Chinese Medicine, 2010, 1, 5-17
doi:10.4236/cm.2010.11002 Published Online June 2010 (
Copyright © 2010 SciRes. CM
Effects of Indole-3-Carbinol and Flavonoids Administered
Separately and in Combination on Nitric Oxide
Production and iNOS Expression in Rats
Evita Rostoka1, Larisa Baumane1, Sergejs Isajevs2, Aija Line3, Karina Silina3, Maija Dzintare1,
Darja Svirina2, Jelena Sharipova1, Ivars Kalvinsh1, Nikolajs Sjakste1,2
1Latvian Institute o f Organic Synthesis, Riga, Latvia
2Faculty of Medicine, University of Latvia, Riga, Latvia
3Latvian Biomedical Research and Study Centre, Riga, Latvia
Received January 20, 2010; revised April 29, 2010; accept ed May 10, 2010
Beneficial effects of natural compounds are often attributed to modulation of NO production; however ef-
fects produced by plant extracts do not correlate with effects of purified components. The goal of our work
was to study ability of flavonoids and indole-3-carbinol, as well as their combinations to modify NO produc-
tion, iNOS gene and protein expression in rat tissues. Baicalein and luteolin decreased NO concentration in
both intact and LPS-treated animals. Baicalein decreased iNOS gene expression. Luteolin decreased NO
production in the liver and heart and number of iNOS-positive cells in the liver of LPS-treated animals.
Combination of the two substances did not decrease the NO synthesis triggered by LPS, although it de-
creased iNOS gene expression. Quercetin decreased NO production in the heart, kidneys and blood of intact
rats, but enhanced the LPS effect in testes, spleen and blood on NO production and iNOS protein expression
in the liver. Indole-3-carbinol decreased NO concentration in the cerebellum, blood, lungs and skeletal mus-
cles. The drug enhanced the LPS-triggered increase of NO production in all rat organs. It increased iNOS
protein expression in intact liver; however it decreased the LPS-triggered outburst of the enzyme biosynthe-
sis. Combination of indole3-carbinol with quercetin decreased NO production in LPS-treated animals how-
ever it slightly increased iNOS gene expression. Taken together our results suggest that modifications of NO
level in tissues by a natural compound cannot be predicted from data about its effects on NOS expression or
activity. Combination of substances can produce an effect differing from that of individual substances. This
stresses importance of direct measurements of NO in the tissues.
Keywords: Nitric Oxide, Baicalein, Luteolin, Indole-3-Carbinol, Quercetin, Inducible Nitric Oxide Synthase
1. Introduction
Natural biologically active compounds of plant origin
including flavonoids are main active substances of tradi-
tional Chinese medicines: herbal extracts and similar
preparations. Nowadays some of these substances are
used in purified form as drugs. Anti-inflammatory activ-
ity, antioxidant activities, anticancer activity of phyto-
genic antineoplastic agents, and neuroprotective effects
of Chinese herbal drugs are in focus of interest of many
researchers worldwide [1]. Chinese traditional medicines
are known to influence also nitric oxide enzymatic pro-
duction and NO synthase activity [2]. It is supposed that
flavonoid intake influences mortality from nitric ox-
ide-dependent processes: ischemic heart disease, stroke,
diabetes mellitus, and cancer [3], NO production is also
modified by chemicals of plant origin [4]. This implies
significance of flavonoid and other natural compound
uptake for functions of cardiovascular, immune and
nervous systems. However biological activity of poly-
phenol-rich food product does not correlate with effects
that could be deduced from effects of individual com-
pounds on NO synthase activity. For example, red wine
is known as vasodilator [5], however purified quercetin,
that is abundant in red wine inhibits iNOS gene expres-
sion [6,7]. It also destabilizes eNOS mRNA [8] and is
even considered to be inhibitor of the NOS enzymatic
activity [9]. Nevertheless the compound produces vaso-
relaxing effects [10] despite the fact that nitric oxide re-
lease in rat aorta is not detected after quercetin admini-
stration [11]. In this work we have studied the ability of
several natural compounds of plant origin administered
separately and in combinations to modify NO production
in rat tissues monitored by ESR spectroscopy of Fe
(DETC)2-NO complexes conducted in parallel to evalua-
tion of iNOS gene expression assayed by real-time RT-
PCR technique. Combination of direct NO detection in
tissues with other approaches characterizing NO produc-
tion at different levels enabled us to reveal unforeseen
effects of presumable NO-donors, anaesthetics and an
anti-ischemic drug [12-14]. The same approach was ap-
plied this time to natural compounds. Flavonoids luteolin,
baicalein and quercetin as well as simple phenolic com-
pound indole-3-carbinol were chosen among many other
compounds after a piloting study.
