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JBiopest, 5 (Supplementary): 250-254 (2012) 250
© JBiopest. 320
Antioxidant property of fresh and marine water cyanobacterial extracts
in Swiss mice
R. Navanietha Krishnaraj1, S. Venkatesh Babu2, B. Ashokkumar3, P. Malliga4and P. Varalakshmi2*
ABSTRACT
Continuous usage of pesticide is the main cause of cellular damage by generation of free radicals. Antioxidants
are intimately involved in the prevention of cellular damage. Hence the present investigation is mainly focused
to study the antioxidant property of cyanobacterial extracts from diverse environments in order to prevent the
free radicals toxicity. The alcoholic extracts of different cyanobacterial isolates including Oscillatoria salina
Synechcococcus, Oscillatoria annae, Oscillatoria chlorina, Spirulina sabsalsa and Spirullina platensis were
analyzed for their antioxidant property by physical body weight change, swimming time and biochemical
parameters (superoxide dismutase activity and total reduced glutathione activity) by using Swiss mice. Stress
was induced by forced swimming test and the antioxidant efficiency of cyanobacterial extracts was determined.
The results showed that Spirulina platensis possess significant antioxidant property and Synechcococcus sp
possess least activity when compared to other cyanobacterial isolates and control.
Key words: Body weight, cyanobacteria, reactive oxygen radicals, super-oxide dismutase
Antioxidant property of cyanobacterial extracts
INTRODUCTION
Pesticides are toxic; they are also potentially hazardous to
humans, animals and other living beings in the environment.
In this present scenario the continuous exposure to pesticides
causes severe cellular and molecular damage to humans and
other animals by generating free radicals. Antioxidants are
substances or nutrients in our food which can prevent or
slow the oxidative damage to our body. Phytonutrients and
pigments present in the cyanobacteria act as antioxidants
which facilitate the formation of the body’s defense against
free radical damage to cells. Antioxidants act as free radical
scavengers and prevent and repair damage done by the free
radicals. Reactive oxygen species (ROS)are often generated
either as byproducts of biological reactions or fro m
exogenous factors (Cerutti, 1991). It includes superoxide
radicals, hydroxyl radicals, singlet oxygen, and hydrogen
peroxide. ROS generallyplay a positive role such as energy
production, phagocytosis, regulation of cell growth and
intercellular signaling, or synthesis of biologically important
compounds (Halliwell, 1997). But, ROS may also play a
negative role; they can attack lipids in cell membranes and
also attack DNA, inducing oxidations that cause membrane
damage such as membrane lipid peroxidation and a decrease
in membrane fluidity, and also cause DNA mutation leading
to cancer (Pietta, 2000). An antioxidant is a substance that
present at low concentrations compared to an oxidizable
substrate has the ability toprevent or delay differenttypes
of cell damage. Theantioxidant defense mechanisms in
biological systems are of two types namely enzymatic and
non-enzymatic reactions. The enzyma tic a nt io xi da nts
includecatalase and hydroperoxidase. The non enzymatic
an tio xid ant s in clu de nut rie nt a nti oxi dants lik e
ca ro tenoids, atocopher ol, ascor bi c ac id, glut athione,
flavonoids, uric acid and plasma proteins such as transferrin,
albumin, metalothionein etc. (Luximon Ramma et al., 2002;
Serena et al., 2010).
There is a great demand throughout the worldin finding new
natural sources for antioxidants to preventoxidative damage
to living cells and to reduce the deterioration of food by
oxidation (Pratt, 1992). Traditionally, some antioxidants such
astea, wine, fruits, vegetables and spices are used from the
ancient days. Cyanobacteria are prokaryotic organism contains
a wide variety of antioxidant pigments than the plants and
mostalgal source (Robbins, 1987). Screening of cyanobacteria
for antibiotics and other pharmacologically active compounds,
has received ever-increasing interest as a potential source for
new drugs. Cyanobacteria are known to produce metabolites
with diverse biological activities such as antibacterial (Jaki et
al., 2000), antifungal (Kajiyama et al., 1998), antiviral (Patterson
et al., 1994), anticancer and antiplasmodial activity (Papendorf
et al., 1998). Recently antioxidant property of cyanobacteria
especially from O.annae has been reported by Rajavel et al.
