J. Env. Bio-Sci., 2016: Vol.

30 (1):87-93
(87) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

AMELIORATION BY SARPAGANDHA ROOT EXTRACT TO COUNTER DNA

DAMAGE INDUCED BY CARBOFURAN IN FISH USING RAPD TOOL
Vidyanand Tiwari, Manoj Kumar and Sunil P. Trivedi*
Environmental Toxicology & Bioremediation Laboratory,
Department of Zoology, University of Lucknow, Lucknow-226007, India
[Corresponding author E-mail*: sat060523@gmail.com]

Received: 14-01-2016 Revised: 10-06-2016 Accepted: 12-06-2016

An assessment of ameliorative potential of Rauvolfia serpentina (Sarpagandha) root extract in fresh water food fish, Channa
punctatus exposed to a carbamate pesticide, Carbofuran (CF) has been done for various haematological and biochemical parameters
and in vivo genotoxicity in terms of RAPD. The fish specimens were exposed to sub-lethal concentration of CF (10% of LC50 i.e. 0.09
gl-1) in a semi-static system and the aforesaid parameters were assessed in exposed and control fish. The DNA damage was
measured in blood cells by RAPD technique using oligo-nucleotide primers of fish specimens exposed to the sub-lethal concentration
of CF. The primers viz., OPA-01, OPA-02, OPA-03, and OPA-13 were used in RAPD pattern assessment. Appearance or disappearance
of bands and their intensities were evident in the RAPD pattern of fish specimens exposed to CF as compared to the control and the
Sarpagandha root extract treated group. Alterations in biochemical and haematological parameters were observed in CF exposed
fishes. However, these values showed that their levels tend to normalise on exposure to Sarpagandha root extract. This study
explores the ameliorative effect of Sarpagandha that can be used for providing healthy environment to fish. Findings of present
study have revealed the protective effect of Sarpagandha root extract against the carbofuran toxicity in fish Channa punctatus.

India is an agrarian country and 16% of its total GDP is
contributed by agriculture. She contributes 20% of all world
rice production 1. For better growth and production, agricultural
crops need a comprehensive cover of crop protection chemicals
(CPCs)-organically based traditional or modern synthetic
pesticides. Being cost effective and more efficient, synthetic
pesticides have competed the traditional methods used to
protect crop damages due to insect, pest, diseases & weeds.
India ranks second in Asia for pesticide production and tenth
in world as its total consumption amounts to about 500 million
tonnes 2. In India, 51% of food supplies are contaminated with
pesticide residues and out of these, 20% have pesticide
residues above the maximum permissible level 3. The
insecticides consumption in India accounts for about 80% of
total pesticide consumption 4. Besides, affecting targeted pests,
pesticides used in agricultural fields also adversally influence
a wide range of non-target aquatic organisms like invertebrates
and fish5-6.
The Carbofuran (CF) (2, 3-dihydro-2, 2-dimethylbenzofuran-7-
yl-methylcarbamate) is an insecticide of carbamate group and
marketed under the trade names Furadan and Curater. It is a
broad-spectrum, ubiquitous, systemic insecticide7 used for
better crop production at large scale8 particularly paddy and
sugarcane9. On account of its widespread use, carbofuran has
NAAS Rating (2016)-4.20
been detected in ground, surface and rain waters, and soils10-
11
and is highly toxic to aquatic animals and fishes in particular12.
It inhibits the acetylcholine esterase enzyme activity at
synapses in the CNS as well as neuromuscular junctions at
sub-acute concentrations13 and produces hypercholinergic
activity of central and peripheral nervous tissues 14. After
entering in the aquatic environment through agricultural runoff,
CF contaminates the water bodies. CF has been reported to
be toxic to several beneficial arthropods 15 and aquatic
organisms including different fish species15-17.
