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Asian Journal of Biochemical and Pharmaceutical Research Therapeutic

efficacy of Pisonia alba and Cardiospermum halicacabum on the biochemical
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 Asian Journal of Biochemical and Pharmaceutical Research Issue 3 (Vol. 4) 2014 ISSN: 2231-2560
CODEN (USA): AJBPAD
Research Article

Asian Journal of Biochemical and Pharmaceutical Research

Therapeutic efficacy of Pisonia alba and Cardiospermum halicacabum on the

biochemical parameters of atrazine intoxicated liver tissue in fresh water fish Labeo
rohita.
Samantham Prabakaran, Kannaiyan Pugazhendy* and Anbazhagan Revathi

*
Department of Zoology, Annamalai University, Annamalai nagar-608 002, Chidambaram.

Received: 05 July 2014; Revised: 19July 2014; Accepted: 03 August. 2014

Abstract: Static bioassay were made to evaluate the therapeutic efficacy of dietary supplementary feed of
Pisonia alba and Cardiospermum halicacabum to counter stress exerted by the herbicide atrazine on fresh water
fish Labeo rohita. In the present experimental study Labeo rohita were exposed to sublethal concentration of
atrazine (20mg/L of atrazine) for 120 hours and certain biochemical parameters were estimated. The variation
of biochemical parameter like protein, glycogen and lipid level were found to be decreased in the liver tissue of
atrazine treated fish, simultaneously the glucose and amino acid level was increased compared to the control.
During the treatment of Pisonia alba and Cardiospermum halicacabum against atrazine exposed fish were
restored near normal level (Group III and IV). The observed results were discussed in detail.

Key words: Atrazine, Labeo rohita, Pisonia alba, Cardiospermum halicacabum, Biochemical, Liver tissue.

INTRODUCTION:

Atrazine is a herbicide and inhibits the photosynthesis in the target plants. It is water-soluble
and can be transported in dissolved form [1]. It has been detected consistently in water bodies [2]. It is
quite susceptible to leaching or runoff. Atrazine has also been reported in precipitation, so it can lead
to contamination of pristine water resources. Approximately 1 to 6% of the applied herbicides are
released to the aquatic environment. Aged and persistent herbicides can become recalcitrant due to
increased sorption and decreased bioavailability over time [3].

Cardiospermum halicacabum (Linn), family Sapindaceae, is a deciduous, branching,

herbaceous climber, which is distributed throughout the India. The whole plant has been used for
several centuries in the treatment of rheumatism, stiffness of limbs, snake bite [4]; its roots for nervous
diseases, as a diaphoretic, diuretic, emetic, laxative, refrigerant, stomachic and sudorific; its leaves and
stalks are used in the treatment of diarrhea, dysentery and headache [5] and as a poultice for swellings
[4]; Phytochemical constituents such as flavones, aglycones, triterpenoids, glycosides and a range of
fatty acids and volatile ester have been reported from the various extracts of this plant [6].

Pisonia grandis (Synmyn: Pisonia alba, Pisonia morindifolia) commonly known as

Leechikottai kerai in Tamil, Velati salet in Hindi [7]. The plant Pisonia grandis, belonging to the
family Nyctaginaceae, is an evergreen glaborous garden tree with young shoots are minutely
puberulous. It is native of Hawai island and naturalized throughout India. In the alternative system of
medicine Pisonia grandis leaves are used as analgesic, antiinflammatory, diuretic [8] hypoglycemic

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agent [9], antifungal [10]. It is also used in the treatment of ulcer, dysentery and snake bite. The leaves
are edible and mostly used to treat wound healing, rheumatism and arthritis [11]. Leaves also
consumed as vegetable and salad, fed to cattle [12].

