IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS)

e-ISSN: 2278-3008, p-ISSN:2319-7676. Volume 10, Issue 5 Ver. I (Sep - Oct. 2015), PP 25-32
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Protective Role Of Alternanthera Sessilis (Linn.) Silver

Nanoparticles and Its Ethanolic Extract against Rotenone
Induced Parkinsonism
Sibi P Ittiyavirah
I. Introduction
Neurodegenerative disorders are characterized by a slow and progressive degeneration of neurons in
specific locations of the central nervous system. It is this topographical arrangement of neuronal death that
accounts for the specific clinical manifestations of each disease. Indeed, a major consequence of this neuronal
loss is to induce changes in the functioning of the neuronal circuits downstream to the lesions and these changes
are responsible for the clinical manifestation of the disease. In this context, changes in neuronal activity in motor
systems will induce motor symptoms such as those seen in Parkinsons disease (PD), Huntington disease (HD),
hemiballism, etc 1.Parkinsons disease (PD) is a progressive, disabling neurodegenerative disorder of unknown
cause, characterized by bradykinesia and at least one of the following symptoms resting tremor, muscle rigidity,
and postural instability 2-3. It is the second most common neurodegenerative disorder, after Alzheimers disease
4
. It is due to Damage or loss of dopaminergic neurons in this brain region results in the depletion of dopamine
from terminals in the striatum/nucleus caudatus. Due to the numerous protective barriers surrounding the CNS,
there is an urgent need for effective treatment for patients living with PD.
Recently, considerable attention has been paid to utilize herbal medicines for the treatment or
prevention of PD. The current study envisages in evaluating the anti parkinsonian effect of ethanolic extract of
Alternanthera sessilis (Linn.) (EEAS) and its silver nanoparticles (ASAgNPs) using oxidative stress model of
parkinsonism using rotenone. Alternanthera sessilis (Linn.) is a commonly known as sessile joy weed, found in
humid and warm regions of the world and it is used to relieve headache and dizziness5. In the present
investigation the preliminary phytochemical tests on ASE and ASAgNPs gave positive results for flavonoids,
steroids glycosides, carbohydrates and sterols, and that may be responsible for its biological activity. The use of
nanoparticles in drug delivery therapy holds much promise in targeting remote tissue. Silver nanoparticles used
as alternative strategies for drug delivery to alzhemier brain are able to cross the Blood brain barrier and
penetrate into the cell cytoplasm and induce underlying cellular change 6.The extracts of Alternanthera sessilis
(Linn.) have shown to exert significant antioxidant activity as in FARP and DPPH radical scavenging assay and
has shown Improved Superoxide dismutase and catalase activities in the livers of ovariectomized mice 7-8.
Hence in the present study an attempt was made to explore the possible anti parkinsonism activity of
Alternanthera sessilis (Linn.), keeping in mind its anti oxidant potential. Pathologically the hallmark of
Parkinson disease are the severe loss of the dopaminergic neurons in the substantia nigra pars compacta and the
presence of proteinaceous inclusion called Lewy bodies, which is mainly composed of fibrillar -synuclein and
ubiquitinated protein within some remaining nigral neurons. The degeneration of dopaminergic neurons results
in threshold reduction of approximately 80% dopamine in the striatum, which lead to the emergence of
neuromuscular executive dysfunction, learning problems and mood disorders. The cause of dopaminergic cell
death in Parkinsons disease remains unknown, but is associated with a number of factors that may cause
programmed cell death including calcium influx, reactive oxygen species (ROS) and mitochondrial complex I
inhibition 9. Rotenone model is one of the most accepted standard model for Parkinsonism. Rotenone, a specific
inhibitor of mitochondrial complex I leading to generation of reactive oxygen species 10. Several studies
suggested that ROS play a crucial role in neurodegenerative diseases. The reduced levels of endogenous
antioxidant molecules such as glutathione (GSH), antioxidant enzymes such as superoxide dismutase (SOD),
increased metabolism of DA and lipid peroxidation product malondialdehyde (MDA) in the brain could
contribute to neuronal death 11.