Continuously increasing concentration of CF in the environment
is found to be associated with several adverse effects18. Once
this pesticide gets entry into fish body it causes various
physical, physiological and biochemical disturbances19. The
alteration of these parameters can be used as sensitive health
indicators of the aquatic environment20. Fish are also excellent
subjects for the study of the mutagenic and/or carcinogenic
potential of contaminants present in water samples since they
are frequently exposed to a variety of toxicants and they have
the ability to metabolize, concentrate, and store waterborne
pollutants quickly21.
Toxic responses in fishes against xenobiotic stressors can
be efficiently recorded and monitored in terms of clinical
 AMELIORATION BY SARPAGANDHA ROOT EXTRACT
hematology and biochemistry besides genotoxic studies.
Blood is extremely sensitive to internal and external environment
perturbations as it is continuously involved in the transport of
various substances including pollutants22-23. Haematological
parameters are one of the most sensitive indicators of
physiological disturbances 24 thus, they can be used as indicator
of fish health status to detect physiological changes following
different stress condition like exposure to pollutant, diseases,
hypoxia25 etc.
Similar to haematological parameters, the biochemical
parameters such as enzyme levels, altered during toxic
exposure, also provide an idea for toxicity assessment 26.
Researches indicate that liver enzymes can be used as
biomarkers of cellular damage in blood plasma, protein
degradation and liver damage itself 27-28 and therefore the
activities of liver marker enzymes viz; Aspartate Transaminase
(AST), Alanine Transaminase (ALT) and Alkaline phosphatase
(ALP) can be used efficiently for assessing the xenobiotic
stress 29 as well as protective role of phytochemicals 27.
In addition to biochemical and heamatological toxicity,
genotoxic risks generated by pesticides in animals are also
significant, so that comprehensive toxicological profile of the
animal under xenobiotic stress can be worked out.
Genotoxicity of toxicants are governed by a variety of factors
viz., their stability or biological accumulation in the environment,
their in vivo metabolism, and their action on biomolecules viz.,
DNA, RNA and proteins 30. Besides the high stability of CF, its
toxicity also results in the production of free radical mediated
neural cells damage31. Furthermore, the free radicals have been
widely reported to generate genotoxicity due to their ability to
bind biomolecules32. Basic molecular technique, Random
Amplified Polymorphic DNA (RAPD) can be effectively utilized
here to detect xenotoxic damage. RAPD depends on
amplification of random segments of genomic DNA using the
polymerase chain reactions 33. Since it is a simple technique
and does not need prior knowledge of target sequence, this
tool has ample application in primary assessment of DNA
damage induced by genotoxic chemicals34-35. Further, the main
advantages of the RAPD technique is its rapidity and high
sensitivity to detect DNA damage and mutations 36 therefore,
this can be effectively use to study genotoxicity in fish induced
by pesticides.
(88)
MATERIAL AND METHODS
Experimental procedure: Fish acclimatization: Healthy
and live fresh water teleostian fish, Channa punctatus (15 ±
1.0 cm and 30 ± 2.0 g) were hand netted from local lentic
habitats in the vicinity of Lucknow. They were given prophylactic
treatment in formalin (0.4%) for 5 min followed by KMnO4 (1
mg l-1) for 30 minutes to keep away dermal infections, if any.
Fish were acclimatized under laboratory conditions
(Temperature, 14 to 22°C, dissolved oxygen, 6.62 to 6.76 mg/
l, alkalinity, 62 to 68 mg/ l) for 10 days in large glass aquaria
(100x40x40 cm3) before experimental exposure. During
acclimatization period, standard methods for fish maintenance
procedure were maintained by following the standard procedure
(APHA et al.,). During experiment they were fed with minced
goat liver and artificial fish food.
