Exp Toxic Pathol 2003; 54: 313318

URBAN & FISCHER

http://www.urbanfischer.de/journals/exptoxpath

Laboratory of Applied Biochemistry, Department of Biology, Faculty of Sciences, University of Ferhat ABBAS, 19000
Setif, Algeria

Anti-inflammatory effect of rutin on rat paw oedema,

and on neutrophils chemotaxis and degranulation
LAID SELLOUM, HAMAMA BOURICHE, CHAFIA TIGRINE, and CHAHRA BOUDOUKHA
With 6 figures
Received: July 19, 2002; Revised: September 16, 2002; Accepted: October 16, 2002
Address for correspondence: Dr. LAID SELLOUM, Laboratory of Applied Biochemistry, Department of Biology, Faculty
of Sciences, University of Ferhat ABBAS, 19000 Setif, Algeria; Tel./Fax: (+213) 36 92 51 22; e-mail: laidsel@yahoo.com
Key words: Rutin; inflammation; rat paw oedema; neutrophils; chemotaxis; degranulation.

Summary

Introduction

Background: Rutin, a natural flavone derivative, is

known for its pharmacological properties. We have previously reported that this flavonol exerted a potent inhibitory
effect on respiratory burst of fMet-Leu-Phe-stimulated
neutrophils, as well as on phosphoinositide 3-kinase activity in a cell free system. In the present study, the anti-inflammatory effect of rutin was investigated in vivo and in
vitro. Methods: rutin or aspirin (100 mg/kg, body weight)
were given orally to rats 1 hour before paw oedema induction, using -carrageenan 1%. The rat paw volume was
measured by mean of plethysmometer, initially and during
6 hours. The chemotaxis of neutrophils towards 107 M
fMet-Leu-Phe was performed using 48-well chemotaxis
chamber. Neutrophils that migrated through 5 m pore size
polycarbonate filter, in presence or in absence of rutin,
were counted microscopically. Elastase exocytosis of either
phorbol 12-myristate 13-acetate or fMet-Leu-Phe/cytochalasin B-stimulated neutrophils was assessed in absence or
in presence of rutin using the synthetic substrate N-SucAla-Ala-Ala-p-nitroanilide. The absorbance of released pnitroaniline was measured at 405 nm using microplate
reader. Results: The maximal swelling in placebo group
was observed at 5 hours, after -carrageenan injection.
Oral administration of rutin reduced rat paw swelling starting 2 hours after -carrageenan injection. Rutin reduced
significantly (p < 0.05) and in a dose-dependant manner the
polymorphonuclear neutrophils chemotaxis to fMet-LeuPhe. Furthermore, elastase exocytosis, induced by both stimuli, was partially inhibited by rutin up to 25 M. Conclusion: The present study revealed that rutin possesses antiinflammatory properties.

Flavonoids are a large class of polyphenolic compounds widely distributed in the plant kingdom. Their
basic nucleus contains 15 carbons arranged in a C6-C3-C6
configuration (HARBORNE 1977). These natural compounds have gained increasing interest as potential therapeutic agents. Years of research have now clearly affirmed the ability of flavonoids to exhibit some pharmacological activities including beneficial effects on inflammation, cancer and cardiovascular diseases (KIM et
al. 1998; STRUCKMANN 1999; ZHAO et al. 2000; MIDDLETON et al. 2000). Interestingly, putative therapeutic effects of many traditional herbal medicine may be ascribed to the presence of flavonoids. The anti-inflammatory effect of flavonoids is assumed to result mainly from
the inhibition of some key enzymes involved in inflammation and/or cell signalling pathways such as cyclooxygenase and lipoxygenase (YOU et al. 1999), protein kinase C (PKC) and phosphoinositide 3-kinase (PI 3-kinase) (VLAHOS 1995; GAMET-PAYRASTRE et al. 1999;
WALKER et al. 2000; SELLOUM et al. 2001). Thus, inhibition of these enzymes may prove beneficial for the treatment of inflammatory conditions. The inflammatory response consists in the increase of blood vessels permeability leading to the migration and activation of polymorphonuclear neutrophils (PMNs). The antimicrobial
function of PMNs is based on their phagocytic capacity
and ability to release proteolytic enzymes and reactive
oxygen species, which play an important role in tissue
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Fig. 1. Chemical structure of rutin.