II. Materials And Methods

Experimental animal
Healthy male Wistar albino rats (150 - 200 gm) were obtained from the animal house of Department of
Pharmaceutical sciences, M.G University, Cheruvandoor, Kottayam. They were housed in well ventilated, large
spacious hygienic cages under standard animal husbandry conditions (22-28C) with relative humidity of 555
% and alternate 12 hour light-dark cycle. The animals were fed with standard food and water ad libitum. All
animals were acclimatized to the experimental environment prior to study.
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Plant
Alternanthera sessilis (Linn.) whole plant were collected from Kanjiramattom village of Ernakulum
district, Kerala, India and were authenticated at CMS College, Kottayam, Kerala. A voucher specimen is
preserved at the Herbarium with collection number. 782.
Drugs and chemicals:
95 % Ethanol, Silver nitrate (3Mm), Rotenone, Syndopa.
Preparation of A. sessilis (Linn.) Silver Nanoparticles
20 ml of the plant extract (2 g) was mixed with 80 ml of 3mM of silver nitrate solution 12. The colour
changed from yellow to reddish brown colour indicates the formation of silver nanoparticles. The ASAgNPs
thus obtained was purified by repeated centrifugation at 7000 rpm for 10 min. The pellet was collected and
dried. The chemical tests were carried out in ASAgNPs for Proteins and Vitamin C.
Characterization of biosynthesized silver nanoparticles of A. sessilis (Linn.)
UV spectra analysis: The reduction of pure silver ions was confirmed by measuring the UV spectrum
of the reaction mixture against distilled water as a blank. The Spectral analysis was done using double beam
Shimadzu 1800, Japan spectrophotometer at a resolution of 1 nm from 250 to 450 nm.
SEM analysis: Morphological characterization of the samples was done using FE-SEM (JEOL JSM
3600). A pinch of dried sample was coated on a carbon tape. It was again coated with platinum in an auto fine
coater and then the material was subjected to analysis.
Particle size measurement: Particle sizing experiments were carried out by means of laser
diffractometry, using Nano- ZS, Malveren Instrument (Zetasizer Ver. 7.03) serial No.MAL1008884.
Measurements were taken in the range between 0.1 and 10,000 nm.
Acute oral toxicity study
The oral acute toxicity study was carried out in adult female albino rats by the fixed dose method of
OECD Guideline No.420, the fixed dose method as in Annex 2d (OECD, 2000).
Rotenone model of Parkinsonism
Male Wistar rats were taken for the study and divided into five groups of six rats each. Group-I:
Normal control (received six subcutaneous injections of the vehicle (DMSO+PEG-300, 1:1 v/v) every 48h for
11days).Group-II: Disease control (received six subcutaneous injections of rotenone (1.5mg/kg, in a max
volume of 5ml/kg) every 48h for 11 days).Group-III: Received rotenone + Syndopa (10mg/kg) p.o daily for 11
days. Group-IV: Received rotenone + ASE (200mg/kg) p.o daily for 11 days. Group-V: Received rotenone +
ASAgNPs (20mg/kg) p.o daily for 11 days 13.The behavioural tests were conducted before the start of the
treatment, then regularly at an interval of 6 days post treatment and final behavioural quantification was done 1
hour of the last dose. The behavioural parameters assessed were locomotor activity with open field apparatus
and catalepsy using bar test 14-15. On 12th day animals were strangulated to death by cervical dislocation and the
brain was dissected out for biochemical estimations.
Catalepsy
The catalepsy was assessed by placing the animals forepaws on a horizontal bar positioned at 9 cm
above the bench surface. The duration of catalepsy, which was defined as an immobile posture, keeping both
forepaws on the bar, was measured up to a maximum of 180s.
Spontaneous locomotor behavior and abnormal involuntary movements (open field apparatus)
Open field apparatus consist of squares (61 61) were used for the study. Blue lines were drawn on the
floor with a marker. The lines divided floor into sixteen squares. A central square was drawn on the middle of
open field. The rats were centrally placed in the open field apparatus and were allowed to walk without restraint
inside the area for 5 minutes and following behavioral aspects were noted:
Ambulation: this was measured in terms of the number of squares crossed by the animal;
Rearing frequency: partial or total elevation on to hind limbs;
Self grooming: number of times animal groomed facial region, and licked /washed/ scratched various part of
the body. Two consecutive days animals were exposed to the apparatus for habituation. The open field was
cleaned with a 5 % water-alcohol solution before behavioral testing to eradicate possible bias due to smells left
by previous rats.
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III. Biochemical Estimations
Lipid peroxidation assay
Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is
used as an indicator of oxidative stress in cells and tissues. It was the process whereby free radicals steal
electrons from the lipids in the cell membrane resulting in cell damage. This process proceeded by a free radical