Technical-grade Carbofuran (CF) 3% G with product name
Furadan 3 G (manufactured by FMC India private Ltd., Hawrah
W. B. India) was purchased from the local dealer. The value of
96 hr-LC50 of CF was determined as 0.9mgl-1 by Trimmed
Spearman-Karber Method38. Based on 96 hr-LC50 value, the
sub-lethal fraction of CF i.e., 96 hr LC50/10 was estimated
0.09 mgl-1 and used for in vivo experiments. The dried roots of
Rauvolfia Serpentina (Sarpagandha) were purchased from
market of Lucknow. 40g of powdered root was extracted in
soxhlet apparatus with ethanol (1 L; 95%) for about 12 hr at
25ºC. After 12 hr, filtrate was concentrated till semi solid state
was achieved by rotary vacuum evaporator. This semi solid
ethanolic root extract was stored in refrigerator (4ºC) and used
for the experiments.
Experimental design: Ten days acclimatized fish were
divided into 3 groups having 10 fish in each group. Briefly,
group 1 was control, group 2 was treated with 96 h-LC50/10
fraction of CF alone and group 3 was treated with 96 h-LC50/
10 fraction of CF along with 10 ppm concentration of
Sarpagandha (SPG). After the end of stipulated exposure
periods viz, 24, 48, 72, and 96 hr. Fish were randomly selected
for collection of blood, from all groups. Triplicates were used
for each group to verify reproducibility of experimental results.
Haematological assay: For the estimation of hematological
parameters blood was collected from caudal vein (OECD 2005)
and stored in 5ml vials coated internally with anticoagulant
0.5% EDTA. Total Leucocyte Count (TLC), Hemoglobin
(89) TIWARI, KUMAR AND TRIVEDI
TABLE -1. Primers used for RAPD analysis
percentage (Hb%), Packed Cell Volume (PCV), Erythrocyte
Sedimentation Rate (ESR), and Clotting Time (CT), were
assessed by employing standard methods 39.
Biochemical assay: The biochemical parameters in terms of
liver marker enzymes such as AST, ALT and ALP were
determined by Ecoline diagnostic kits (Merck
No.11767400011730; 11766300011730; and 11767700011730,
respectively).
Random Amplified Polymorphism (RAPD) Analysis: For
RAPD analysis, genomic DNA was isolated from fresh blood
by using the phenol-chloroform extraction protocol.The
concentration and purity of DNA was determined by measuring
the absorbance of diluted DNA solution at 260nm and 280nm
by spectrophotometer (UV/visible-1800 Shimadzu). Random
primers with 10 bases were used for DNA amplification by
thermocycler (eppendorf flexlid mastercycler). PCR
amplification was carried out in 25µl reaction volume
containing 50ng genomic DNA, 100µM dNTPs, 40 nM
primer (Operon), 2.5 units of Taq DNA polymerase and 5µl
promega 10X Taq DNA polymerase buffer. PCR cycling
conditions were initial denaturation of 5 minutes at 94°C,
followed by 35 cycles of 1 minute at 94°C, 1 minute at 36°C
and 2 minutes at 72°C and finally, one cycle at 72°C for 5
minutes. Amplifed products resolved on 1.5% agarose gel,
were observed under UV illumination and images were captured
with a Gel Documentation System (Vilber Lourmat).
RESULTS AND DISCUSSION
Heamatological parameters viz. clotting time (CT), TLC, Hb%,
ESR and PCV were evaluated for different experimental groups
and were presented in Figs.- 1.A to 1.E. The present study
demonstrated a time dependent reduction in the clotting time
of blood of group 2 fish which were exposed to 1/10th fraction
96 hr LC50, in comparison to control group fish which were
not exposed to any toxicant. This reduction in CT of blood
was found to be highest (i.e. from 56±2.70 sec. in control
group fish to 52±1.2 sec. in group 2 fish) at 96 hr of exposure
period. However, this reduction of blood CT in fish of group 3
has been found be less evident and it is 55±1.11 sec., quite
close to CT value recorded in blood of fish of control group.