damage during inflammation (WEISS 1989; SCHILLER

et al. 2000; HII et al. 2001).
Rutin, quercetin-3-O-rhamnosylglucoside, is a natural
flavone derivative (fig. 1). Some biological and pharmacological effects have been attributed to this flavonol
such as inhibition of human phospholipase A2 (PLA2)
from synovial fluid (LINDAHL and TAGESSON 1997). The
gastric ulcer protecting effect of rutin has been reported
by LA CASA et al. (2000). These authors suggested that
rutin exerts its gastroprotective effect through an antilipoperoxidant and by enhancement of the antioxidant
activity of glutathione peroxidase. Recently, we have reported that rutin, which has no scavenger effect on superoxide anion radicals, inhibited the respiratory burst of
fMet-Leu-Phe-stimulated neutrophils as assessed by
Pholasin enhanced luminescence. We have also observed
that this flavonol inhibited PI 3-kinase activity in a cell
free system (SELLOUM et al. 2001).
To study the therapeutic effectiveness of rutin, we investigate, in the present work, some anti-inflammatory
properties of this compound in vivo and in vitro. The
anti-inflammatory activity of rutin was evaluated using
-Carrageenan induced rat paw oedema, as a standard
model of acute inflammation. The effect of rutin on
fMet-Leu-Phe-activated human neutrophils chemotaxis
and degranulation was also investigated.

Material and methods

Chemicals: Rutin, -carrageenan, carboxymethyl cellulose (CMC), aspirin, formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), phorbol 12-myristate 13-acetate
(PMA), cytochalasin B (CB), ovalbumin, Histopaque1077, N-Suc-Ala-Ala-Ala-p-nitroanilide (N-Suc-(Ala)3-pNA) and Hanks balanced salt were obtained from Sigma
(Germany). All other reagents were of the highest grade
available from Sigma (Germany).
Animals: Male and female Albino Wistar rats, weighting between 150250 g, were obtained from Institute Pasteur, Algeria. Animals were acclimatised at least for one
week, under constant environment condition with free access to a standard diet and water.

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Exp Toxic Pathol 54 (2003) 4

Anti-inflammatory activity: To investigate the anti-inflammatory activity of rutin in vivo, -carrageenan as a

useful agent to induce rat paw oedema was used. Drugs,
rutin or aspirin, were suspended in CMC 1% (w/v) and
then, administered with a single dose of 100 mg/kg body
weight, one hour before the injection of 0.1 ml of -carrageenan 1% (w/v) in the subplantar region of the right
hind paw (WINTER et al. 1962). Rats of placebo or control
group were injected with -carrageenan 1% or sterile chloride saline, respectively. Both groups received orally only
the vehicle (CMC 1%). The oedema was assessed by measuring the injected paw initially and 1, 2, 3, 4, 5, and 6
hours after carrageenan injection, using a plethysmometer
(UGO Basile, Germany). Oedema volume was expressed
for each animal as the percentage change in rat paw volume
after carrageenan injection, compared with placebo group.
Human neutrophils isolation: Polymorphonuclear
(PMNs) were isolated from freshly heparinized (5 U/ml) venous blood from healthy volunteers, as previously reported
(SELLOUM et al. 2001). After dextran sedimentation at room
temperature, PMNs were obtained by centrifugation at 4 C
through Histopaque. Contaminating erythrocytes were removed by a briefly hypotonic lysis. Cells were washed three
times with Hanks balanced salt solution (HBSS) pH 7.4,
and kept on ice until use. Cell suspension contained
9598% of neutrophils. The viability of the cells was more
than 95%, as assessed by Trypan blue exclusion test.
Neutrophils chemotaxis: The chemotaxis assay was
performed using 48-well chemotaxis chamber (Neuro
probe, inc., USA) as described by FALK et al. (1980).
Briefly, 25 l of rutin at various concentrations, or 1.5 M
cytochalasin B, were added to the bottom wells of the
chamber in presence of fMet-Leu-Phe (107 M). Then, 45 l
of PMNs (1.5 106 cells/ml), suspended in HBSS, supplemented with 0.25% ovalbumin, were placed in the upper
wells of the chamber. Compartments were separated by a 5
m pore size polycarbonate filter (Neuro probe, inc.,
USA). The chemotaxis chamber was incubated for 90 min
at 37 C in a humidified atmosphere containing CO2.
Thereafter, the filter was removed, fixed in absolute
methanol and stained with Wrights stain. Positive control
(lower wells contained fMet-Leu-Phe 107 M) and negative
control (lower wells contained medium only) were conducted in parallel with experimental groups. PMNs, that
migrated through the filter, were counted microscopically
by 400 magnification in five random fields for each of
three replicate wells. A mean value was obtained for each
well. The average number of cell migration in the negative
control wells was subtracted as background from the number migrating in test wells, to yield the net number of neutrophils migrating per field. Chemotaxis in presence of test
compounds was expressed as a percentage of the maximal
chemotaxis to fMet-Leu-Phe in the same experiment. Each
experiment was repeated at least three times.
Neutrophils degranulation: Neutrophils (5 106
cells/ml) were equilibrated in HBSS for 10 min at 37 C.
Various concentrations of rutin (1, 5, 10 and 25 M), dissolved in DMSO 0.1% (v/v), were added for an additional
10 min. Appropriate stimulus, 107 M PMA or 106 M fMetLeu-Phe supplemented with 105 M cytochalasin B, was
then added and the incubation was continued for 30 min.

