PROTECTIVE ROLE OF PHYLLANTHUS
AMARUS ON ENDOSULFAN INDUCED
NEUROTOXICITY: A PRE-CLINICAL
STUDY.
Principle investigator – Mr. Hashim. A
Content
 Introduction
 Review of literature
 Need for the study
 Aim and Objectives
 Methodology
 Expected outcome
 References
Endosulfan
 Endosulfan, a chlorinated hydrocarbon pesticide is active
against a wide range of insects and mites and is used in
many parts of the world
 Endosulfan is a neurotoxic pesticide belonging to the group
of cyclodienes. Endosulfan aerially sprayed on the crops
 Causes mild headache to severe poisoning functional
abnormalities of central nervous system and may even
result in death.
Phyllanthus amarus
 Phyllanthus amarus is a common herbaceous plant of
Euphorbiaceae family.
 It is commonly called as “stonebreaker.” It is commonly
seen in central and southernIndia and is found in many
other countries.
 This herb which nurtures up to 10–60 cm tall, with elliptic
leaves is used in India for curing a ailments like jaundice,
urogenital problems, dysentery, dyspepsia, arthritis,
ulcers, genito urinary tract infections, hemorrhoids.
Review of literature
 Tanuja Singh et al (2014) studied the the potential of
medicinal plant Eclipta alba to counter pesticide
endosulfan induced biochemical and haematological
alterations in Swiss albino mice. The biochemical analysis
showed that serum levels of SGPT, ALP, total protein,
albumin, urea, creatinine, total cholesterol and
triglycerides, statistically significant decreased in treated
group.
 Vanessa et al. (2012) did study and it shows that Endosulfan exposure
inhibits brain AChE activity and impairs swimming performance in
adult zebrafish
 Bharath BK et al. (2011) significant improvement in the immunity was
observed in the Ocimum sanctum treated groups indicates the
immuno modulatory property.
 Nathiya S et al. (2013) studied the effect of Hybanthus enneaspermus
to recover the infertility in endosulfan induced toxicity in male albino
rats.
Need for the study
 Since endosulfan has the potential to cause various ill effects
on humans, there are no effective drugs to mitigate the
deleterious effect of this pesticide.
 Various studies have proved that Phyllanthus amarus possess
hepatoprotective, antioxidant, antidiabetic, anticonvulsant
and antiamnesic activity.
 Hence this plant is chosen for the study to investigate on its
neuroprotective activity against endosulfan induced toxicity
in animals.
Aim
 To evaluate the protective role of Phyllanthus amarus
leaves against Endosulfan induced neurotoxicity in
experimental animal models.
Objectives of the study
 To study the protective role of Phyllanthus amarus
ethanolic extract on Endosulfan induced neurotoxicity
 To identify the active phytoconstituent of Phyllanthus
amarus responsible for the neuroprotective action by
HPLC-MS analysis.
Hypothesis
 Phyllanthus amarus plays an important role in alleviating
endosulfan induced neurotoxicity
Materials and methods
Materials:
 Animals: wistar albino rats
 Endosulfan:
 Endosulfan will be obtained from Sigma Aldrich. It will be
dissolved in groundnut oil and will be given at a dose of 10
mg/kg body weight orally for 60 days. Endosulfan dose is
selected based on earlier pre-clinical study.
 Plant material:
 Phyllanthus amarus plants will be cultivated during the
monsoon season
Group Drugs
Number of animals in
each group
Number of days,
each animal will be
housed
1
Normal [Distilled
water]
6
28
2.
Endosulphan (10
mg/Kg weight)
6
28
3.