(2011). Carotenoids are the most widely distributed and
structurally diverse classes of natural pigments predominantly
251
Navanietha Krishnaraj et al.
produced by cyanobacteria and that are doing important
functions in photosynthesis and nutrition. Also they have
potent anti oxidant activity. With this background of this
present study mainly focused to screen are the antioxidant
property offive diff erent cyanobacte ri al isolates like
Oscillatoria annae,O. chlorina, Spirullinasabsalsa,
Synechococcus andS. platensis in order to prevent the
oxidative damage caused by the pesticides because that are
absorbed on the surface of vegetables and fruits would cause
severe damage to the health of the animals while they consume
the fruits and vegetables.
MATERIALS AND METHODS
Swiss mice were the animal model used for this experiment.
Mid log phase culture of different cyanobacterial isolates
including O. salina, Synechcococcus,O .annae, O. chlorina ,
S. sabsalsa and S. platensis were collected from National
Facility for Marine Cyanobacteria, Bharathidasan University,
Tiruchirappalli. The cultures were grown BG11 and ASN
medium in a culture flask separately. The cultures were allowed
to grow till they reached the mid log phase. Five different
strains of cyanobacteria (1 g fresh weight) were homogenized
separately with glass powder and 75% alcohol using Mortar
and Pestle. The homogenized extracts were centrifuged at
5000 rpm for 10 minutes. The clear extract was separated and
dried using speed vac concentrator. Antioxidant effect of
different cyanobacterial extracts were analyzed by measuring
the level of antioxidantactivity before and after the stress
induction to the experimental animal on 1st, 14th and on 28th
day.
Swimming test
Stress was induced by forced swimming test. Induction of
Stress (Nagaraja and Jeganathan, 1999) was carried out in
polypropylene tub 90 cm height, 90 cm diameter and 60 cm
depth of water. The water was maintained at 18°C by adding
ice cubes to the container. Male albino rats of Swiss strain
(130 to 200g) were isolated into 19 groups and each group
contains 6 animals.
Analysis of superoxide dismutase and tota l reduced
glutathione activity
Animals were examined carefully, weighed and placed at room
temperature (30°C) in normal environmental conditions. They
were fed with normal diet (pellet) directly into the oesophagus
using curved feeding tube daily at 11:00 am. On 1st, 14th and
28th days the animals were weighed and were given stress.
The blood samples (2 mL) were taken for the analysis of
antioxidant effect by puncturing the retro orbital plexus directly
into heparinised micro capill ary tubeint o a tes t tub e
containing 0.1 ml of heparin. The physiological parameters
(bodyweight changes), the biochemical parameters super
oxide dismutase activity in haemolysate (Marklund and
Marklund, 1974) and total reduced Glutathione activity in
haemolysate (Patterson and Lazarow, 1975; Gul and Kutay,
2000) were analyzed.
Experimental animal groups
Five groups of animals were used for this study. They were:
Group A - A1 = 0.5 µg/L of Spirullina sabsalsa; A2 = 1.0 µg/
L of Spirullina sabsalsa; A3 = 1.5 µg/L of Spirullina sabsalsa;
Group B - B1 = 0.5 µg/L of Synechococcus; B2 = 1.0 µg/L of
Synechococcus; B3 = 1.5 µg/L of Synechococcus; Group C-
C1 = 0.5 µg/L of Spirullina platensis; C2 = 1.0 µg/L of
Spirullina platensis; C3 = 1.5 µg/L of Spirullina platensis;
Group D - D1 = 1.0 µg/L of Oscillatoria annae; D2= 1.0 µg/L
of Oscillatoria annae; D3 = 1.5 µg/L of Oscillatoria annae;
Group E - E1 = 0.5 µg/L of Oscillatoria chlorina; E2 = 1.0 µg/
L of Oscillatoria chlorina; E3 = 1.5 µg/L of Oscillatoria
chlorina; Group F - F1 = 0.5 µg/l of Oscillatoria salina; F2 =
1.0 µg/L of Oscillatoria salina F3 = 1.5 µg/L of Oscillatoria
salina.