Rohu (Labeo rohita) is a fish of the carp family Cyprinidae, found commonly in rivers and
freshwater lakes in and around South Asia and South-East Asia. It is an herbivore. It is treated as a
delicacy on Orissa, Bihar and Uttar Pradesh. In fact, the Kayastha community of Uttar Pradesh treats it
as one of their most sacred foods: to be eaten on all auspicious occasions. An attempt has been made to
evaluate the toxicity of atrazine and therapeutic efficacy of Pisonia alba and Cardiospermum
halicacabum on biochemical parameter such as protein, amino acid, glucose, glycogen and lipid in
liver tissue of Labeo rohita.

MATERIALS AND METHODS:

Experimental animal collection and maintenance: The freshwater fish Labio rohita were collected
from the VGM fish farm located in Kurinjipadi, Cuddalore district. The fish were brought to the
laboratory and transferred to the rectangular cement tanks (100 × 175) of 500 liters capacity containing
chlorine free aerated well water. The fishes measuring 14-16 cm in length and 70-80 g in weight were
selected irrespective of their sex for the experiments. During this time they were fed every 24 hour
with a commercial diet. The physico-chemical parameters of the water were monitored throughout the
acclimation period and remained constant (pH: 7.18 ± 0.5, conductivity: 118.25 ± 8.7 µS cm-1,
dissolved oxygen: 8.49 ± 0.9 mg O2 L-1, temperature: 21.96 ± 2.7 0C).

Experimental chemical: Experimental chemical atrazine was purchased from (TATA Atrataf 50%
WP) manufacture by Rallis India Limited, Mumbai.

Supplementary feed: Healthy disease free leaves of Cardiospermum halicacabum and Pisonia alba
were collected from in and around Chidambaram and Thiruvenkadu, the plant was identified. The
leaves were washed in running tap water for 10 minutes leafs were dried, aerial parts (1kg) of
Cardiospermum halicacabum and Pisonia alba were macerated thrice at room temperature and
prepared in powdered condition and equal amount of rice brane mixed well and small amount water
added and prepared small pellet as feed.

Biochemical analysis in liver: Liver tissue was dissected out and homogenized (10 × volumes) in
potassium phosphate buffer (0.1 M, pH 7.0) and centrifuged (20 min, 13000 g, 4 0C) for biochemical
analysis. The protein content of the sample was determined according to the method of [13] using
crystalline bovine serum albumin standard. The glycogen and glucose was estimated by the method of
[14]. The free amino acids were determined by the method of [15]. The total lipids were extracted
based on the procedure of [16].

Experimental design

Group- I: Fish exposed to tap water (control)

Group- II: Fish exposed to atrazine
Group-III: Fish exposed to atrazine along with Cardiospermum halicacabum
Group- IV: Fish exposed to atrazine along with Pisonia alba

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Group- V: Fish exposed to Cardiospermum halicacabum alone

Group- VI: Fish exposed to Pisonia alba alone

STATISTICAL ANALYSIS: The data obtained in the present work were expressed as means ± SE,
percentage changes and were statistically analyzed using student t-test [17] to compare means of
treated for the various biochemical parameters studies data against their control ones and the result
were considered significant at (P <0.05), (P<0.01) level.

OBSERVATION: In the present study observed that liver tissue biochemical parameters such as
glucose and amino acid levels are increased significantly at 5 % level (p<0.05) in the treated group II
(Table 2 and 3). At the end of 120 hours glucose and amino acid levels are increased when compared
to control group I. In the group III and IV glucose and amino acid levels are near to normal when
compared to group II. In the group V and VI glucose levels are decreased significantly at 120 hours
compared to group II and which was near to control group I.

Protein, glycogen and lipid levels were significantly decreased in the group II when compared
to control group I (Table 1, 4 and 5). In the group III and IV protein, glycogen and lipid level was
increased compared to group II. In the group V and VI protein, glycogen and lipid level increased
significantly at 120 hours compared to group II and which was near to control group I.