Luteolin, 3’, 4’, 5, 7-tetrahydroxyflavone is abundant
in vegetables: roots of celery, rutabaga, red pepper, spin-
ach and flowering plants: Ajuga decumbens, Taraxacum
officinale (dandelion), Medicago sativa (alfalfa). Luteo-
lin is known as dietary compound with antioxidant activ-
ity [15]. Baicalein (5, 6, 7-trihydroxyflavone) is found in
Scutellaria baicalensis Georgi roots. Quercetin (penta-
hydroxyflavonol) is found in numerous higher plants.
This flavonol is abundant in onions, apples, leaf vegeta-
bles, beans, tea, red wine, clover, pollen. Indole-3-carbinol
(3-indolmetanol) is found in Mustard family plants:
(Brassica sp.): cabbage, broccoli, Brussels sprouts. The
compound is widely studied as chemotherapeutic agent
for cancer treatment [16]. Chemical structures of the
compounds are given in Figure 1. Literature data indi-
cated possible impact of all the three substances on NOS
expression and/or NO production [15-22]. The chosen
compounds are active substances in several drugs used in
Chinese medicine. Quercetin is in important component
of Shaofu Zhuyu decoction active extract [23], Saururus
chinensis, a herb used traditionally in Chinese medicine
for treatment of urological diseases [24], together with
luteolin it is found in tree peony yellow flowers also
widely used in Chinese medicine [25]. Baicalein is an
active component of numerous Chinese medicines in-
cluding Niu Huang Jie Du Pill [26].
The aims of the present work were: 1) To study effects
NO production in several organs of intact rats and in LPS
model of sepsis; 2) To reveal modifications of NO pro-
duction by luteolin, baicalein, quercetin and indole-3-
carbinol given separately and in combinations in both
intact and LPS-treated animals; 3) To study contribution
of changes in iNOS gene and protein expression in
modifications of NO production by natural compounds
and their modifications.
Figure 1. Chemical structures of luteolin, baicalein, quer-
cetin and indole-3-carbinole.
2. Material and Methods
2.1. Chemicals
Indole-3-carbinol, quercetin, baicalein and luteolin were
purchased from Dayang Chemical Co., LTD (Hangzhou,
China). Lipopolysaccharide, diethylthiocarbamate, fer-
rous sulfate, sodium citrate, TRI reagent and all other
chemicals were from Sigma-Aldrich Chemie GmbH
(Taufkirchen, Germany).
2.2. Experiment Design and Drug
Animals were obtained from the Laboratory of Experi-
mental Animals, Riga Stradins University, Riga, Latvia.
All experimental procedures were carried out in accor-
dance with guidelines of the Directive 86/609/EEC
“European Convention for the Protection of Vertebrate
Animals Used for Experimental and other Scientific
Purposes” (1986) and were approved by the Animal Eth-
ics Committee of the Food and Veterinary Service (Riga,
Wistar male rats, each weighing 215.00 ± 5.63 g at the
beginning of the experiments, were used in all the work.
The environment was maintained at a temperature of 22
± 0.5˚C with a 12-h light/dark cycle. The animals were
fed a standard laboratory diet. Description of the experi-
mental groups is given in Table 1. In NO production
experiments substances were administered per os in con-
centrations indicated in the Table 1. 3.5 hours after sub-
stance administration spin trap was injected, after 30 min
Copyright © 2010 SciRes. CM
Copyright © 2010 SciRes. CM
Table 1. Description of groups and exper i ment design.
