer from frequent
or severe infections with this microorganism [3]. Addition of the
MPO inhibitor ABAH to the medium significantly decreases the
73
capacity of neutrophils to kill C. albicans. We did not observe this
negative effect of the coumarins in macrophages, which are cells
that contain low levels of MPO [22]. In fact, coumarin 1 improves
the in vitro and in vivo phagocytic and microbial killing ability of
murine monocytes and macrophages against Salmonella typhimuri-
um[22]. Compounds 1 and 2 do not kill C. albicans directly, and
they do not exhibit expressive antimicrobial activity against many
Gram-positive and Gram-negative bacteria [65,66].
In addition, inhibition of the effector functions of neutrophils is
not mediated by cytotoxicity of the four coumarins, at least under
the assessed conditions. In the same range of concentration used in
this study, coumarin 1 is not toxic to J774 macrophages or to hu-
man and rabbit neutrophils [5,21,23].
The prooxidant effect of hydroxycoumarins, at low levels, in-
duces the apoptosis of U-937 cancer cells but not of normal cells
[67]. The intracellular oxidative stress, at sub-lethal levels, can in-
duce the synthesis of protective enzymes, such as glutathione syn-
thetase and AKR enzymes [17]. On the other hand, the antioxidant
effect of these coumarins protects the host against oxidative injury.
7-Hydroxycoumarin scavenges the O 2 , hydroxyl, and alkylperoxyl
radicals [4,5,16], inhibits the production of NO by different macro-
phage cell lines [19–21], and diminishes the secretion of O 2 and
H2O2 by macrophages obtained from healthy and Salmonella
typhimurium-infected mice [22]. This coumarin improves the anti-
oxidant status of diabetic rats by enhancing both nonenzymic and
enzymic antioxidants [11,18], and protects human lymphocytes
from radiation-induced generation of ROS [16] and human astrocy-
toma 1321N1 cells against H2O2 and aldehyde toxicity [17]. It also
protects mice from neurotoxicity caused by exposure to 1-methyl-
4-phenyl-1,2,3,6-tetrahydropyridine, a compound that leads to the
production of ROS [68]. Moreover, the production of adequate
amounts of ROS is important to regulate the course of acute and
chronic inflammatory responses and autoimmune inflammation
[3]. Hence, compounds that promote the formation of ROS can be
useful to treat rheumatoid arthritis and other inflammatory dis-
eases [1,69].
Most of the biological effects of the coumarin derivatives re-
ported herein take place in a range of concentrations considered
relevant for therapeutic purposes (1–10 lM) [53]. In physiological
buffer, the hydroxylated coumarin derivatives display significant
MPO-mediated prooxidant effect at concentrations as low as
1 lM. Recommended doses of coumarin range from 8 mg/day for
the treatment of venous constriction to 7 g/day in antineoplastic
therapies [7,8]. After oral administration, the absorption rate of
coumarin from the gastrointestinal tract can reach up to 83%,
and 60–75% of the dose is converted to the glucuronide of
7-hydroxycoumarin [8]. It is not clear whether these glucuronide
conjugates are pharmacologically active. However, mammalian
b-glucuronidases released from neutrophils at inflammatory
sites may deconjugate 4-methylumbelliferyl-b-D-glucuronide and
many flavonoid glucuronides and then deliver the free parent
drugs [70]. Therefore, it is possible that the 7-hydroxycoumarin
derivatives achieve therapeutic levels at inflammatory sites, but
further studies are needed to establish the best dosage form and
administration route.
In summary, the coumarins investigated here modulate the oxi-
dative metabolism and degranulation but not the elastase activity
of human neutrophils. These compounds display antioxidant ef-
fects that prevent neutrophils from generating O 2 , thereby scav-
enging HOCl and protecting Asc from oxidation. On the other
hand, the hydroxylated compounds have prooxidant effects after
MPO-catalyzed reactions convert them to oxidizing species. These
species are highly reactive and oxidize Asc, the chemiluminescent
probe luminol, and the spin traps 4-POBN and DMPO. Considering
that the inflammatory site is a significant source of neutrophil
MPO, formation of coumarin-derived reactive products is probably
74 L.M. Kabeya et al. / Chemico-Biological Interactions 206 (2013) 63–75
part of the overall anti-inflammatory effect of 7-hydroxycoumarin
(1). Therefore, it is possible that both the anti- and prooxidant ef-
fects of these compounds culminate in beneficial pharmacological
effects in vivo and contribute to suppressing the inflammatory re-
sponse. 7-Hydroxycoumarin displays anti-inflammatory effect in
the murine model of allergic airway inflammation [10] and paw
edema [4,13–15]. However, the impaired C. albicans killing ability
of neutrophils is an important side-effect of 7-hydroxycoumarin
that needs to be considered during further research. In addition,
the low cost and low in vitro and in vivo toxicity of these coumarins
make them promising immunomodulating and anti-inflammatory
drugs.
5. Conclusion
7-Hydroxycoumarin (1), 7-hydroxy-4-methylcoumarin (2), and
their acetylated analogs (3 and 4, respectively) modulate the effec-
tor functions of human neutrophils studied: oxidative metabolism,
degranulation and microbial killing. They act through different
mechanisms, by interfering in intracellular signaling pathways,
scavenging ROS, interacting with MPO and generating oxidizing
species. In general, the pro- and antioxidant effect of 7-hydroxy-
coumarin in cellular and cell-free systems, as well as its ability to
modulate the effector functions of neutrophils here investigated,
are not significantly affected by adding the 4-methyl group but
are impaired by acetylating the free hydroxyl group. The balance
between the anti- and prooxidant properties of these compounds
is essential to achieve beneficial pharmacological effects without
suppressing the microbial killing ability of neutrophils. Thus, it is
relevant to understand how these coumarins modulate the effector
functions of neutrophils, because these cells play important roles
in the regulation of inflammatory processes.
Acknowledgements
The authors thank Mr. Alcides S. Pereira and Mrs. Nadir
Mazzucato for technical assistance, and Prof. Dr. Gilberto U.L.
Braga (FCFRP-USP, Brazil) for providing the C. albicans strain
used in the microbial killing assay. This study was supported
by the Brazilian agencies: Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP, Grants 2000/06233-7, 2000/
10641-3, 2002/09518-8, 2005/60596-8, 2007/02487-3, 2007/
00840-8), Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq, grant 150302/2007-0), Instituto
do Milênio Inovação e Desenvolvimento de Novos Fármacos e
Medicamentos (IM-INOFAR), Instituto do Milênio Redoxoma,
Instituto Nacional de Ciência e Tecnologia (INCT) de Processos
Redox em Biomedicina, INCT de Inovação Farmacêutica and INCT
de Bioanalítica.
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