RESULTSAND DISCUSSION
Total reduced glutathione activity
The biological antioxidant system has several enzymes to
protect the body from free radicals. Reduced glutathione is
one of the enzymes which can be considered as a marker for
anti oxidant act iv it y. Reduce d g luta thione is direc tl y
proportional to the amount of biological antioxidant activity.
GSH based antioxidant study shows that S. platensis has
higher reduced glutathione activity when compared with
O. salina,O. annae,O. chlorina Synechococcus sp and
S. sabsalsa.O. chlorina and Synechococcus sp has the least
GSH activity. O. salina, O. annae and S. sabsalsa have
intermid ia te act ivit y. So the exper iment reveal s that
S. platensis extract may prevent the oxidative damage caused
by pesticides.
Body weight changes
A body weight change is one of the physical parameters to
study the oxidative stress. Induction of stress increases the
body weight of mice. On supplementing the antioxidants, the
body weight decreases. The body weight has significantly
reduced in S. platensis when compared to O. salina,O. annae,
O.chlorina and S. sabsalsa. Obviously, S. platensis has higher
antioxidant activity. O. chlorina and Synechococcus sp. has
least GSH activity. O. salina, O. annae and S. sabsalsa have
intermidiete activity.
252
Swimming effects
Swimming time is one of the physical parameters to study
antio xidant activity. Induct io n of stress increases the
swimming time of mice. Use of antioxidant decreases the
swimming time. O. salina (0.5µg) has the lowest swimming
time and has the highest antioxidant activity. S. platensis
(1.5µg) also has a moderately higher antioxidant activity. O.
0
1 0
2 0
3 0
4 0
5 0
6 0
7 0
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C y a n o b a c t e r i a l e x t r a c t s
Figure 1. Effect of cyanobacterial extracts in the total reduced glutathione of Swiss Albino mice
salina,O. annae and S. sabsalsa take more time to swim and
have least antioxidant activity.
Analysis of Super Oxide Dismutase
Like reduced glutathione, SOD is another important natural
free radical scavenging antioxidant enzyme. So, the amount
of SOD expressed is directly proportional to the antioxidant
activity. S. platensis has higher SOD activity. Synechococcus
0
50
100
150
200
250
B
o
d
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w
e
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Cyanobacterial Extracts
Figure 2. Influence of cyanobacterial extracts in body weight of Swiss Albino mice
Antioxidant property of cyanobacterial extracts
Super oxide dismutase activity (µg/ml)
Body weight (g)
253
Navanietha Krishnaraj et al.
shows least SOD activity. O. salina,O. annae,O. chlorina
and S. sabsalsa exhibit moderate SOD activity.
The extract of S. platensis has the potent anti oxidant activity
in swiss albino mice. Hence S. platensis can be a new
pharmaceutically valuable source for the animals ingested
the with toxic pesticides in order to reduce the free radicals
formation.
REFERENCES
Ce rut ti, P. A. 19 91. Oxidant s tre ss and
ca rci nog ene sis. E uro pean J ourn al o f C lin ica l
Investigation,21: 1-11.
Figure 3. Influence of cyanobacterial extracts in swimming effect of Swiss Albino mice
Gul, M. and Kutay, F. Z. 2000. Cellular and clinical implications
of glutathione. Indian Journal of Experimental Biology,
38: 625-634.
Halliwell, B. 1997. Antioxidants and human diseases: a general
introduction.Nutrition Review,55: 44-52.
Jaki, B., Heilmann J. and Sticher, O. 2000. New antibacterial
me tab oli tes from the cyano bac ter ium Nos toc
co mmu n e( EAWAG 122 b). Jou rna l of N atu ral
Products,63: 1283-1288.
Kajiyama, S., Kanzaki, H., Kawazu, K. and Kobayashi, A. 1998.