of animals Substance/dose LPS Parameter studied Organs studied
1. 24 - - NO production Brain cortex, liver, heart, kidney,
blood, lungs
2. 9 Indole-3-carbinol (50 mg/kg) - NO production Brain cortex, liver, heart, kidney,
blood, lungs
3. 5 Luteolin (50 mg/kg) - NO production Brain cortex, liver, heart, kidney,
blood, lungs
4. 6 Quercetin (50 mg/kg) - NO production Brain cortex, liver, heart, kidney,
blood, lungs
5. 5 Baicalein (50 mg/kg) NO production Brain cortex, liver, heart, kidney,
blood, lungs
6. 28 - 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
7. 12 Indole-3-carbinol (50 mg/kg) 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
8. 6 Luteolin (30 mg/kg) 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
9. 6 Quercetin (50 mg/kg) 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
10. 6 Baicalein (30 mg/kg) 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
11. 8
Baicalein (30 mg/kg)
Luteolin (30 mg/kg) 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
12. 6
Quercetin (50 mg/kg)
Indole-3-carbinol (50 mg/kg) 10 mg/kgNO production
Brain cortex, cerebellum, liver,
heart, kidney, blood, lungs, testes,
spleen, skeletal muscles
13. 20 - - iNOS mRNA and protein expressionLiver, brain cortex
14. 3 Indole-3-carbinol (50 mg/kg) iNOS mRNA and protein expressionLiver, brain cortex
15. 3 Luteolin (50 mg/kg) iNOS mRNA and protein expressionLiver, brain cortex
16. 3
Quercetin (50 mg/kg)
iNOS mRNA and protein expressionLiver, brain cortex
17. 3 Baicalein (50 mg/kg) iNOS mRNA and protein expressionLiver, brain cortex
18. 3
Quercetin (50 mg/kg)
Indole-3-carbinol (50 mg/kg) iNOS mRNA and protein expressionLiver, brain cortex
19. 3
Baicalein (50 mg/kg)
Luteolin (50 mg/kg) iNOS mRNA and protein expressionLiver, brain cortex
20. 21 - 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
21. 6 Indole-3-carbinol (50 mg/kg) 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
22. 6 Luteolin (50 mg/kg) 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
23. 6 Quercetin (50 mg/kg) 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
24. 6 Baicalein (50 mg/kg) 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
25. 6
Quercetin (50 mg/kg)
Indole-3-carbinol (50 mg/kg) 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
26. 6
Baicalein (50 mg/kg)
Luteolin (50 mg/kg) 10 mg/kgiNOS mRNA and protein expressionLiver, brain cortex
Copyright © 2010 SciRes. CM
rats were decapitated under slight ether narcosis. In sev-
eral groups lipopolysaccharide (10 mg/kg) was intrap-
eritoneally injected to rats, substances or their combina-
tions were administered per os in the same time, spin
traps were administered 3.5 hours later, 30 minutes after
spin trap injection rats were decapitated under slight
ether narcosis. In additional piloting series of experi-
ments (not shown) iNOS inhibitor AMT (2 mg/kg), was
administered intraperitoneally shortly before spin-trap
administration, 30 minutes later rats were decapitated.
For real-time PCR and immunochemistry rats were de-
capitated under slight ether narcosis; liver tissue was
taken for RNA extraction and immunohistochemical
examination. Brain cortex tissue was also taken from
some animals for immunohistochemistry. Natural com-
pounds and LPS were administered following the above
time schedule.
2.3. Administration of Spin Trap Agents
To determine production level of nitric oxide in the tis-
sues we used ESR spectroscopy of paramagnetic Fe-
diethylthiocarbamate–nitric oxide complex (Fe (DETC)2-
NO) [27]. Spin traps were administered 30 minutes be-
fore the sacrifice. Rats were administered 400 mg/kg of
the nitric oxide scavenger diethylthiocarbamate via in-
traperitoneal injection and ferrous citrate subcutaneously
(40 mg/kg ferrous sulphate + 200 mg/kg sodium citrate).
Diethylthiocarbamate binds ferrous ion, the resulting
complex traps nitric oxide converting to the paramag-
netic Fe (DETC)2-NO complex that is detected by ESR
2.4. Sacrifice, Organ Dissection and Sample
Preparation for Electron Paramagnetic
Resonance Spectroscopy
Following the drug and spin trap administration the rats
were decapitated under slight ether anesthesia, samples
of brain cortex, cerebellum, myocardium tissue, liver,
kidney, testes, skeletal muscles, lungs and blood were
compacted in a glass tube 30 mm in length with inner
diameter 4 mm and immediately frozen in liquid nitrogen.
Before recording the ESR spectra, the specimen was
placed in a quartz finger Dewar flask ER 167 FDS-Q
(Bruker, Karlsruhe, Germany) filled with liquid nitrogen.