Supernatants were separated by centrifugation at 400 g for

10 min at 4 C. Elastase exocytosis was measured using the
synthetic substrate N-Suc-(Ala)3-p-NA (BIETH et al. 1974).
75 l of each supernatant were mixed, in a microtiter plate
well, with 4.5 g of substrate dissolved in 50 l of 0.1 M
HEPES buffer, pH 7.4 containing 0.5 M NaCl. The plate
was incubated at 37 C for 2 hours. All concentrations are
final ones. The absorbance of released p-nitroaniline was
measured at 405 nm with a microplate reader (Stat Fax
2100, USA), against the appropriate enzyme free reference.
Results were expressed as the percentage of released elastase compared with control without drug set to 100%.
The effect of rutin on elastase activity was determined in
a cell free system. Elastase was produced by incubating
PMNs (5 106 cells/ml) for 30 min at 37 C in presence of
fMet-Leu-Phe/CB. Then, the activity of the released elastase was determined in the cell free supernatant in presence
or absence of rutin.
Cytotoxic effect of rutin: Cytotoxic effect of rutin on
neutrophils was determined by the release of the cytoplasmic enzyme lactate dehydrogenase LDH (EC 1.1.1.27).
PMNs (2.5 106 cells/ml) were incubated with various
concentrations of rutin ranged between 50 and 500 M for
30 min at 37 C. The LDH activity was measured in the supernatant at 340 nm using 1.6 mM pyruvate and 0.2 mM
NADH. All concentrations are final ones. Enzyme release
was expressed as the percentage of the maximum value,
obtained after treatment of cells with 0.2% Triton-X100
(KOMURA et al. 1999).

Fig. 2. Time course of the paw oedema induced by subplantar injection of 0.1 ml of -carrageenan 1% in rat pre-treated
orally with vehicle (r, placebo), rutin (, 100 mg/kg body
weight) or aspirin (d, 100 mg/kg body weight). The control
group is injected with 0.1 ml of saline solution and pre-treated orally with the vehicle (h). Each point represents the percentage increase in volume of the injected paw compared
with the maximal volume of placebo group set to 100%.
Symbols are means SEM of 7 animals for each group.

Data analysis: Results are expressed as the mean SEM,

and were tested for significance using Students t-test.
Probability values (p) of less than 0.05 were taken to indicate statistical significance.

Results
Effect of rutin on rat paw oedema
Rat paw oedema, as a standard model of acute inflammation, was used for testing the anti-inflammatory activity of rutin. Subplantar injection of 0.1 ml of -carrageenan 1% induced a progressive swelling of the rat
paw, reaching a maximal volume in the placebo group at 5
hours. In the control group, rat Paws showed a slight
swelling, and recovered their initial volume after 1 hour.
Figure 2 showed that oral administration of 100 mg/kg of
rutin has no effect after 1 hour, and only a comparable effect was observed at 2 hours. Thereafter, rutin showed a
significant (p < 0.05) effect on rat paw oedema, compared
with the placebo group. Similar results were observed
with aspirin, used as a standard non-steroidal anti-inflammatory drug (NSAID). However, the activity profile of
rutin differs from that of aspirin, since this NSAID induced inhibitory effect close to the maximal activity already 1 hour following the injection of the phlogistic
agent. Whereas, the effect of both drugs remained steady
during all measurement time. Rutin exhibited a significant
(p < 0.05) inhibition activity (58.3 6.4%) determined at
5 hours after oedema induction. This inhibition was less
than that obtained with aspirin, 73.64 4% (fig. 3).