Endosulp+ PAEE
(100 mg/kg for
next 14 days
6
28
Work plan
Phase Study
I
Procurement of Endosulfan
To confirm the neurotoxicity of Endosulfan
II
Basic pharmacological experiments for assessing the level of anxiety,
depression, cognitive impairment and seizurogenic potential
 Estimation of neurotransmitters and antioxidant enzymes in the brain
homogenate
 Brain histopathology
 Ladder assay for assessing the DNA damage
Further phases of this proposed work will be proceeded only after the results of Phase II
III
Cultivation of Phyllanthus amarus
IV
Preparation of Phyllanthus amarus ethanolic extract
V
To assess the protective role of Phyllanthus amarus ethanolic extract on Endosulfan
induced neurotoxicity
 Basic pharmacological experiments for assessing protective role of plant extract
on Endosulfan induced CNS disorders like anxiety, depression, cognitive
impairment and seizures.
 Estimation of neurotransmitters and antioxidant enzymes in the brain
homogenate
 Brain histopathology
 Ladder assay for assessing the DNA damage.
VI
Phytochemical analysis of Phyllanthus amarus ethanolic extract.

Expected Outcome
 The study is expected to provide vital information regarding the
neuroprotective action of the indigenous medicinal plant, Phyllanthus
amarus.
 Further studies can be taken up to test the efficacy of identified key
phytoconstituents as therapeutic agents to mitigate the neurotoxicity
caused by Endosulfan
Timeline
S. No.
Activities
1.
Review of
Literature
2.
Tool validation and
designing
3.
Pilot study
4.
Modification of tool
5.
Data collection
6.
Analysis &
interpretation
7.
Report writing
JanMar
I year
April-Jun July-Sep
Oct-Dec
Jan- Mar
II year
AprilJuly-Sep
Jun
Oct-Dec
15
Budget
Category
JRF salary for 2 years @ 16,000 X 24
months
Consumables [Endosulfan, chemicals for
estimation of neurotransmitters, ladder
assay, HPLC analysis etc.]
Contingency
Travel
Total
Year 1
1,92,000
Year 2 Total
1,92,000 3,84,000
1,00, 000
1,00,000 2,00,000
20,000
20,000
3,32,000
20,000 40,000
20,000 40,000
3,32,000 6,64,000
References
 1. Tanuja Singh, Anjali Singh, Nivedita, Sanjay Kumar Singh, Singh JK. Efficacy of
Eclipta alba (L.) against sub lethal dose of endosulfan induced biochemical
and haematological alterations in Swiss albino mice. Int. J. Pharm. Sci. Rev. Res
2014; 27(2): 216-221.
 2. Vanessa Maynart Pereira et al. Endosulfan exposure inhibits brain AChE
activity and impairs swimming performance in adult zebrafish (Danio rerio).
NeuroToxicology 2012; 33 : 469–475.
 3. Bharath BK, Anjaneyulu Y, Srilatha. Imuuno-modulatory effect of Ocimum
sanctum against endosulfan induced immunotoxicity. Veterinary World 2011, 4
(1):25-27.
 4. Nathiya S and Senthamil Selvi R. Anti‐infertility effect of Hybanthus
enneaspermus on endosulfan induced toxicity in male rats. Int J Med Biosci.
2013; 2(1): 28 ‐ 32.
References
 5. Kishor Haloi, Monikankana Kalita, Ramesh Nath, Aparajita Borkotoki. A
study on histopathological impact of endosulfan on liver of Channa
punctatus Bloch. International Journal of Research in Zoology 2014; 4(2): 1923.
 6. Ezhil Vendan S. Endosulfan ban in India: for good or not? Current Science
2011, 101 (11): 1398.
 7. Valérie Preud’hommea, Sylvain Milla, Virginie Gillardin, Edwin De Pauw,
Mathieu Denoël, Patrick Kestemonta. Effects of low dose endosulfan
exposure on brain neurotransmitter levels in the African clawed frog
Xenopus laevis. Chemosphere 2015;120: 357–364.
 8. Alva S, Damodar D, D'Souza A, D'Souza UJ. Endosulfan induced early
pathological changes in vital organs of rat: A biochemical approach.
Indian J Pharmacol 2012;44:512-515.
 9. Sabiha Khan. Histopathological effects of endosulfan in liver of albino
rats. World J. Biol. Med. Science 2014;1(2):1-6.
Thank you….