DISCUSSION:

Proteins are one of the important biochemical parameters which have been used to understand
the general state of health and biological mechanism of metabolism under pollutant stress. In the
present experimental result shows that the, levels of total protein were decreased when the fish
exposed to atrazine. But the group IV (atrazine along with Cardiospermum halicacabum) the protein
level was gradually increases when compared to the group II. The increases were may be due to the
presence of beneficial bioactive molecules having in C. halicacabum. Moreover, the group III
(atrazine along with Pisonia alba) also enhance the protein level very slowly than the group IV. Fish
under stress condition may also mobilize protein to meet energy demand to maintain increased
physiological activity [18]. Decreased protein levels may be due to starvation, malnutrition and acute
hepatic diseases [19]. Decrease in protein content of Clarias batrachus exposed to fenvalerate was
reported [20]. A reduction in protein content was also observed in Labeo rohita exposed to
cypermethrin [21].

Glycogen is the main reserve source of energy for animals during normal metabolism and their
content in liver of fish exposed to chemical may indicate the health condition of the fish. During
unfavorable environmental situation the normal metabolism is affected which in turn leads to
alteration in the glycogen reserve of fish. The present observation, glycogen level was decreased in
group II. But the group IV (atrazine along with Cardiospermum halicacabum) the glycogen level was
gradually increased when compared to the group II. Because of the increases was may be the presence
of beneficial bioactive molecules having in C. halicacabum. Moreover, the group III (atrazine along
with Pisonia alba) also enhance the glycogen level very slowly than the group IV. Glycogen depletion
in liver after toxic stress has been reported in several studies with aquatic animals [22]. Decrease in

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liver glycogen content of gold fish (Carassius auratus) exposed to pesticide was reported [23].
Situation such as toxic substances, including pesticides have been reported to decrease glycogen
content in tissues of fish [24]. Glycogen, because of its easy availability for energy production, is
rapidly catabolized resulting in loss of energy reserve. Similar reduction in glycogen content in
Clarias batrachus was observed after fish were exposed to pesticide dimethoate and Rogor [25].

Amino acids are essential intermediates in the process of protein synthesis and its degradation
products appear in the form of different nitrogenous substances. Amino acid and some nitrogenous
compounds play an important part during osmotic stress hence increase or decrease in free amino acid
content provide valuable information during stress phenomenon at the tissue level. In the present
investigation the amino acid level was increased in the liver tissue of L. rohita by the exposed of
atrazine. But the group IV (atrazine along with C. halicacabum) the amino acid level was gradually
decreased when compared to the group II. Because of the decreases may be the presence of beneficial
bioactive molecules having in C. halicacabum. Moreover, the group III (atrazine along with Pisonia
alba) also reduce the amino acid level very slowly than the group IV. [26] reported that increase in
concentration of amino acid attributed to stepped up proteolysis or increase synthesis of free amino
acid by transaminase reaction. The increase in amino acid level of tissue indicates stepped up proteases
activity and fixation of ammonia into keto acid [27]. [28] reported that the enhanced amino acid may
be due to depletion of reserved glycogen so that the fish can try to yield metabolic energy by
glueoneogenesis process.

Glucose is the major energy source in the body. Glycogen is the storage form of glucose and
glycogen is stored in liver. Pursuant to the present observation, level of glucose was increased in group
II at 120 hrs of sub lethal concentration of atrazine. But the group IV (atrazine along with
Cardiospermum halicacabum) the glucose level was gradually decreased when compared to the group
II. Because of the decreases may be the presence of beneficial bioactive molecules having in C.
halicacabum. Moreover, the group III (atrazine along with Pisonia alba) also reduce the glucose level
very slowly than the group IV. The storage or mobilization of metabolic substrates such glucose is
disrupted by exposure to several trace metals, including cadmium [29], manganese [30], nickel [31]
and metal mixtures in a polluted habitat [32]. Glucose might be rapidly converting into energy rich
adenosine triphosphate bonds (ATP) for sequestering the effect of variety of toxicants in the aquatic
habitat [33].

