Russian Journal of Marine Biology, Vol. 29, No. 4, 2003, pp. 259261.

Original Russian Text Copyright 2003 by Biologiya Morya, Khasina, Kolenchenko, Sgrebneva, Kovalev, Khotimchenko.

PHARMACOLOGY

Antioxidant Activities of a Low Etherified Pectin

from the Seagrass Zostera marina
E. I. Khasina, E. A. Kolenchenko, M. N. Sgrebneva,
V. V. Kovalev, and Yu. S. Khotimchenko
Institute of Marine Biology, Far East Division, Russian Academy of Sciences, Vladivostok 690041, Russia
e-mail: inmarbio@mail.primorye.ru
Received February 5, 2003

AbstractIn experiments on male white mice we initiated the induction of peroxidation of lipids by lead ace-
tate, tetrachloromethane, sodium nitrite, or SOVOL (a mixture of polychlorinated biphenyls). Then the animals
received intragastral injections of pectin isolated from the seagrass Zostera marina, in the form of a 1% gel, at
100 mg/kg for 2 weeks. It has been found that the low etherified pectin normalizes the level of malonic dialde-
hyde and the activity of glutathione reductase and glutathione peroxidase in the liver.

Key words: pectin, zosterine, malonic dialdehyde, glutathione reductase, glutathione peroxidase.

The development of many human diseases is
accompanied by activation of free radical peroxide oxi-
dation of lipids (POL), considered as a mechanism of
the destruction of biological membranes [16]. The
induction of POL occurs during inflammatory pro-
cesses, intoxications, cardiovascular problems, cancer,
allergic reactions, and many other diseases [17]. The
protection of cells and tissues against the aggressive
effects of free radicals is provided by the endogenous
antioxidant system. However, the endogenous antioxi-
dants cannot always prevent the development of oxi-
dant stress. For this reason, it is of interest to search for
some exogenous substances with antioxidant properties
that might be used as a drug for prophylactics or ther-
apy of diseases accompanied with increased reactions
of free radical oxidation [18].
In recent years some papers have appeared provid-
ing evidence about the high antioxidative activities of
polysaccharides isolated from marine brown algae [19,
20]. Our attention has been called to a polysaccharide
from the seagrasses of the family Zosteraceae called
zosterine and belonging to pectins. Like other pectins,
zosterine is characterized by the presence in the chem-
ical structure of polysaccharide chains constructed of
residua of galacturonic acid united by -1 4-glyco-
side bonds. One of specific feature of this pectin is the
low degree of etherification in its molecule [13]. Exper-
imental investigations with zosterine revealed in the lat-
ter some pharmacological effects, like antivirus, anti-
bacterial, antitumor, and hypocholesterolemic ones.
Clinical tests have demonstrated the effectiveness of
this substance in complex treatment of stomach ulcers,
cancer and urological diseases, and immunodeficient
conditions [2].
The aim of this project is to prove experimentally the
presence of antioxidant effects in the pectin-zosterine.
MATERIALS AND METHODS
The seagrass Zostera marina was collected in Peter
the Great Bay (Sea of Japan) from areas with clean
water. The isolation of the polysaccharide was per-
formed using a technique including acid hydrolysis of
the raw material, extraction with ammonium oxalate,
and precipitation of pectin by ethanol [14]. The concen-
tration of galacturonic acid in the pectin was deter-
mined using a colorimetric hydroxydiphenyl method
[7]. The degree of etherification was determined with a
titrimetric method [1]. The characteristic viscosity was
determined in a mixture of 0.05 M solution of sodium
chloride and 0.005 M solution of sodium oxalate at a
temperature of 25.0C and pH 6.0 using a viscosimeter
of Ubbelod [10]. The physical-chemical properties of
the examined sample of the isolated pectin were as fol-
lows: the concentration of hyaluronic acid equaled 74.8%,
the degree of etherification was 5.7%, and the characteris-
tic viscosity equaled 340 mg/g of galacturonan.
The experiments were performed on 63 male white
mice with a body weight of 2022 g. The activation of
POL was induced by different substances, like lead ace-
tate (20 mg/kg of body weight, if recalculated for the
metal), tetrachloromethane (300 mg/kg), sodium nitrite
(50 mg/kg), or SOVOL (5 mg/kg). The tested sub-
stances were injected intragastrally, on an empty stom-
ach, 60 min before the animals were fed, for 14 days.
Lead acetate and sodium nitrite were dissolved in dis-
tilled water; tetrachloromethane and SOVOL, in olive
oil. The SOVOL represented a mixture of tetra-, penta-,
hexa, and heptachlorobiphenyls. Fourteen days after,

