Antioxidantien
Antioxidants
Effects of Phycocyanin Extract on
Tumor Necrosis Factor-α and Nitrite
Levels in Serum of Mice Treated
with Endotoxin
Cheyla Romay a, René Delgadob, Diadelis Remirez a, Ricardo González a, and Armando Rojasb
Department of Pharmacology, National Center for Scientific Researcha, Havana (Cuba),
and Department of Pharmacology, Center for Pharmaceutical Chemistry b, Havana (Cuba)
Summary
Phycocyanin is a biliprotein which exerts
antioxidative and anti-inflammatory ef-
fects in various in vivo and in vitro expe-
rimental models. In this study phycocy-
anin effects on tumor necrosis factor-α
(TNFα) and nitrite levels in serum of
mice treated with lipopolysaccharide
(LPS) was examined. TNFα was measured
by cytotoxicity on L-929 cells and nitrite
by the Griess reaction, after reduction of
all nitrates to nitrites by nitrate reduc-
tase. 1 h after LPS injection (0.5 mg/kg
i.p) there was a significant increase in
Zusammenfassung
Wirkung von Phycocyanin-Extrakt auf
die Serumspiegel von Tumornekrosefak-
tor-α und Nitrit bei Endotoxin-behandel-
ten Mäusen
Phycocyanin, ein aus Mikroalgen ge-
wonnenes Biliprotein, besitzt antioxida-
tive und entzündungshemmende Eigen-
schaften, wie in verschiedenen In-vivo-
und In-vitro-Modellen festgestellt werden
konnte. In der vorliegenden Studie
wurde die Wirkung von Phycocyanin auf
Tumornekrosefaktor-α (TNFα)- und Ni-
trit-Spiegel im Serum von mit Lipopoly-
saccharid (LPS) behandelten Mäusen un-
tersucht. TNFα wurde anhand der Zytoto-
xicität an L 929-Zellen gemessen, und die
Nitrite wurden mit Hilfe der Griess-Reak-
tion bestimmt, nachdem alle Nitrate un-
Arzneim.-Forsch./Drug Res. 51 (II), 733−736 (2001)
ArzneimForschDrugRes
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TNFα levels in mouse serum. Phycocy-
anin (50−300 mg/kg p.o), administered
1 h before LPS, reduced dose-depen-
dently the TNFα concentration in serum.
After 18 h, LPS (30 mg/kg i.p) also indu-
ced a substantial increase in serum ni-
trite levels, which were reduced dose-de-
pendently by phycocyanin pretreatment
(100−300 mg/kg p.o). The results indicate
that phycocyanin exerts inhibitory ef-
fects on TNFα and NO production which
might be ascribed to the antioxidative
properties of the biliprotein.
Key words
ter Beteiligung der Nitratreduktase zu Ni-
triten reduziert worden waren. 1 h nach 䊏 Endotoxic shock, mouse
der Injektion von LPS (0.5 mg/kg i.p.) 䊏 Nitric oxide
war ein signifikanter Anstieg der TNFα- 䊏 Phycocyanin
Spiegel im Serum der Mäuse zu verzeich- 䊏 Tumor necrosis factor-α
nen. Wurde Phycocyanin (50−300 mg/kg
p.o.) 1 h vor der Verabreichung von LPS Arzneim.-Forsch./Drug Res.
gegeben, nahm die TNFα-Serumkonzen-
51 (II), 733−736 (2001)
tration dosisabhängig ab. 18 h nach der
Gabe von LPS (30 mg/kg i.p.) wurde ein
Anstieg der Nitrit-Serumspiegel beobach-
tet, welche durch Vorbehandlung mit
Phycocyanin (100−300 mg/kg p.o.) dosis-
abhängig reduziert werden konnten. Die
Ergebnisse zeigen, daß Phycocyanin die
Produktion von TNFα und NO hemmt,
was vermutlich den antioxidativen Eigen-
schaften dieses Biliproteins zuzuschrei-
ben ist.
Romay et al. − Phycocyanin extract 733
Antioxidantien

1. Introduction All the experiments were conducted in accordance with the

Septic shock is a complex disease state characterised by
significant hemodynamic, cardiovascular and meta-
bolic disturbances which may result in multi-organ fail-
ure [1]. Experimental and clinical studies demonstrate
that exposure to endotoxin (lipopolysaccharide, LPS)
results in the release of various inflammatory mediators
although particular attention has been paid to the pro-
inflammatory cytokines e.g tumor necrosis-α (TNFα)
and the reactive oxygen (ROS) and nitrogen species,
such as nitric oxide (NO), which currently are con-
sidered to be key mediators of tissue injury and mortal-
ity in septic shock [2−5].
It has been also demonstrated that endotoxin and
TNFα represent the most potent stimulus for inducible
nitric oxide synthase (iNOS), which contributes to the
enhanced generation of related nitrooxy radicals and
subsequent alteration of organ and cellular physiology
ethical guidelines for investigations in laboratory animals and
were approved by the Ethical Commitee for Animal Experi-
mentation of the National Center for Scientific Research
(CNIC), Havana (Cuba).
2.3. TNFα assay
TNF was measured by cytotoxicity on L929 cells [13]. TNF
levels were determined using recombinant human TNFα
(BASF/Knoll, Ludwigshafen, Germany; specific activity 107 U/
mg) and expressed as ng/ml. Six mice per group were used and
results were compared by Student’s t-test. Results are expressed
as per cent inhibition of TNF production. All experiments were
repeated at least three times.
2.4. Measurement of serum nitrite
The amount of nitrate/nitrite in deproteinized serum was
measured by the Griess reaction [14], after reduction of all ni-
trate to nitrite by the enzyme nitrate reductase. The samples