Pursuant to the present consequence, level of lipid was decreased in group II at 120 hrs of sub
lethal concentration of atrazine. But the group IV (atrazine along with Cardiospermum halicacabum)
the lipid level was gradually increased when compared to the group II. Because of the increases was may
be the presence of beneficial bioactive molecules having in C. halicacabum. Moreover, the group III
(atrazine along with Pisonia alba) also improve the lipid level very slowly than the group IV. Lipids
may be very transient body materials but they are an important source of potential chemical energy
and their presence or absence reflects the physiological capacity of fish [34]. The various reporter
pointed out that the decrease of liver total lipids in fish exposed to acute and chronic concentrations of
thiobencarb may be the value of energy production to satisfy the increasing demand of energy in the
studied fish on exposure to thiobencarb [35]. [36] studied the biochemical changes induced by nickel
and chromium in the liver of Cyprinus carpio and observed significant decline in the lipid level of
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liver and stated this may be due to toxicity stress which suppresses the activity of a number of
enzymes responsible for lipid transformation ultimately causing disturbance in lipid metabolism.

C. halicacabum plant having prodigious of bioactive molecules but among the molecules
the “Luteolin” has been studied in several preliminary in vitro scientific investigations. Proposed
activities include antioxidant activity (ie. scavenging of free radicals), promotion of carbohydrate
metabolism and immune system modulation. Other in vitro studies suggest luteolin has anti-
inflammatory activity [37] and that it acts as a monoamine transporter activator [38], a
phosphodiesterase inhibitor [39] and an interleukin 6 inhibitor [37]. At the same the P. alba having the
certain important medicinal properties but it compared to the C. halicacabum was less.

The preliminary phyto chemical studies of P. alba showed the presence of Vitamin A,
Vitamin C, thiamine, riboflavin, nicotinic acid (Vitamin B3), alkaloids, proteins and fats. Vitamin C is
one of the four dietary antioxidants, the others being Vitamin E, Vitamin A precursor β-carotene and
Selenium [40]. The chemical profile of C. halicacabum L. is relatively complete there is some
variability in the content of specific chemicals.[41] Reported the chemical profile: specified fatty acids
98.8 % of lipids; Oil content 31.60% by weight; Iodine value 71% by weight. However, noticed that
leaves contain considerable amounts of saponins, alkaloids, (+)-pinitol, apigenium, luteolin and
chrysoeriol. The major cyano lipid (49%) is a diester having two fatty acid moieties esterfied with 1-
cyano-2- hydroxymethyl-prop-2ene-l-ol followed by a diester derived from 1-cyano- 2-
hydroxymethyl-prop-2-ene-3-ol (6%). Of the fatty acids, 11- eicosenoic acid is the major component
(42%), other chief components of the oil include oleic acid (22%), arachidic acid (10%), linolenic acid
(8%), palmitic acid (3%) and stearic acid (2%) including small proportions (1- 2%) of a low-molecular
weight acid, and several C22 acids. Other minerals such as Ca (1.30%), K (4.01%), Mg (0.43%), P
(0.83%), Organic-N (5.19%), Total-N (7.16%), and C (48.1%) were recorded by [42]. These bioactive
compounds present in Cardispermum halicacabum and Pisonia alba which may give recovery to fish
in the presence of toxic stress.

CONCLUSION:

The present study concludes the exposure of Labeo rohita to sublethal concentration of
atrazine caused alterations in biochemical parameter such as protein, amino acid, glucose, glycogen
and lipid in liver tissues. These alterations in exposed fish suggest the operation of mechanism to cope
with the toxic stress of atrazine. Results of the recovery data suggest that the C. halicacabum may
protect on certain biochemical parameter such as protein, amino acid, glucose, glycogen and lipid is
reversible in nature than the P. alba. The C. halicacabum possess certain significant properties that
support its role in medicinal field. At the same the P. alba having the certain important medicinal
properties but it compare to the C. halicacabum was less. When compared to both plants, the C.
halicacabum having the valuable therapeutic properties was more.