The group 3 fish were exposed with 96 hr-LC50/10 fraction of
CF along with 10 ppm concentration of SPG. The total
leucocyte count (TLC) was gradually enhanced in time
dependent manner on exposure of cabofuran in group 2 in
comparison to control group fish from 68±2.22 per cubic mm
to 86±3.2 per cubic mm, while after Sarpagandha treatment
along with CF in group 3, TLC of blood of fish (70±2.32 per
cubic mm) was less affected and was recoreded to control
value. Hb% was recorded decreasing throughout the exposure
periods in Carbofuran treated fish from group 2 in comparison
to Hb % of control group fish i.e. 10.6±0.22 and the highest
decrease (7±0.21) was recorded after 96hr exposure period.
This decrease was not pronounced i.e. 9±0.54 and was
recorded close to control value in blood of fish of group 3 in
which fish were treated with CF and SGF simultaneously. ESR
values were recorded increasing throughout the exposure
periods in Carbofuran treated fish from 8.0±0.27mm/hr to
9.1±0.23mm/hr but with simultaneous treatment with
Sarpagandharoot extract. ESR values were estimated
8.55±0.34mm/hr i.e. close to normal value (8.0±0.27). PCV
values were recorded decreasing to 28.4±0.22 in blood of fish
of Carbofuran treated group 2 from 34.3 ± 0.21 in blood of fish
of control group 1. On the other hand, simultaneously
Sarpagandha root extract and Carbofuron treated group 3, PCV
values were observed close to normal value 30.6±0.42. Thus,
some haematological parameters viz., Hb%, PCV, and CT
were reduced while other parameters like TLC and ESR were
increased in CF treated fish. The alteration in haematological
indices may be due to an appreciable decline in the
haematopoiesis endorsed by CF toxicity.
Hematological parameters are directly related to the response
of the animal to the pollution status of habitats where fishes
live Gabriel et al.,40 has stated that the fall in haemoglobin
concentration is due to decreased erythrocyte count on
exposure of pesticides. Similar reduction in Hb% and TLC in
carbofuran treated group was also recorded in the present
study that may be due to the toxic effect of carbamate
pesticides. Similar perturbations in haematological parameters
 AMELIORATION BY SARPAGANDHA ROOT EXTRACT (90)

FIGURE-1. A-E: Representation of different haematological parameters viz., CT, TLC, Hb%, ESR, and PCV of blood
of C. punctatus in different experimental groups after different exposure periods viz., 24hr., 48hr., 72hr.
and 96hr.

on exposure of quinalphos pesticide in silver barb fish have
also been documented41. Changes in these haematological
constraints are directly related to toxicity induced by toxic
chemicals 29, pollutants and other environmental factors 42. The
toxic effects of carbofuran were not pronounced in group 3
where fish were treated by carbofuran along with Sarpagandha
root extract and this can be attributed by its medicinal effects
on fish health. Likewise, Azmi and Qureshi22 have documented
the ameliorative potential of Sarpagandha root extract on blood
parameters of Alloxan treated mice.
in terms of activities of liver enzymes viz., AST, ALT and ALP
were found increased after exposuretocarbofuranin group 2 in
a time dependent manner. However, insignificant increase was
recorded in group 3 fish, exposed to carbofuran along with
Sarpagandha (fig.- 2.A to 2.C). The observed increase in the
activity of AST was recoded from 17.16 ± 1.068 IU/L in blood
of control group fish to 37.74 ± 0.697 IU/L in blood of carbofuran
treated group 2 fish. However, this increase was insignificant,
near to control value (26.61 ± 1.069 IU/L) in fish treated with
Sarpagandha along with carbofuran in group 3.

Several biochemical and physiological alterations occur when
a toxicant enters into an organism43. Carbofuran mainly works
by inhibiting the activity of the enzyme acetylcholinesterase
44
, which is an important enzyme in nerve impulse transmission.