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at all levels [6].
Recently it has been reported that phycocyanin, a bi-
liprotein obtained from microalgae Spirulina (Arthos-
pira) maxima, has antioxidant and ROS scavenging
properties [7] and also exerts anti-inflammatory activity
in in vivo experimental models of colitis in rats [8] and
arthritis in mice [9], in which NO, TNF-α and arachi-
donic acid metabolites are strongly involved in the in-
flammatory response [10].
Thus taking into account the former results we de-
cided to study a phycocyanin extract in endotoxin-
treated mice in order to determine its potential effects
on TNFα and NO levels in blood serum.
2. Materials and methods
2.1. Preparation of phycocyanin extract
Phycocyanin was extracted from microalgae Spirulina (Arthos-
pira) maxima by freeze-thawing of the cellular biomass in an
aqueous acidulated medium, as described for a Cuban patent
[11]. The blue powder thus obtained showed a peak in the vis-
ible absorption spectrum of 617 nm, which is very close to the
one reported for phycocyanin [12].
were mixed with equal amount of Griess reagent and thereafter
were incubated at 25 °C for 10 min and absorbance was meas-
ured at 550 nm using a microplate reader. Nitrite concentra-
tions were calculated by comparison with DO550 of a standard
solution of sodium nitrite (3−500 (mol/l). Six mice per group
were used and results were compared by Student’s t-test. Re-
sults are expressed as per cent inhibition of nitrite production.
All experiments were repeated at least three times.
3. Results
3.1. Effect of phycocyanin extract on TNFα levels
in mouse serum
1 h after LPS injection there was a significant increase
in TNF-α levels (Table 1). Phycocyanin dose-depen-
dently inhibited the TNF-α induction by LPS. This re-
duction was statistically significant at doses above 100
mg/kg. Chlorpromazine (4 mg/kg), used as control
drug, reduced almost totally the TNFα release. When
phycocyanin (300 mg/kg) was administered alone, no
effect on TNFα induction was observed (Table 1).
Table 1: Effects of phycocyanin extract on TNF-α levels in serum
of mice treated with LPS.

Treatment TNFα (ng/ml)

2.2. Animals and treatments
(mg/kg) Mean ± SE Inhibition (%)
Male OF1 mice obtained from the National Center for Labora-
tory Animal Production (Havana, Cuba) weighing 20-25 g were Saline 0.2 ± 0.055 −
used. LPS (Sigma, Serotype 055.B5 from E. coli) was given i.p Phycocyanin (300) 0.3 ± 0.07 −
LPS 79.2 ± 6.48 −
at doses of 0.5 mg/kg or 30 mg/kg for TNFα or iNOS induction, Phycocyanin
respectively, in 0.2 ml of sterile pyrogen-free saline. Phycocy- 50 + LPS 59.3 ± 8.6 25.1
anin was administered orally at doses of 50, 100, 200 and 300 100 + LPS 51.7 ± 4.2* 34.7
200 + LPS 46.1 ± 3.9** 41.7
mg/kg in 0.2 ml saline 1 h before LPS. A control group receiving
300 + LPS 11.9 ± 1.66** 84.9
LPS and two other groups receiving saline or phycocyanin CPZ (4 mg/kg) + LPS 0.4 ± 0.08** 99.4
alone were also included. Chlorpromazine (4 mg/kg) or dexa-
* p < 0.05, ** p < 0.01 vs. respective control (LPS + vehicle) by Stu-
metasone (30 mg/kg) used as reference drugs were adminis-
dent’s t-test (n = 6). The animals were treated with phycocyanin
tered i.p in 0.2 ml saline 30 min before LPS. The mice were (50, 100, 200, 300 mg/kg p.o.) 1 h before i. p. administration of LPS
bled from the retro-orbital plexus under light anaesthesia and (0.5 mg/kg). 1 h later blood samples were taken to measure TNFα
serum TNF and nitrites were measured 1 h and 18 h after ad- levels in serum. Chlorpromazine (4 mg/kg i.p, 30 min before LPS)
was used as control drug.
ministration of LPS, respectively.