ACKNOWLEDGEMENT:

We thank to the Head of Department of Zoology, Annamalai University for providing

necessary facilities for carried out this research work.

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Table 1. Variations of Protein levels (mg/g wet wt. of tissue) in the liver tissue of fresh water fish
Labeo rohita exposed to atrazine followed by the supplementary feed of Pisonia alba and
Cardiospermum halicacabum for 120 hours.
Hours of exposure
Groups
24 48 72 96 120
Group-I Control 80.486 ± 1.008 80.401 ± 1.112 80.340 ± 1.681 80.291 ± 0.986 80.198 ± 1.224
Group-II 76.980** ± 1.106 73.272** ± 0.874 70.110** ± 1.370 67.884** ± 1.110 63.112** ± 1.001
ATRAZINE -4.356 -8.866 -12.733 -15.452 -21.304
% COC 2.343 5.041 4.718 8.360 10.807
Group-III
77.654** ± 1.008 75.861** ± 1.112 72.610** ± 1.540 69.980** ± 0.888 66.083** ± 1.604
ATRAZINE+P.
-3.518 -5.646 -9.621 -12.842 -17.600
alba
0.875 3.533 3.565 3.087 4.707
% COC
1.909 2.888 3.391 7.776 6.998
% COT
Group-IV C. 79.180** ± 1.171 77.687** ± 1.076 75.273** ± 1.153 73.612** ± 1.440 70.983** ± 1.273
halicacabum -1.622 -3.375 -6.306 -8.318 -11.490
% COC 2.857 6.025 7.364 8.437 12.471
%COT 1.037 1.754 2.486 3.827 5.220
80.480NS ± 1.800 80.406NS ± 1.540 80.540NS ± 1.420 80.761NS ± 1.210 80.890NS ± 1.090
Group-V P. alba
0.007 0.006 0.248 0.301 0.862
% COC
0.002 0.002 0.090 0.301 0.422
Group-VI C. 80.516NS ± 1.371 80.610NS ± 1.254 80.680NS ± 1.117 80.864NS ± 1.084 80.991NS ± 1.063
halicacabum 0.037 0.259 0.423 0.713 0.988
% COC 0.017 0.124 0.168 0.391 0.489

Values are mean ± S.E- Mean of six individual observations and student t-test. Significant at *P< 0.05, significant at
**P<0.01 levels, (+,-) denotes decreased and increased. % COC (change over control); % COT (change over treated), NS-
Non-significant.

Table 2. Variations of Amino acid (µg/mg/wet wt. of tissue) content in the liver tissue of fresh
water fish Labeo rohita exposed to atrazine followed by the supplementary feed of
Pisonia alba and Cardiospermum halicacabum for 120 hours.

Hours of exposure
Groups
24 48 72 96 120
Group-I Control 10.880 ± 0.058 10.887 ± 0.073 10.895 ± 0.066 10.904 ± 0.043 10.915 ± 0.054
Group-II 11.009* ± 0.040 11.370** ± 0.078 12.086** ± 0.089 12.963** ± 0.072 13.877** ± 0.096
ATRAZINE % +1.185 % +4.436 % +10.931 % +18.882 % +27.136
% COC 1.842 4.556 10.827 24.807 26.927
Group-III 10.933NS ± 0.094 11.084** ± 0.040 11.316** ± 0.088 11.747** ± 0.220 12.024** ± 0.117
ATRAZINE+P. alba % +0.487 % +1.809 % +3.864 % +7.731 % +10.160
% COC % -0.690 % -2.515 % -6.371 % -9.380 % -13.353
% COT 0.481 2.373 3.827 3.763 8.664
Group-IV C. 10.954NS ± 0.042 11.117NS ± 0.096 11.806** ± 0.072 12.230** ± 0.077 12.884** ± 0.113
halicacabum % +0.680 % +2.112 % +8.361 % +12.160 % +18.055
% COC % -0.499 % -2.225 % -2.316 % -5.654 % -7.155
%COT 1.042 1.916 9.391 15.069 15.752
10.895NS ± 0.370 10.912NS ± 0.219 10.926NS ± 0.096 10.939NS ± 0.083 10.954NS ± 0.375
Group-V P. alba
% +0.137 % +0.229 % +0.284 % +0.320 % +0.357
% COC
0.040 0.108 0.267 0.376 0.103
Group-V IC. 10.899NS ± 0.377 10.926NS ± 0.115 10.939NS ± 0.273 10.952NS ± 0.110 10.965NS ± 0.098
halicacabum % +0.174 % +0.358 % +0.403 % +0.440 % +0.458
% COC 0.049 0.286 0.157 0.406 0.450

