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Antioxidantien ArzneimForschDrugRes

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),

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and Department of Pharmacology, Center for Pharmaceutical Chemistry b, Havana (Cuba)

Summary

Phycocyanin is a biliprotein which exerts TNFα levels in mouse serum. Phycocy-


antioxidative and anti-inflammatory ef- anin (50−300 mg/kg p.o), administered
fects in various in vivo and in vitro expe- 1 h before LPS, reduced dose-depen-
rimental models. In this study phycocy- dently the TNFα concentration in serum.
anin effects on tumor necrosis factor-α After 18 h, LPS (30 mg/kg i.p) also indu-
(TNFα) and nitrite levels in serum of ced a substantial increase in serum ni-
mice treated with lipopolysaccharide trite levels, which were reduced dose-de-
(LPS) was examined. TNFα was measured pendently by phycocyanin pretreatment
by cytotoxicity on L-929 cells and nitrite (100−300 mg/kg p.o). The results indicate
by the Griess reaction, after reduction of that phycocyanin exerts inhibitory ef-
all nitrates to nitrites by nitrate reduc- fects on TNFα and NO production which
tase. 1 h after LPS injection (0.5 mg/kg might be ascribed to the antioxidative
i.p) there was a significant increase in properties of the biliprotein.

Zusammenfassung
Key words
Wirkung von Phycocyanin-Extrakt auf ter Beteiligung der Nitratreduktase zu Ni-
die Serumspiegel von Tumornekrosefak- triten reduziert worden waren. 1 h nach 䊏 Endotoxic shock, mouse
tor-α und Nitrit bei Endotoxin-behandel- der Injektion von LPS (0.5 mg/kg i.p.) 䊏 Nitric oxide
ten Mäusen war ein signifikanter Anstieg der TNFα- 䊏 Phycocyanin
Spiegel im Serum der Mäuse zu verzeich- 䊏 Tumor necrosis factor-α
Phycocyanin, ein aus Mikroalgen ge- nen. Wurde Phycocyanin (50−300 mg/kg
wonnenes Biliprotein, besitzt antioxida- p.o.) 1 h vor der Verabreichung von LPS Arzneim.-Forsch./Drug Res.
tive und entzündungshemmende Eigen- gegeben, nahm die TNFα-Serumkonzen-
51 (II), 733−736 (2001)
schaften, wie in verschiedenen In-vivo- tration dosisabhängig ab. 18 h nach der
und In-vitro-Modellen festgestellt werden Gabe von LPS (30 mg/kg i.p.) wurde ein
konnte. In der vorliegenden Studie Anstieg der Nitrit-Serumspiegel beobach-
wurde die Wirkung von Phycocyanin auf tet, welche durch Vorbehandlung mit
Tumornekrosefaktor-α (TNFα)- und Ni- Phycocyanin (100−300 mg/kg p.o.) dosis-
trit-Spiegel im Serum von mit Lipopoly- abhängig reduziert werden konnten. Die
saccharid (LPS) behandelten Mäusen un- Ergebnisse zeigen, daß Phycocyanin die
tersucht. TNFα wurde anhand der Zytoto- Produktion von TNFα und NO hemmt,
xicität an L 929-Zellen gemessen, und die was vermutlich den antioxidativen Eigen-
Nitrite wurden mit Hilfe der Griess-Reak- schaften dieses Biliproteins zuzuschrei-
tion bestimmt, nachdem alle Nitrate un- ben ist.

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


Romay et al. − Phycocyanin extract 733
Antioxidantien

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


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

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

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734 Romay et al. − Phycocyanin extract
Antioxidants

Table 2: Effects of phycocyanin extract on nitrite levels in serum dation induced by Fe+3-ascorbic acid. Also phycocyanin
of mice treated with LPS.
exerted inhibitory effects in various experimental
Treatment NO2 (µmol/l) models of inflammation in rodents [8, 9, 18] and evi-
(mg/kg)
Mean ± SE Inhibition (%) dence has been provided that besides ROS scavenging
properties, the inhibition by phycocyanin of prosta-
Saline 65.7 ± 2.86 −
Phycocyanin (300) 76.2 ± 4.47 − glandin E2 (PGE2) and leukotriene B4 (LTB4) production
LPS 543.9 ± 46.3 − is also involved in its mode of action as anti-inflamma-
Phycocyanin tory agent [19, 20].
100 + LPS 482.5 ± 35.4 11
200 + LPS 226.7 ± 39.5* 58 In our opinion, the anti-inflammatory activity and
300 + LPS 119.2 ± 15.3* 78 the inhibitory effects of phycocyanin on TNFα and NO
Dexamethasone 65.3 ± 4.12* 100
(30 mg/kg) + LPS levels in the serum of mice treated with endotoxin are
consequence primarily elicited by the antioxidant prop-
* p < 0.05, versus respective control (LPS + vehicle) by Student’s t-
test (n = 6). The animals were treated with phycocyanin (100, 200, erties of the biliprotein, although its inhibitory effects
300 mg/kg p.o.) 1 h before i. p. administration of LPS (30 mg/kg). on eicosanoids production may contribute to the ef-
18 h later blood samples were taken to measure nitrate/nitrite le- fects on endotoxic shock. This view is scientifically sup-
vels in serum. Dexametasone (30 mg/kg i.p., 30 min before LPS)
was used as control drug. 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
3.2. Effect of phycocyanin extract on nitrite levels hand, various antioxidants with ROS scavenging prop-
in mouse serum erties, leukotriene antagonists and lipoxygenase inhib-
LPS (30 mg/kg i.p) induced a remarkable increase in itors protected mice against endotoxin-mediated organ
nitrite concentration in mouse serum (543.9 ± 46.3 injury and reduced TNFα and NO· levels in blood serum
µmol/l) with respect to nitrite levels of untreated mice [22]. The effects of antioxidant agents have been
(65.7 ± 2.86 µmol/l) when they were measured 18 h after ascribed by some authors to inhibition of activation of
LPS injection. the nuclear transcription factors (NFkB) which is activ-
Phycocyanin (100, 200, 300 mg/kg p.o) dose-depend- ated by ROS with the subsequent induction and expres-
ently decreased the nitrite levels in serum of mice sion of various cytokines and enzymes such as TNFα
treated with LPS. However, there was no significant and iNOS, respectively [16, 17], which are involved in
change in nitrite levels in the serum of mice treated the induction and development of endotoxic shock.
with phycocyanin alone. Thus taking into account our results and the former
Dexametasone (30 mg/kg i.p) produced a remarkable findings of other authors described above it is conceiv-
reduction in nitrite concentration up to control values able that, as occurs with other antioxidants and ROS
in the mice treated with endotoxin (Table 2). 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
4. Discussion progress in our laboratory in order to elucidate these as-
pects.
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 5. References
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736 Romay et al. − Phycocyanin extract