2.5. ESR Measurements
ESR spectra were recorded in liquid nitrogen using an
ESR spectrometer “Radiopan” SE/X2544 (Radiopan,
Poznan, Poland). The conditions of the electron paramag-
netic resonance measurements were: operation at X-band,
25 mW microwave power, 100 kHz modulation frequency,
5 G modulation amplitude, receiver gain 0.5 × 104, and
time constant 1 s. Spectra were recorded for 4 minutes.
The nitric oxide content in the samples was evaluated
from the height of the third component of the NO signal
at g = 2.031.
The NO concentration (ng/g of tissue) was calculated
on the basis of calibration curves as described previously.
Briefly, different quantities of NaNO2 (final concentra-
tions 10, 20, 30, 40, 60, 100 M) were mixed with DETC
(33 mg/mL) and FeSO4·7H2O (3.3 mmol/L), an excess of
Na2S2O4 (2 mol/L) was added to the mixture. The EPR
spectra were taken as described above.
Further details are given in our previous publications
2.6. RNA Extraction and cDNA Preparation
Total RNA was isolated from liver and brain cortex us-
ing TRI reagent (Sigma Aldrich, Taufkirchen, Germany).
DNA contaminations were removed with RNA-free kit
(Ambion, Austin, TX, USA). The resulting RNA quan-
tity and purity were determined by spectrophotometry,
integrity of RNA molecules was monitored by gel elec-
trophoresis, and only specimens with well-pronounced
rRNA bands were taken for reactions. RNA (2 μg) was
reverse-transcribed using a random hexamer primer
(RevertAid™ First Strand cDNA Synthesis Kit, Fermen-
tas, Vilnius, Lithuania) to obtain cDNA.
2.7. Real Time RT-PCR
The mRNA expression rates of the brain cortex, liver
iNOS and reference gene were determined using the
SYBR® Green PCR Master Mix (Applied Biosystems,
Foster City, CA, USA) according to the instructions pro-
vided by the manufacturer. Amplification and detection
of specific products were performed on a StepOne™
Real-Time PCR System (Applied Biosystems, Foster
City, CA, USA) using the following temperature-time
profile: one cycle of 95˚C for 10.00 min; and 40 cycles
of 95˚C for 0.15 min, 60˚C for 1.00 min. To check speci-
ficity of amplification products, the dissociation curve
mode was used (one cycle at 95˚C for 0.15 min, 60˚C for
1 min and 95˚C for 0.15 min). To evaluate the suitability
of candidates as reference genes, we applied the GeNorm
program [31]. Primers were designed using Primer3
software. The primers were supplied by Metabion inter-
national AG, Germany. The 2∆∆CT method was applied
for analysis of the results. Primer sequences for iNOS
gene were 5’-GCTACACTTCCAACGCAACA-3’ for
forward and 5’-CATGGTGAACACGTTCTTGG for rev-
erse primer, the expected size of the product was 116 bp.
RNA-polymerase II [32] was used as reference gene (5’-
GACTCTGTTTGT-3’, 135 bp).
2.8. Histological and Immunohistochemical
Paraffin-embedded tissue was cut in 4-micron-thick sec-
tions and stained with haematoxylin and eosin for mor-
phological examination. Infiltration of inflammatory cells
in brain tissue was assessed in subcortical perivascular,
subcortical parenchymal, and intracortical peri-vascular
regions (magnification × 400). Perivascular infiltrates were
defined as inflammatory cells, which are located not fur-
ther than three cell layers from blood vessels. Inflamma-
tory cells further than three layers from a blood vessel
wall were defined as parenchymal infiltrates. Infiltration
of inflammatory cells was assessed according to four
score scale: 0no infiltration; 1light infiltration; 2
medium infiltration; 3marked infiltration; 4very
marked infiltration (more than 25% of the total field of
The morphology of liver tissue was evaluated by
evaluating the histological activity index (HAI), as de-
scribed [33]: infiltration of inflammatory cells (0-4
scores); necrosis of hepatocytes around a central vein
(0-6 scores); necrosis of hepatocytes and apoptosis in
periphery lobules (0-4 scores); inflammatory changes of
portal tracts (0-4 scores).
Tissue sections were stained for visualization of iNOS
positive cells by an immunochemical approach as previ-
ously described [34]. Briefly, antigen retrieval was
achieved by treatment in a microwave oven for 20 min at
300 W in citrate buffer, pH = 6.0. Endogenous peroxi-
dase activity was blocked by 0.5 % H2O2 for 10 min.