Values are mean ± S.E-Mean of six individual observations and student t-test. Significant at *P<0.05; significant at
**P<0.01 levels. (+,-) denotes decreased and increased. % COC (change over control); % COT (change over treated), NS-
Non-significant.

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Table 3. Variations of glucose (mg/g wet wt. of tissue) content in the liver tissue of fresh water
fish Labeo rohita exposed to atrazine followed by the supplementary feed of Pisonia
alba and Cardiospermum halicacabum for 120 hours.

Hours of exposure
Groups
24 48 72 96 120

Group-I Control 4.096 ± 0.044 4.195 ± 0.036 4.273 ± 0.022 4.290 ± 0.056 4.377 ± 0.047

4.883** ± 0.033 5.373** ± 0.044 5.974** ± 0.036 6.540** ± 0027 6.990** ± 0.043
Group-II ATRAZINE
19.213 28.081 39.808 52.447 59.698
% COC
14.309 20.910 40.05 36.290 41.476
Group-III 4.244** ± 0.066 4.290** ± 0.054 4.344** ± 0.037 4.396** ± 0.038 4.271** ± 0.028
ATRAZINE+ P. alba 3.613 2.264 1.661 2.470 2.421
% COC 13.086 20.484 27.284 32.782 38.898
% COT 1.873 1.484 1.651 1.582 1.962
Group-IV C. 4.370** ± 0.047 5.006** ± 0.056 5.112** ± 0.033 5.744** ± 0.027 5.980** ± 0.018
halicacabum 6.689 19.332 19.634 33.892 36.623
% COC 10.505 6.830 14.429 12.171 14.449
%COT 4.281 12.287 21.512 23.451 32.06
4.110NS ± 0.031 4.119NS ± 0.037 4.128NS ± 0.053 4.135NS ± 0.047 4.141NS ± 0.035
Group-V P. alba
0.341 1.811 3.393 3.613 5.391
% COC
0.264 1.490 2.543 2.123 4.068
Group-VI C. 4.112NS ± 0.047 4.210NS ± 0.057 4.224NS ± 0.063 4.298NS ± 0.065 4.380NS ± 0.073
halicacabum 0.390 0.357 1.146 0.186 0.068
% COC 0.25 0.223 0.742 0.094 0.034
Values are mean ± S.E-Mean of six individual observations and student t-test. Significant at *P<0.05; significant at
**P<0.01levels. (+,-) denotes decreased and increased. % COC (change over control); % COT (change over treated), NS- Non-
significant.

Table 4. Variations of glycogen (mg/g wet wt. of tissue) content in the liver tissue of fresh water
fish Labeo rohita exposed to atrazine followed by the supplementary feed of Pisonia
alba and Cardiospermum halicacabum for 120 hours