Trotter16 have reported that carbofuran is extremely toxic to
fish and its 96 hr LC50 is less than 1 mg/L. Biochemical values
Likewise, inductive pattern of ALT activities from 22.48 ± 1.004
IU/L in control group fish to 39.24 ± 0.708 IU/L in carbofuran
exposed group 2 were recorded. This increase was more
evident in the group 2 where no Sarpagandha treatment was
given as compared to fish of group 3 treated with Sarpagandha
along with Carbofuran (32.71 ± 1.004 IU/L). Furthermore, ALP
(91) TIWARI, KUMAR AND TRIVEDI
FIGURE-2. A-C: Representation of different Liver
marker enzyme viz., AST, ALT and ALP of
blood of C. punctatus in different experimen
tal groups after different exposure periods
viz., 24hr., 48hr., 72hr. and 96hr.
FIGURE-3 : Randomly amplified polymorphic DNA
(RAPD) patterns in fish Channa punctatus;
Lane 1 represents patterns of marker DNA;
Lane 2-3 represent DNA patterns of fish of
control group 1; Lane 4-5 represent DNA
patterns of fish of group 2 treated with
carbofuran; Lane 6-7 represent DNA patterns
of fish of group 3 treated with Sarpgantha
root extract along with carbofuran.
values were recorded increasing in Carbofuran exposed group
2. The increase in ALP was from 20.114 ± 0.754 IU/L in control
group to 43.204 ± 0.818 IU/L in group 2 as compared to 29.447
± 0.748 IU/L recorded in fish treated with Sarpagandha along
with Carbofuran in group 3. Notable biochemical findings of
the present study are graphically presented in Fig.- 2.A to
2.C. Similarly, the ameliorative potential of Sarpagandha root
extract on ALT activity in Alloxan treated mice has been
reported by Azmi and Qureshi22.
The hepatotoxic chemical such as pesticide disturbs the
 AMELIORATION BY SARPAGANDHA ROOT EXTRACT
normal hepatic physiology but they can be maintained by
hepatoprotective agents 40. Phytochemicals are partly
responsible for the health benefits through a variety of
mechanisms. Present study justifies that the ethanolic extract
of Sarpagandha root extract possesses ample hepatoprotective
effect. In fact, the antioxidant phytochemical compounds
present in Sarpagandha root extract are responsible for its
hepatoprotective effect. Similar biochemical findings are also
observed by Khan et al. 27.
The differential genomic patterns of the amplified DNA generated
by using random primers was evaluated for the fish Channa
punctatus in control as well as treated groups. Primers were
used to amplify the random sequences and clearly detectable
bands were recorded on agarose gel (Fig.-3). These random
primers amplified and results obtained were varied from 130 to
1250 base pairs in the form of bands. On average 8.3±0.9
bands per primer were obtained. Maximum score-able bands
were similar and monomorphic for control and treated groups.
Moreover, most of these bands were monomorphic for fish of
control group and group 3 in which fish were co-exposed with
Sarpagandha and Carbofuran, whereas bands were not
similarly monomorphic in case of carbofuran treated group 2
fish. The DNA of the samples of fishes this group treated with
carbofuran reveals the appearance of average 30% new bands
(polymorphic) at 96 hr of exposure period which did not appear
in the samples of control group. These new bands could be
considered as genetic diagnostic markers which attributed by
Carbofuran toxicity. In addition, the results clearly reflect that
primers used herein show appearance of different polymorphic
bands in a time dependent manner in different treated groups.
The appearance of bands were obtained in group 3 is almost
close to the control group which indicates the protective effects
of Sarpgandha root extract in terms of genetic diagnostic marker
against the CF induced impairments in fish. Azami and
Quereshi22 reported the presence of alkaloids, carbohydrates,
flavonoids, glycosides, cardiac glycosides, phlobatannins,
resins, saponins, steroids, tannins, and triterpenoids in root
extract of Rauwolfia Serpentina. These compounds may be
attributed to impart ameliorative role in counteracting toxicity
generated by CF in C. punctatus. Khan and Sinha have also
reported vitamin C induced dose-dependent amelioration of
genotoxicity induced by the pesticides in mice. Similar findings
are also reported for DNA damage in terms of comet assay in
(92)
rat against carbofuran toxicity and their recovery by secondary
metabolites present in Ipomoea aquatica 46.