Nonspecific primary antibody binding was blocked by
serum-free protein block for 10 min. Rabbit polyclonal
active iNOS antibody Abcam Inc. (Cambridge, MA,
USA) was applied in 1:200 dilution and incubated for 1h
at room temperature in a humidified chamber. Detection
of primary antibody binding was performed using spe-
cific peroxidase conjugated polyclonal goat anti-rabbit
IgG (1:100 for 30 min) and subsequently peroxidase
conjugated polyclonal rabbit anti-goat IgG (1:100 for 30
min). The immunoperoxidase color reaction was devel-
oped by incubation with diaminobenzidine (7 min). A
negative control without primary antibody was included
in each staining run. iNOS positive cells were counted in
twenty high-powered fields at magnification × 400. All
cell counts were expressed as cells per square millimeter.
For morphological examination, at least 3 replicate
measurements of iNOS positive cells were performed by
the same observer in 10 randomly selected slides, and the
intraobserver reproducibility was assessed with the coef-
ficient of variation and with the interclass correlation
coefficient. The intraobserver coefficient of variation
was 4%, and the intraobserver correlation coefficient was
2.9. Statistical Analysis
Results were expressed as mean ± SD. The significance
of differences in NO concentration and iNOS expression
between groups was evaluated according to Student's
unpaired t-test, the Mann-Whitney U test was used for
quantification of immunohistochemical experiments. Re-
sults were considered to be significant when P was less
than 0.05.
3. Results
3.1. Effects of Natural Compounds and their
Combinations on NO Production in Intact
and LPS-Treated Rats
In order to test the ability of the natural compounds to
modify NO production in animals the radical concentra-
tion was monitored in several rat organs and tissues.
Data are summarized in Figure 2. ESR spectra of the
different organs had a typical Cu-DETC spectrum shape
with a superposed Fe(DETC)2-NO peak, spectra were
published previously [14]. The NO production reached
the highest levels in the brain cortex, liver, lungs, and
blood Figure 2. The NO production in heart and kidneys
was an order of magnitude lower.
When control group of animals was compared to ani-
mals treated with natural substances it turned out that
baicalein decreased NO concentration in heart, kidney,
liver and lungs (Figure 2). Luteolin decreased NO pro-
duction in the liver and heart. Quercetin induced signifi-
cant decrease of NO production the heart, kidneys and
blood. Indole -3-carbinol caused a significant decrease of
NO production in the cerebellum, spleen, blood, lungs
and skeletal muscles.
In the following set of experiments the eventual activ-
ity of the compounds as modifiers of NO production was
tested against the background of the iNOS induction.
Intraperitoneal injection of LPS to the animals caused a
drastic increase of NO production levels in all tissues
studied (Figure 2). The highest production of nitric ox-
ide was detected in liver, whereas very strong increases
in nitric oxide accumulation (50-100 fold compared to
control) were observed in heart, blood and kidney.
However, the effects of LPS were less pronounced in
brain tissues where nitric oxide increased 4-6 times only.
Nitric oxide production increase in testes was of compa-
rable magnitude.
Baicalein (30 mg/kg) decreased NO concentration in
brain cortex, liver, heart and kidneys. Luteolin (30 mg/kg)
decreased NO outburst in all organs except skeletal mus-
cles. In contrast, administration of the indole-3-carbinol
(50 mg/kg) enhanced the LPS-induced increase of NO
production in all organs except spleen and testes, Figure 2.
Quercetin (50 mg/kg) produced similar effect: NO
Copyright © 2010 SciRes. CM
Figure 2. Effects of indole-3-carbinol, luteolin, baicalein
quercetin and combinations of indole-3-carbinole + querc-
etin, baicalein + luteolin on NO production in rat organs of
intact (a) and LPS-treated rats (b, c). Stars indicate statis-
tically significant differences (P < 0.05) with control (a) or
LPS (b, c) groups.
production increased in testes blood and spleen (Figure 2).
The observed ability of individual compounds to mod-
ify the LPS-triggered NO production raised the question
about maintenance of the effects in presence of other
substances. This approach modeled to some extent ad-
ministration of flavonoid-containing herbal extracts.
Baicalein as NO down-regulating and indole-3-carbinol
as NO up-regulating substances were supplemented by
luteolin and quercetin that produced NO-decreasing and
NO-increasing effects correspondingly. Synergism of the
effects was anticipated. Surprisingly, the results indi-
cated adverse effects in both cases. Combination of in-
dole3-carbinol with quercetin (50/50) decreased NO
concentration up-regulated by LPS in many tissues.