1063-0740/03/2904-0259$25.00 2003 AI Nauka /Interperiodica

260 KHASINA et al.
Effects of low etherified pectin from the seagrass Zostera marina on peroxide oxidation of lipids and the glutathione-depen-
dent antioxidant system in the liver of mice exposed to the effects of destructive agents
Malonic aldehyde,
Group of animals
nmol/mg of protein
The control 5.4 0.3
Lead acetate 12.7 0.9
Lead acetate + pectin 8.0 0.5*
Tetrachloromethane 13.6 1.0
Tetrachloromethane + pectin 9.3 0.5*
Sodium nitrite 9.5 0.7
Sodium nitrite + pectin 6.0 0.5*
SOVOL 10.8 0.7
SOVOL + pectin 7.5 0.5*
P < 0.05, in comparison with the respective group that did not receive pectin.
the animals that took one of the inductors of POL were
split into two groups. The first group received standard
food used in the vivarium. The second group, once a
day, 60 min prior to the feeding, got intragastrally an
injection of pectin-zosterine in a dose of 100 mg/kg of
body weight of the animal, in the form of a 1% gel, for
14 days. The euthanasia of the animals, when the exper-
iments were completed was performed under an ether
anesthesia. In the liver we determined the concentration
of malonic dialdehyde [9], and the activity of glu-
tathione reductase [12] and glutathione peroxidase
[15]. The concentration of protein was determined with
a reagent of Folin [11].
The statistical treatment of the results was per-
formed with calculation of the arithmetic mean and the
mean error. The results are presented here as M m,
where M is the mean values from 7 animals and m is the
mean error. The reliability of differences has been esti-
mated with Students t-test.
RESULTS AND DISCUSSION
All tested substances used in this project caused in
the liver of the mice an increase in the level of malonic
dialdehyde at an average of 1.82.5 times and a
decrease in the activity of glutathione reductase and
glutathione peroxidase by 3154%. Fourteen days after
the cessation of the introduction of the inductors of
POL, the registered indices were still significantly dif-
ferent from those in the control animals. At the same
time, the injection of pectin caused normalization,
although not complete, in both the level of malonic
dialdehyde and the activities of both the enzymes (see
table).
The results of the experiments showed that after
enteric injection of the low etherified pectin isolated
from Zostera prooxidant processes are suppressed and
antioxidant mechanisms are restored. These effects of
the pectin were observed against the background of the
RUSSIAN JOURNAL OF MARINE BIOLOGY
Glutathione reductase, Glutathione peroxidase,
nmol/mg of protein/min nmol/mg of protein/min
51.2 4.7 296.5 19.4
34.8 2.7 198.3 12.7
48.8 3.1* 256.2 15.8*
23.6 2.2 162.2 14.3
39.8 3.0* 200.5 19.0
35.2 3.5 203.3 15.8
47.5 3.6* 280.4 20.0*
26.7 2.5 172.4 15.2
34.7 3.0 250.0 20.4*
effects of lead acetate, sodium nitrite, tetrachlo-
romethane, and chlorobiphenyls. Each of these sub-
stances has a particular mechanism of initial destruc-
tive effects, but in the end they all result in the activa-
tion of POL [5, 6, 8, 21].
The mechanism of antioxidant effects of the pectin-
zosterine is still unclear. It might be due not only to its
sorption properties that are the most expressed in the
range of pharmacological effects of low etherified pec-
tins [4]. It is possible that the pectin directly interacts
with oxidants and free radicals. This conclusion fol-
lows from in vitro studies of antioxidant properties of
polysaccharides isolated from brown algae Laminaria
japonica [20] and Fucus vesiculosus [19]. The possibil-
ity of resorptive effects of the low etherified pectin has
been confirmed by pharmokinetic studies on 3H-zoster-
ine [3]. The authors of this paper suppose that low
molecular fragments of zosterine (MW within 10000 D),
which constitute about 1520% of the entire polysac-
charide, could be absorbed into the blood from the
small intestine of the experimental animals and be
accumulated in the internal organs. In these cases a
direct antiradical effect of the pectin is quite possible.
REFERENCES
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2. Loenko, Yu.N., Artyukov, A.A., Kozlovskaya, E.P.,
et al., Zosterin (Zosterine), Vladivostok: Dalnauka,
1997.
3. Popov, A.M., Lyamkin, G.P., Artyukov, A.A., et al.,
Studies of Pharmakokinetics of zosterine, A Pectin from
a Seagrass Zostera asiatica, Dokl. AN SSSR, 1990,
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4. Khotimchenko, Yu.S., Kropotov, A.V., and Khotim-
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Vol. 29 No. 4 2003