Nostifungicidine, an antifungal lipopeptide from the field
0
5
10
15
20
25
30
35
40
S
u
p
e
r
o
x
i
d
e
d
i
s
m
u
t
a
s
e
a
c
t
i
v
i
t
y
(
µ
g
/
m
l
)
Cyanobacte rial Extracts
Fig 4. Effect of cyanobacterial extracts in scavenging of SOD in Swiss Albino mice
Super oxide dismutase activity (µg/ml)
254
grown terrestrial blue-green alga, Nostoc commune.
Tetrahedron Letters,39: 3737-3740.
Luximon Ramma, A., Bahorun, T., Soobrattee, M. and Aruoma,
O. I. 20 02. Anti oxi dan t ac tiv it ies of ph eno lic ,
proanthocyanidin andflavonoid components in extracts
of Cassia fistula.Journal of Agriculture Food Chemistry,
50: 5042–5047.
Marklund, S. and Marklund, G. 1974. Involvement of
superoxide anion radical in autooxidation of pyrogallol
and convenient assay for superoxide dismutase. Europian
Journal of Biochemistry,47: 469-474.
Nagaraja,H. S.and Jeganathan,P. S. 1999. Comparative study
of different types of stress on some physiological and
biochemical parameters in albino rats. Biomedicine,19(2):
137-149.
Papendorf, O., König, G. M., Wright, A. D. and Hirridin, B.
1998. 2,4-dimethoxy-6 epta decylphe nol, secondary
me ta bol ite s fr om the cyanob act eri um
Phormidium ectocar piwi th antipla smo dial activity.
Phytochemistry,49: 2383-2386.
Patterson, J. W. and Lazarow, A. 1975. Methods of biochemical
analysis. Interscience publishers inc. New York, 259 PP.
Patterson, G. M. L., Larsen, L. K. and Moore, R. E. 1994.
Bioactive natural products from blue-green algae. Journal
of Applied Phycology, 6: 151-157.
Pietta, P. G. 2000. Flavonoids as antioxidant.Journal of Natural
Products, 63: 1035-1042.
Pratt, D. E. 1992. Natural antioxidants from plant material,
Phenolic compounds. In: Food and their effects on health.
Ameri can Ch emi cal So c ie t y, Wa shing ton , (AC S
Symposium Series, 507): 54-71.
R. Navanietha Krishnaraj 1, S. Venkatesh Babu2,
B. Ashokkumar3, P. Malliga4and P. Varalakshmi2*
1Department of Biotechnology, Kalasalingam University,
Krishnan Koil, Tamil Nadu, India.
2Department of Molecular Microbiology, School of
Biotechnology, Madurai Kamaraj University, Madurai,
Tamil Nadu, India.
3Department of Genetic Engineer ing, School of
Biotechnology, Madurai Kamaraj University, Madurai,
Tamil Nadu, India.
4Department of National Facility Marine Cyanobacteria,
Bharathidasan University, Tiruchirappalli, Tamil Nadu
India.
*E.Mail: vara5277@gmail.com
Rajavel, R., Sivakumar, T., Jagadeeswaran, M., Rajesh, V. and
Malliga, P. 2011. Evaluation of in vitro and in vivo
antioxidant activity of Oscillatoria annae.The Internet
Journal of Pharmacology,9 (2).
Robbins, J. 1987. Anti-inflammatory and Antioxidant effects.
Diet for A new America: 20.
Schuler, P. 1990. Natural antioxidants exploited commercially,
In: Food Antioxidants (Hudson, B. J. F. ed.). Elsevier,
London. 99 -170 PP.
Serena, M. M., Balasubramani, M., Rajan, K. and Gerald, I. A.
J. 2010. Evaluation of the larvicidal activity of the leaf
extracts of Duranta erecta Linn. (Verbenaceae) on the
larvae of Culex quinque fascitatus (Say) (Culicidae).
Journal of Biopesticides,3(3): 582 – 585.
Received: September 04, 2011 Revised: October 10, 2011 Accepted: February 12, 2012
Antioxidant property of cyanobacterial extracts