Fig. 3. Effects of rutin and aspirin (100 mg/kg, body

weight) on the inflammatory response measured 5 hours
after subplantar injection of -carrageenan 1% into the right
hind paw. Drugs were given orally 1 hour before -carrageenan injection. Results are expressed as the percentage
of oedema inhibition. Values are means SEM of 7 animals
for each group. significant dose response is seen (p < 0.05).

Effect of rutin on neutrophils chemotaxis and

degranulation
The chemotaxis of neutrophils towards fMet-Leu-Phe,
and PMA or fMet-Leu-Phe-induced degranulation was
investigated in presence and absence of rutin. Treatment
of human PMNs with 10, 25, 50, 75, 100 and 150 M
Exp Toxic Pathol 54 (2003) 4

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Exp Toxic Pathol 54 (2003) 4

rutin reduced significantly (P < 0.05), and in a dose-dependent manner, their migration towards 107 M fMetLeu-Phe (fig. 4). The IC50 value was 97.8 M. At a concentration of 150 M, the percentage of inhibition was
77.24 0.1%. Cytochalasin B, used as a standard inhibitor of chemotaxis, abolished almost completely neutrophils migration (fig. 5).
In the same way, rutin caused a significant (p < 0.05) inhibitory effect on elastase release in PMA or fMet-LeuPhe/CB-stimulated neutrophils. Whatever the stimulus,
rutin inhibitory effect was important at low concentrations,
and remained steady up to 25 M (fig. 6). Indeed, rutin, at
1 M final concentration, exerted an inhibitory effect of
54.99 2.6% and 23.23 3.1% on fMet-Leu-Phe/CB and
PMA-induced degranulation, respectively. We observed
that fMet-Leu-Phe/CB mediated-elastase release was fourteen times higher than that induced by fMet-Leu-Phe alone
(results not shown). Under our assay conditions, rutin, up
to 500 M final concentration, did not induce cell toxicity
as assessed by LDH release. Therefore, these drug effects
are not due to a cytotoxic action.
To confirm that rutin effect did not reflect functional
inhibition of the released enzyme, the same quantity of
neutrophils elastase was directly treated with this compound. No inhibition of elastase activity was observed in
this cell free system.
Fig. 4. Effect of rutin on neutrophils chemotaxis. The
chemotaxis assay was performed in 48-well chemotaxis
chamber, using a 5 m pore size polycarbonate filter.
PMNs (1.5 106 cells/ml) were incubated in presence of
the chemoatractant fMet-Leu-Phe (107 M) with or without
rutin. After incubation for 90 min at 37 C, cells adherent to
the lower surface of the filter were counted at 400 magnification. Results are expressed as the percentage of neutrophils migrating per field compared with neutrophil
chemotaxis to fMet-Leu-Phe without rutin set to 100%.
Values represent the mean SEM of three independent experiments performed in triplicate. A significant dose response is seen (p < 0.05).When they do not appear, error
bars are smaller than the symbol size.
Fig. 5. Effect rutin (50 and 150 M) and 1.5 M cytochalasin B (CB) on neutrophils chemotaxis to 107 M fMetLeu-Phe. All other experimental conditions are the same as
in fig. 4. Results are expressed as the inhibition percentage
of neutrophils migration. Each histogram is the mean
SEM of three separate experiments performed in triplicate.
When they do not appear, error bars are smaller than the
symbol size.
Fig. 6. Effect of rutin on elastase release in PMA or fMetLeu-Phe/CB-stimulated neutrophils. PMNs (5 106
cells/ml) were preincubated for 10 min at 37 C with indicated concentrations of rutin, and then exposed to appropriate stimulus PMA (100 nM) or fMet-Leu-Phe (106 M)/CB
(105 M). The activity of elastase released in the supernatant was measured at 405 nm. Elastase release induced
by each stimulus in the absence of rutin was defined as
100%. All concentrations are final ones. Values represent
the mean SEM of three experiments. A significant dose
response is seen (p < 0.05).