Hours of exposure
Groups
24 48 72 96 120
Group-I Control 50.563 ± 0.870 50.970 ± 0.973 51.116 ± 1.063 51.370 ± 0.777 51.417 ± 0.651
47.660** ± 0.117 44.387** ± 0.980 41.210** ± 0.888 37.878** ± 0.960 35.117** ± 0.770
Group-II ATRAZINE
-5.741 -12.915 -19.379 -26.272 -31.701
% COC
3.310 4.770 7.152 10.924 16.170
Group-III
48.370** ± 1.073 46.370 ± 1.123 43.870** ± 0.610 40.375** ± 0.873 38.873** ± 1.002
ATRAZINE+
-4.337 -9.024 -14.175 -21.403 -24.396
P. alba
+1.489 +4.467 +6.454 +6.592 +10.505
% COC
1.587 3.097 5.915 9.413 10.505
% COT
Group-IV
48.983** ± 1.003 47.544** ± 0.983 46.112** ± 0.917 44.373** ± 0.783 42.773** ± 0.783
ATRAZINE +
-3.124 -6.721 -9.789 -13.620 -16.811
C. halicacabum
+2.775 +7.112 +11.895 +17.147 +21.801
% COC
1.190 2.477 3.568 6.343 8.491
%COT
50.561NS ± 1.063 50.984NS ± 0.980 50.996NS ± 1.217 51.600NS ± 1.310 51.810NS ± 0.796
Group-V P. alba
+0.003 +0.027 +0.234 +0.447 +0.764
% COC
1.456 0.010 0.074 0.151 0.382
Group-VI 50.961NS ± 0.980 51.063NS ± 1.023 51.273NS ± 1.008 51.540NS ± 1.370 51.796NS ± 0.953
C. halicacabum +0.787 +0.182 +0.307 +0.330 +0.737
% COC 0.303 0.065 0.107 0.107 0.328
Values are mean ± S.E-Mean of six individual observations and student t-test. Significant at *P>; significant at **P<0.01 levels.
(+,-) denotes decreased and increased. % COC (change over control); % COT (change over treated), NS- Non-significant.

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Table 5. Variations of lipid (mg/g wet wt. of tissue) content in the liver tissue of fresh water fish
Labeo rohita exposed to atrazine followed by the supplementary feed of Pisonia alba
and Cardiospermum halicacabum for 120 hours.

Hours of exposure

Group 24 48 72 96 120

Group I Control 25.231±0.241 25.270±0.336 25.294±0.523 25.356±0.433 25.408±0.251

Group II 23.286**±0.354 20.541**±0.660 18.617**±0.227 17.158**±0.351 15.544**±0.236

Atrazine 7.708 18.713 26.397 32.331 38.822
% COC 4.544 6.390 11.714 14.718 28.674
Group III 23.916±0.658 22.336**±0.311 21.457**±0.314 20.556**±0.415 18.369**±0.283
Atrazine+ P. alba 5.211 11.618 15.169 18.930 27.703
% COC 2.705 8.738 15.254 19.804 18174
% COT 1.878 6.424 6.290 8.013 18.621
Group IV 24.220**±0.314 23.270**±0.412 21.936**±0.124 21.145**±0.214 19.654**±0.414
Atrazine+C. 4.006 7.914 13.275 16.607 22.643
halicacabum 4.010 13.285 17.827 23.236 26.441
% COC 2.559 3.766 6.253 8.736 11.888
% COT
Group V 25.248±0.314 25.397±0.418 25.452±0.371 25.527±0.416 25.541±0.261
P. alba 0.067 0.502 0.644 0.674 0.602
% COC 0.043 0.229 0.254 0.285 0.472
Group VI 25.251±0.313 25.386±0.196 25.422±0.317 25.459±0.618 25.565±0.287
C. halicacabum 0.079 0.459 0.506 0.406 0.613
% COC 0.050 0.298 0.209 0.136 0.409

Values are mean ± S.E-Mean of six individual observations; and student t-test. Significant at *P<0.05; Significant at ** P<0.01
levels. (+,-) denotes decreased and increased. % COC (change over control); % COT (change over treated), NS- Non-significant.

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*Correspondence Author: Kannaiyan Pugazhendy, Department of Zoology, Annamalai University,

Annamalai nagar-608 002, Chidambaram. INDIA.

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