The hematological, biochemical and genotoxic investigations
are indicative of the fact that adverse effects of Carbofuran
pesticide on fish health can be effectively encountered by
remedial action of Sarpagandha root extract. Thus, this study
provides toxicological responses of Carbofuran and remedial
role of Sarpagandha root extract on three major physiological
departments of fish, viz.,haematological, biochemical and
molecular.Since, there is much concern about the toxicity of
pesticides to the native fauna, it is expected that the
optimization and cultivation of plant species that produce active
ingredients as secondary metabolites (Alkaloids, flavonoids
and polyphenols etc.,) will be beneficial for the aquaculture
industry and consequently be more profitable and safe for fish
production. We can conclude that Carbofuran has harmful
effects on fish metabolism and its genetic integration. Several
studies indicate that some plant extracts including Rauvolfia
serpentina showed an ameliorative activity due to its richness
in secondary metabolites. Thus, generated data and inferences
in present study will be helpful not only for pollution biologists
but also for fishery farmers to protect their fishery resources
against carbofurantoxicity by using naturally occurring plant
products. Outcome of the study may settle a milestone for
fishery biologists and policy makers in their efforts to conserve
fish biodiversity which is the need to overcome the challenges
of population explosion and concomitant shrinking of traditional
agricultural resources, viz., land farming.
ACKNOWLEDGMENTS
The first author gratefully acknowledge the UGC, New Delhi,
India for providing financial support in form of Major Research
Project (Award no. F-37-426/2009 (SR). Authors are thankful
to the Head, Department of Zoology, University of Lucknow,
U.P. India, for providing necessary facilities.
REFERENCES
1. Arjun, K. M.(2013). In: Indian agriculture-status, importance and
role in Indian Economy. 4: 343.
2. Shende, N. V, Section, S. & Section, S. (2013). Am. Int. J. Res.
Humanit. arts Soc. Sci. 25:80.
3. Gupta, P. K.(2004). Toxicology 198: 83.
4. Devi, N. L. & Raha, P.(2013). Bull. Environ. Sci. Res. 2: 9.
(93) TIWARI, KUMAR AND TRIVEDI

5. Burkepile, D. E., Moore, M. T. & Holland, M. M.(2000). Bull. Environ. 358: 131.
Contam. Toxicol. 64:114. 28. Vutukuru, S. S., Prabhath, N. A., Raghavender, M. & Yerramilli,
6. Peter, V. S., Babitha, G. S., Bonga, S. E. W. & Peter, M. C. S.(2013). A. (2007). Int. J. Environ. Res. Public Health 4: 224.
Aquat. Toxicol. 126: 306. 29. Blahova, J. Modra, H., Sevcikova, M., Marsalok, P., Zelnickova,
7. Gupta, R. C. (1994). J. Toxicol. Environ. Health 43: 383. L., Skoric, M., Svobodova,Z. (2014). Biomed Res. Int. 980948.
8. Cinar, O., Semiz, O. & Can, A. 1-9 (2015). doi:10.1093/toxsci/kfu 30. Crosby, D. G.(1982) In: Genetic Toxicology 201-218 (Springer
317. US, 1982). doi:10.1007/978-1-4684-4352-3_18
9. Battu, R. S., Singh, B. & Kang, B. K.(2004). Ecotoxicol. Environ. 31. Sharma, R. K. & Sharma, B. (2012). Dis. Markers 32: 153.
Saf. 59: 324. 32. Wu, D. & Cederbaum, A. I. (2003). Alcohol Res. Health 27: 277.
10. Arráez-Román, D., Segura-Carretero, A., Cruces-Blanco, C. & 33. Ali, B. A., Huang, T. H., Qin, D.-N. & Wang, X. M. (2004) Rev.
Fernández-Gutiérrez, A.(2004). Pest Manag. Sci. 60: 675. Fish Biol. Fish. 14: 443.
11. Caldas, S. S., Demoliner, A., Costa, F. P., D 'oca, M. G. M. & Primel, 34. Cenkci, S., Yildiz, M., Cigerci, I. H., Konuk, M. & Bozdag, A.