Discussion
In this study, the anti-inflammatory activity of rutin
was investigated in vivo and in vitro. Using a standard
model of acute inflammation (WINTER et al. 1962), we
observed that the subplantar injection of -carrageenan
induced a local oedema, reaching a maximal intensity
within 5 hours in the placebo group. Whereas, upon reabsorption of chloride saline solution, the control group
paws recovered their initial volume after 1 hour. It is well
known that the early phase of carrageenan-induced oedema is related to the production of inflammatory mediators such as arachidonic acid metabolites. While, the delayed phase of inflammatory response has been linked to
neutrophil migration and accumulation within inflammatory site where they release reactive oxygen species and
proteolytic enzymes (CUZZOCREA et al. 1998). Our results showed that rutin exhibited a significant (p < 0.05)
inhibitory effect on rat paw oedema formation. Effectively, BORISSOVA et al. (1994) reported that rutin prevented oedema formation in rats induced by histamine
and serotonin. The anti-inflammatory effect of rutin may
be explained, at least in part, by the inhibition of inflammatory mediators production, which play an important
role in neutrophil recruitment and activation. Indeed, it
has been reported that rutin inhibited PLA2 activity, the
initial enzyme in arachidonic acid cascade, from human
synovial fluid (LINDAHL and TAGESSON 1997). Furthermore, flavonoids exerted an inhibitory effect on cyclooxygenase and lipoxygenase activities (YOU et al.
1999). The inhibitory effect of rutin in the present study
started after 2 hours, this might be due to an absorption
delay of the drug (WILLIAMSON et al. 2000).
As neutrophils play an important role in the inflammatory process, inhibition of their migration and/or degranulation may account for parts of the anti-inflammatory activity. For this reason, we examined the effect of
rutin on human neutrophils chemotaxis and degranulation. Our results showed that rutin exerted a significant
(p < 0.05) inhibitory effect on PMNs migration towards
fMet-Leu-Phe, and in a dose-dependent manner. In a
control experiment, the addition of 1.5 M cytochalasin
B to fMet-Leu-Phe abolished drastically neutrophils migration. It is known that cytochalasin B, which binds to
the barbed ends of actin filaments, inhibits actin polymerization/depolymerization in fMet-Leu-Phe-stimulated neutrophils (HARVATH 1990).
On the other hand, rutin exerted a partial inhibitory effect on degranulation of fMet-Leu-Phe/CB-stimulated
neutrophils. A similar effect was shown for wortmannin,
a specific inhibitor of PI 3-kinase (HII et al. 2001). These
latter authors reported that degranulation of fMet-LeuPhe-stimulated neutrophils was partially suppressed in
presence of 150 M wortmannin. Whereas, at 10 M this
compound abolished most totally superoxide anion radicals generation in fMet-Leu-Phe-stimulated PMNs.
Since, mechanisms underlying rutin effects are still unclear, we suggest that this flavonoid may exert its effect

by inhibiting enzyme(s) involved in neutrophils response

such as PI3-kinase. Effectively, we previously reported
that rutin inhibited PI 3-kinase activity in a cell free
system (SELLOUM et al. 2001). This enzyme plays an important role in the reorganization of actin cytoskeleton ,
as well as in degranulation (HII et al. 2001; MIETTINEN et
al. 1998). However, many studies reported the involvement of other enzymes in these processes such as protein
kinase C (CABANIS et al. 1996) and tyrosine kinases
(MOCSAI et al. 2000). Thus, we examined the effect of
rutin on PMA-mediated PMNs degranulation. Results
showed that this flavonol inhibited PMA-mediated elastase exocytosis in the same way as observed with fMetLeu-Phe stimulation, but slightly less effective. This
could be explained by an additional inhibitory effect of
rutin on PI 3-kinase activity which is directly stimulated
by the G-protein-linked fMet-Leu-Phe receptor. Since
PKC has been reported to be insensitive to rutin action
(FERRIOLA et al. 1989), other enzymes involved in PMNs
degranulation may be possible candidates for inhibition
by this flavonol, such as mitogen-activated protein kinases (MAPK). MOCSAI et al. (2000) reported that fMetLeu-Phe-induced degranulation of primary and secondary granules of neutrophils is mediated by p38
MAPK activated via Src tyrosine kinases.
Taken together, our observations, based on in vivo and
in vitro approaches, revealed that rutin possesses anti-inflammatory properties. However, cellular mechanisms
underling these properties merit to be more elucidated.
Other possible target enzyme(s) involved in neutrophil
signalling pathways could be identified using specific inhibitors.
Acknowledgements: Authors are most grateful for the
encouragement and facilities provided by Dr. J. ARNHOLD,
University of Leipzig (Germany). This study was supported by grants from the International Foundation for Science
(ifs), Sweden (F/ 2825-1), from ANDRS, Algeria (05/ 01/
02/ 98 006) and from the Ministry of High Education and
Scientific Research, Algeria (F 1901/01/96).

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