Arzneim.-Forsch./Drug Res. 51 (II), 733−736 (2001)

734 Romay et al. − Phycocyanin extract

Table 2: Effects of phycocyanin extract on nitrite levels in serum
of mice treated with LPS.
Treatment NO2 (µmol/l)
(mg/kg)
Mean ± SE Inhibition (%)
Saline 65.7 ± 2.86
Phycocyanin (300) 76.2 ± 4.47
LPS 543.9 ± 46.3
Phycocyanin
100 + LPS 482.5 ± 35.4
200 + LPS 226.7 ± 39.5*
300 + LPS 119.2 ± 15.3*
Dexamethasone 65.3 ± 4.12*
(30 mg/kg) + LPS
* p < 0.05, versus respective control (LPS + vehicle) by Student’s t-
test (n = 6). The animals were treated with phycocyanin (100, 200,
300 mg/kg p.o.) 1 h before i. p. administration of LPS (30 mg/kg).
18 h later blood samples were taken to measure nitrate/nitrite le-
vels in serum. Dexametasone (30 mg/kg i.p., 30 min before LPS)
was used as control drug.
3.2. Effect of phycocyanin extract on nitrite levels
in mouse serum
LPS (30 mg/kg i.p) induced a remarkable increase in
nitrite concentration in mouse serum (543.9 ± 46.3
µmol/l) with respect to nitrite levels of untreated mice
(65.7 ± 2.86 µmol/l) when they were measured 18 h after
LPS injection.
Phycocyanin (100, 200, 300 mg/kg p.o) dose-depend-
ently decreased the nitrite levels in serum of mice
treated with LPS. However, there was no significant
change in nitrite levels in the serum of mice treated
with phycocyanin alone.
Dexametasone (30 mg/kg i.p) produced a remarkable
reduction in nitrite concentration up to control values
in the mice treated with endotoxin (Table 2).
4. Discussion
Currently there is a growing body of evidence that in-
crease of ROS such as superoxide (O2·-), hydrogen per-
oxide (H2O2), hydroxyl (OH·), nitric oxide (NO·) and per-
oxynitrite (ONOO-) are strongly involved in the patho-
genesis of septic and endotoxic shock and certainly they
are considered mediators of induction and develop-
ment of it in animals and humans [5, 15]. Also an in-
crease in malondialdehyde (MDA), conjugate dienes
and other products of lipid peroxidation have been
found in septic shock [5]. In agreement with these find-
ings some antioxidants and ROS scavengers exert a pro-
tective action against endotoxic shock in rodents inhi-
biting both TNF-α and NO· production [16, 17].
Very recently, we demonstrated that phycocyanin is
an antioxidant and ROS scavenger agent [7]. Thus, it
was able to scavenge OH· and alkoxy (RO·) radicals as
well as to inhibit luminol-enhanced chemilumines-
cence from zymosan-activated human polymorphonu-
clear leukocytes (PMNL) and microsomal lipid peroxi-
Arzneim.-Forsch./Drug Res. 51 (II), 733−736 (2001)
Antioxidants
dation induced by Fe+3-ascorbic acid. Also phycocyanin
exerted inhibitory effects in various experimental
models of inflammation in rodents [8, 9, 18] and evi-
dence has been provided that besides ROS scavenging
properties, the inhibition by phycocyanin of prosta-
−
− glandin E2 (PGE2) and leukotriene B4 (LTB4) production
− is also involved in its mode of action as anti-inflamma-
tory agent [19, 20].
11
58 In our opinion, the anti-inflammatory activity and
78 the inhibitory effects of phycocyanin on TNFα and NO
100
levels in the serum of mice treated with endotoxin are
consequence primarily elicited by the antioxidant prop-
erties of the biliprotein, although its inhibitory effects
on eicosanoids production may contribute to the ef-
fects on endotoxic shock. This view is scientifically sup-
ported by the fact that ROS are strongly involved in the
induction and development of the inflammatory pro-
cess, in arachidonic acid metabolism (e.g prosta-
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glandins and leukotrienes production) and in the pa-
thogenesis of endotoxic shock [15, 21]. On the other
hand, various antioxidants with ROS scavenging prop-
erties, leukotriene antagonists and lipoxygenase inhib-
itors protected mice against endotoxin-mediated organ
injury and reduced TNFα and NO· levels in blood serum
[22]. The effects of antioxidant agents have been
ascribed by some authors to inhibition of activation of
the nuclear transcription factors (NFkB) which is activ-
ated by ROS with the subsequent induction and expres-
sion of various cytokines and enzymes such as TNFα
and iNOS, respectively [16, 17], which are involved in
the induction and development of endotoxic shock.
Thus taking into account our results and the former
findings of other authors described above it is conceiv-
able that, as occurs with other antioxidants and ROS
scavengers, phycocyanin might exert its effects on en-
dotoxic shock by inhibition of activation of NFκB. This
may explain its inhibitory effects on TNFα, NO and
lipid mediators production. Currently research is in
progress in our laboratory in order to elucidate these as-
pects.
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Correspondence:
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Cheyla Romay,
42, 2295 (1999) Department of Pharmacology,
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on anti-inflammatory activity of phycocyanin in some animal Post Box 6412, Havana (Cuba)
models of inflammation. Inflamm. Res. 47, 334 (1998) E-mail: ozono@infomed.sld.cu

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736 Romay et al. − Phycocyanin extract