E. G. Artic.(2010). J. Braz. Chem. Soc 21: 642. (2009). Chemosphere 76: 900.
12. Begum, G. (2004). Aquat. Toxicol. 66: 83. 35. Danylchenko, O. & Sorochinsky, B. (2005). BMC Plant Biol. 5:
13. Singh, R. K. & Sharma, B. (1998). Pestic. Sci. 53: 285. S9.
14. Gupta, R. C., Milatovic, S., Dettbarn, W.-D., Aschner, M. & Milatovic, 36. Enan, M. R. (2006). Biotechnol. Appl. Biochem. 43: 147.
D. (2007). Toxicol. Appl. Pharmacol. 219: 97. 37. APHA, A. and W.(2012). In: Standard Methods for the
15. Saglio, P., Trijasse, S. & Azam, D. (1996). Arch. Environ. Contam. Examination of Water and Waste water. (American Public Health
Toxicol. 31: 232. Association, 2012).
16. Trotter, D. M., Kent, R. A. & Wong, M. P. (1991). Crit. Rev. Environ. 38. Hamilton, M. A., Russo, R. C. & Thurston, R. V. (1978). Environ.
Control 21: 137. Sci. Technol. 12: 417.
17. Ramesh, M., Narmadha, S. & Poopal, R. K. (2015). Beni-Suef 39. Sood Ramnik.(2010). In: Hematology for Students and
Univ. J. Basic Appl. Sci. 4: 314. Practitioners. (Jaypee brothers, 2010). doi:10.5005/jp/books/
18. Gupta, R. C. J. (1994). Toxicol. Environ. Health 43: 383. 11142
19. Saravanan, M., Prabhu Kumar, K. & Ramesh, M. (2011). Pestic. 40. Manna S., Bhattacharyya D., Mandal T. K., D. S. (2004). J Vet
Biochem. Physiol. 100: 206. Sci. 5(3):241.
20. Pimpão, C. T., Zampronio, A. R. & Silva de Assis, H. C. (2007). 41. Mohammod Mostakim, G., Zahangir, M. M., Monir Mishu, M.,
Pestic. Biochem. Physiol. 88: 122. Rahman, M. K. & Islam, M. S. (2015) J. Toxicol. , 41:59.
21. Bhatnagar, A.,Yadav, A., Cheema, N., (2016). Adv. Biol. , 1:6. 42. Tavares, M. J. Tavares, J. M., Ranzani, P., Ishikawa, M.C., Eilas,
22. Azmi, M. A., Naqvi, S. N. H.,Akhtar, K. M., Parveen, S.,Parveen, C. D., Risabffi, A., Da, V.S. (2004). Brazilian Arch. Biol. Technol.
R., Aslam, M. (2009). J. Environ. Biol. 30: 747. 47: 945.
23. Mayeux, R. (2004). NeuroRx 1: 182. 43. Dobsikova, R. (2003). Plant Prot. Sci. - UZPI (Czech Republic)
24. Al-Asgah, N. A., Abdel-Warith, A.W. A., Younis, E. S. M. & Allam, 39: 103.
H. Y. (2015). Saudi J. Biol. Sci. 22: 543. 44. Usmani, K. A., Hodgson, E. & Rose, R. L. (2004). Chem. Biol.
25. Walencik, J. & Witeska, M. (2007). Comp. Biochem. Physiol. C. Interact. 150: 221.
Toxicol. Pharmacol. 146: 331. 45. Khan, P. K. & Sinha, S. P. (1996). Mutagenesis. 11(1): 33.
26. Hernández, A. F. et al. (2013). Food Chem. Toxicol. 61: 144. 46. Datta, S., Sinha, M., Das, D., Ghosh, S. & Dhar, P. (2013). Indian
27. Khan, S. M., Sobti, R. C. & Kataria, L. (2005). Clin. Chim. Acta. J. Exp. Biol. 51: 1109.