Similarly, combination of baicalein and luteolin lost the
NO-decreasing activity characteristic of individual sub-
stances (Figure 2).
To test possible involvement of iNOS in the observed
modifications natural substances were combined with
iNOS inhibitor AMT (not shown). In intact animals the
inhibitor markedly decreased NO production in all or-
gans except the heart, AMT inhibiting action was attenu-
ated by indole-3-carbinol in skeletal muscles. Quercetin
also attenuated AMT effects in brain cortex, testes, blood
and muscles. Luteolin slightly interfered with AMT ac-
tion. These results indicated possible involvement of
iNOS in effects of the compounds; this encouraged us to
test ability of the substances to modify iNOS gene ex-
pression in rat liver.
3.2. Effects of Natural Compounds on iNOS
Gene mRNA Expression in Intact and
LPS-Treated Rats
No influence of indole-3-carbinol on iNOS expression in
liver was observed (Figure 3). Surprisingly, luteolin up-
regulated the gene expression. Baicalein decreased level
of the gene expression. Level of transcription was still
decreased when baicalein was given in combination with
luteolin. Quercetin did not produce any significant effect
on iNOS gene transcription, a tendency for increase was
observed when it was given in combination with in-
dole-3-carbinol (Figure 3(a)).
The LPS effect on the gene expression in the liver was
drastic (Figure 3(b)) as it could be predicted from the
increase of NO production (3442.82 761.24). However
the effect was not well-reproducible between individual
animals. Quercetin (50 mg/kg) significantly decreased
the LPS-triggered iNOS mRNA expression. Enhance-
ment of the iNOS mRNA expression by indole-3-carbinol
was observed (7715.01 1877.35% compared to control,
the result is not statistically significant). The same trend
was observed when indole-3-carbinol was supplemented
by quercetin (50 mg/kg). Baicalein somewhat decreased
the gene expression triggered by LPS, the effect was
better pronounced if it was combined with luteolin (Fig-
ure 3(b)).
3.3. Effects of Natural Compounds on iNOS
Protein Expression in Intact and
LPS-Treated Rats
Data on effects of the tested compounds on number of
iNOS positive cells in rat liver and brain cortex are given
in Figures 4 and 5. Interestingly, indole-3-carbinol pro-
Copyright © 2010 SciRes. CM
ControlBaicaleinQuercetinIndole- 3-
Indole- 3-
carbin ol
iN O S gene exp ression vs cont rol
LPS Baicalein
Indole- 3-
Luteolin ,
Ba icalein
Indole- 3-
Querce tin
iN OS gene exp ression vs cont rol
Figure 2. Effects of indole-3-carbinol, luteolin, baicalein
quercetin and combinations of indole-3-carbinole + quer-
cetin, baicalein + luteolin on iNOS gene expression in rat
liver. Results are presented as percentage vs average of the
control. (a) intact animals; (b) LPS-treated animals. All
compounds were administered in dose 50 mg/kg. *–P < 0.05
versus control gr oup, #–P < 0.05 versus LPS group.
duced statistically significant increase of the protein ex-
pression; an effect is coherent with ESR data (Figure 2),
the substance increased iNOS expression also in brain
cortex (Figure 5(f)). Quercetin (50 mg/kg) and luteolin
did not modify the protein expression level. Baicalein
manifested a tendency to decrease number of iNOS-
positive cells in the liver.
LPS significantly increased the number of iNOS posi-
tive cells in liver and brain cortex tissue. Indole-3-carbinol
decreased outburst of iNOS protein translation triggered
by LPS both in liver tissue (predominantly in Kupfer
cells) and brain cortex tissue (Figures 5(c) and (g)), in
this case immunohistochemistry data are in contradiction
with ESR data indicating enhancement of LPS effects by
the compound. Baicalein significantly decreased the LPS
effect. On the contrary, quercetin enhanced expression of
the enzyme in the liver of LPS-treated animals; this ef-
fect followed the trend observed in ESR experiments.
ControlIndole- 3-
Quercetin LuteolinBaicalein
% of iNOS positive cells
LPSIndole- 3-
Lute o lin
Baic al ein
% of iNO S pos it ive c ells
Figure 3. Effects of indole-3-carbinol, luteolin, baicalein
and quercetin on number of iNOS-positive cells in rat liver.
Results are presented as percentage vs average of the con-
trol. (a) intact animals; (b) LPS-treated animals. All com-
pounds were administered in dose 50 mg/kg. *–P < 0.05
versus control gr oup, #–P < 0.05 versus LPS group.
4. Discussion
4.1 Baicalein and luteolin
According published data baicalein suppresses iNOS
gene expression in glia [35-37] and macrophages [38, 39]
the inhibiting effect is achieved by decrease of lipoxine
synthesis. In blood vessels this compound acts as antago-
nist of nitric oxide, it inhibits the soluble guanylate cy-
clase [40]. Baicalein depresses also the smooth muscle
iNOS [41]. Luteolin is also is known as a weak inhibitor
of iNOS expression, but it is not capable to inhibit the
enzyme activity [17-20]. Some authors find that luteolin
stimulates eNOS gene expression [21]. Apparently our
results about decrease of NO concentration in some tissues
after luteolin and baicalein administration are in good
agreement with literature data, as this effect was obser-
ved both in intact and LPS-treated animals. Unexpect-
edly combination of the two compounds did not produce
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Copyright © 2010 SciRes. CM
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 5. Photomicrographs of in rat liver tissue stained immunohistochemically with iNOS antibody (a-d), and brain cortex
tissue (e-h). (a,e)-control, (b,f)–indole-3-carbinol, (c,g)–LPS at dose 10 mg/kg; (d,h)–simultaneous administration of in-
dole-3-carbinol and LPS. Arrows indicate iNOS positively stained cells. Magnification at ×100 (a-d) and at × 400 (e-h).
this effect. iNOS gene and protein expression was de-
creased by baicalein, this effect persisted when luteolin
was administered to the same animal. Interestingly,
Scutellaria baicalensis extract, i.e. combination of bai-
calein with other compounds increases NO synthesis in
induced macrophages [42]. Interesting explanation can
be proposed on the basis of hypothesis about nitrite pro-
tonation in acidic medium with generation of nitric acid.
In this case flavonoids could reduce the acid to NO and
stomach could turn into NO generator [43]. Simpler ex-
planations can be also proposed. We have observed that
higher dose of baicalein (100 mg/kg, not shown) pro-
duced less pronounced NO-lowering effect compared to
a lower dose (50 mg/kg). Probably cytotoxic effect of the
substances interferes with NO-modulating activity. Both
baicalein and luteolin are inducers of apoptosis, the sub-
stances intercalate into DNA, inhibit topoisomerases I
and II and DNA polymerase, induce TNF [44]. Thus
apoptosis process can be triggered in many cells, NO
level would increase as protection reaction against apop-
tosis [45].
4.2. Indole-3-Carbinol and Quercetin
Indole-3-carbinol is a weak iNOS induction inhibitor
[22]. The substance interferes with several signaling
pathways [46]. Decrease of NO concentration in blood,
lungs and skeletal muscles caused by the substance can
also indirectly indicate iNOS-inhibiting activity of the
compound. Surprisingly, inverse effect was observed
when we assessed number of iNOS-positive cells in the
liver of intact rats. The compound enhanced increase of
the gene transcription triggered by LPS in the liver, NO
production in liver of intact animals and the LPS-induced
increase of NO production in all the organs studied,
amazingly, number of iNOS-positive cells in the liver of
LPS-treated animals dropped down after administration
of the substance. Thus the substance can either increase
or decrease NO production in rat tissues. The effect de-
pends on the tissue and physiological state of the animal.
The NO decreasing activity of indole-3-carbinol can be
explained by its capability to reduce NF-kB DNA bind-
ing activity [16,46]. Moreover the substance was shown
to inhibit Akt-kinase activity, this leads to decrease of
NF-kB expression [16,46]. Ability of the compound to
increase the NO production in some organs, especially in
LPS-treated animals could be rather ascribed to antioxi-
dant activity of indole-3-carbinol [22]. Scavenging of
reactive oxygen species prevents involvement of NO in
interaction with these radicals increasing its bioavailabil-
ity. This effect is produced by several natural compounds
including cocoa polyphenols [47] and resveratrol [48].
Increase of enzyme expression by the drug indicates ex-
istence of some mechanism for regulation on transcrip-
tion level. Enhancement by the substance of nitric oxide
production induced by lypopolysaccharide upregulation
of protein kinase C is also quite possible [49,50].
Quercetin decreased NO concentration in the heart,
kidneys and blood; however it enhanced the LPS effect
in testes, blood and spleen. The substance attenuated the
LPS effects on the level of iNOS gene expression, how-
ever it enhanced these effects on protein expression level.
Data about quercetin impact on NO synthesis are rather
contradictory. The substance inhibits iNOS induction,
the effect is better detectable in in vitro cultured cells
[6,7,51]. In vivo quercetin did not decrease NFkB activa-
tion in kidney cortex [52]. In systems, where iNOS ex-
pression decrease by quercetin was observed, the flavon-
oid did not down-regulate NFkB activation [7,53]. Proba-
bly, quercetin interferes with tyrosin kinase-mediated
pathways [54], and decreases TNFα expression [55].
However other authors observed the quercetin-induced
inhibition of IkBα (inhibitor of kappa B alpha) degrada-
tion via depression of IkB kinase activity, this leads to
inhibition of NFkB [56]. Other reports inform about
prevention and/or inhibition of IkB phosphorylation [57,
58], depression of NFkB activation by interleukin [59]
and hydrogen peroxide [60] produced by the flavonoid.
Inhibition of iNOS expression by quercetin is often as-
cribed to inhibition of the NFkB pathway [61,62]. Inter-
estingly, some authors [63] find out that quercetin does
not inhibit iNOS gene expression; however it inhibits
iNOS enzyme expression. We came up to quite an oppo-
site conclusion; however both the cited and our data in-
dicate separate mechanisms for regulation of the iNOS
gene and protein expression by this substance. Quercetin
also destabilizes eNOS mRNA [8]. There are even data
about ability of the substance to inhibit NOS enzymatic
activity [9]. In the same time quercetin protects epithe-
lium against lesions produced by NOS inhibitors [64]
and stimulates NO synthesis in leukemia cells as protec-
tion reaction against induction of apoptosis [45]. Quercetin
is considered to be the main active compound of red
wine; some authors have observed NO-dependent vaso-
relaxation induced by quercetin [10]. Supplementation of
diet with quercetin favors NO production in endothelium
[5]. However the nitric oxide release from endothelium
was not detected in special studies [11]. Quercetin is also
one of the active substances of the Gingko biloba ex-
tracts, both purified quercetin and Gingko biloba extracts
(i.e. mixture of quercetin with other natural compounds)
decrease LPS-induced iNOS expression, however the
extract acts via NF-kB inhibition, but quercetin inhibits
TNFa pathway [65,66]. In our studies quercetin in-
creased the LPS-triggered NO outburst in lungs, testes
and lungs. Apparently, the NOS-inhibiting effect was not
detectable on organism level. Our data contradict for-
merly published reports about decrease of nitrite produc-
tion in brain [67] and blood plasma of LPS-treated ani-
mals [68] or streptozotocin-treated animals [69] by
quercetin. However nitrite and NO production levels do
not always correlate. When NO production was assessed
by an approach similar to ours the quercetin-induced
increase of NO production in rat brain was also observed
[70]. Moreover the substance did not modify much iNOS
gene expression in both healthy and LPS-treated animals.
This effect can be associated with apoptosis-promoting
activity of quercetin [71]. Probably simultaneous ad-
ministration of indole-3-carbinol and quercetin favored
manifestation of NOS-inhibiting activity of quercetin and
abolished increase of NO bioavailability produced by
indole-3-carbinol, as no decrease of iNOS expression
was observed in this case. NO scavenging activity of
quercetin [69-73] also could manifest itself in this case.
5. Conclusions
Taken together our results suggest that modifications of
NO level in tissues by the studied natural compounds
cannot be predicted from data about its effects on NOS
expression or activity. Effects of individual compounds
are not additive when these are administered in combina-
tion. This stresses importance of direct measurements of
NO in the tissues using ESR method.
6. Acknowledgements
This study was supported in part by the National Re-
serach Program 2010.10.-4/VPP4 “Creation of novel
means and methods for prophylaxis, treatment and diag-
nostics, elaboration of biomedical technologies for im-
provement of social health” and the grant 04.1317
“Pathological production of nitric oxide, possibilities of
its pharmacological correction” awarded to N. Sjakste by
the Latvian Council of Science. We thank L. Lauberte
for technical assistance. Participation of D. Meirena in
experiment design and discussion is greatly appreciated.
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