often affected poly unsaturated fatty acids because they contain multiple double bonds in between which lies
methylene- CH2 groups that poses especially reactive hydrolysis. Polyunsaturated fatty acid peroxides generate
malondialdehyde (MDA) upon decomposition. Malondialdehyde (MDA) formed from the breakdown of
polyunsaturated fatty acids serves as an index for the determination of the extent of peroxidation reaction.
MDA, a product of lipid peroxidation reacts with TBA (thiobarbituric acid) to give a pink coloured product
having absorption maxima at 535nm. 14
he tissue homogenate was prepared in 0.1N Tris-HCl buffer.1ml of the homogenate was combined with
2ml of TCA-TBA-HCl reagent and mixed thoroughly. The solution was heated in a boiling water bath for 15
minutes. After cooling the flocculent precipitate was removed by centrifugation at 1000xg for 10 min. The
absorbance of the sample was read at 535nm against a blank that contained no homogenate. 15
Reduced glutathione assay
GSH, the primary non-protein sulfhydryl in aerobic organisms is synthesized in most cells. The
ubiquitous tripeptide is formed by the ATP dependent condensation of glutamic acid and cysteine, catalyzed by
gamma glutamyl cysteinyl synthentase. Glycine is then added by glutathione synthetase to form GSH. In
addition to donating an electron during the reduction of hydroperoxides to the respective alcohols, GSH
functions as a co-substrate in the metabolism of xenobiotics catalyzed by glutathione-s-transferases. It is also a
co-factor for several metabolic enzymes and is involved in metabolic transport, functions as an antioxidant and
radioprotectant and facilitates protein folding and degradation.
Tissue was homogenized in 5ml precipitating solution (1.6g of glacial meta phosphoric acid, 0.2g of
disodium or dipotassium ethylene diamine tetra acetic acid (EDTA) and 30g of NaCl were placed in a 100 ml
flask and brought to volume with distilled water). The tubes were incubated for 5 min at room temperature and
then filtered through course grade filter paper. To 0.2ml filtrate, 3ml of 0.3M phosphate solution and 1ml of
0.04% DTNB was added. The tubes were capped, mixed by inversion and contents were read at 412nm within 4
min.16
Statistical analysis
The results of studies were expressed as mean SEM. The difference between control and treated means
were analyzed using one way analysis of variance (ANOVA). P-values < 0.05 were taken to be statistically
significant. Tukeys multiple comparisons test was used for multiple comparisons. The statistical analysis was
done by using graph pad prism version 6.05.
IV. Results
Characterization of biosynthesized Silver nanoparticles of Alternanthera sessilis (Linn.)
UVvis spectra analysis
Extracts from whole plants under study (A.sessilis Linn) showed rapid conversion of silver nitrate into
silver nanoparticles indicated by color changes from pale yellow to red-brown within few min of extract
addition in 3mM AgNO3 solution. A representative scheme of biosynthesis and UV-Vis spectrum is given in
Figure 1. Synthesized silver nanoparticles primarily characterized by UV-visible spectroscopy. ASAgNPs give
typical spectrum having maximum absorption in range of 420-450 nm. This absorption is unique property of
metal nanoparticles called SPR (Surface Plasmon Resonance) arises due to conduction of electrons on surface of
AgNPs. 17.
Scanning electron microscopy
Characterization of ASAgNPs under the study by Scanning electron microscopy revealed that
nanoparticles formed by A.sessilis Linn. are spherical in shape. (Figure 2)
Particle Size Analysis
Characterization of plant nanoparticles under the study by Particle size analysis revealed that
nanoparticles formed by A.sessilis (Linn.) had an average size of 122 - 396 nm. A well dispersed ASAgNPs
were found with respect to intensity in this range. (Figure 3)

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Catalepsy Test
Rotenone induced a significant increase in duration of catalepsy compared to vehicle group. EEAS and
ASAgNPs were orally administered to male Wistar rats and duration of catalepsy was measured. The duration of
catalepsy was significantly decreased on 12th day in EEAS (P<0.001) treated groups; 6th and 12th day in standard
treated group compared to Rotenone induced Parkinsonism group. There observed non significant decrease in
duration of catalepsy on 6th and 12th day in ASAgNPs group (Figure 4)
Open field Test
The open field test was done in order to determine the effect of EEAS and ASAgNPs upon spontaneous
locomotor activity. Rotenone group showed significant reduction in locomotor parameters compared to vehicle
group. Locomotor parameters were significantly (P<0.001) enhanced on 12 th day in EEAS, 6th and 12th day in
standard group when compared to Rotenone group. EEAS treated group was seen to produce a significant
increase in the ambulation (P<0.01), rearing (P<0.01) and self grooming activity (P<0.05) compared to
Rotenone group. ASAgNPs treated group also showed an improvement in locomotor activity compared to
Rotenone group (Figure 5a, 5b, 5c)
Biochemical estimation
Lipid peroxidase assay
The oxidative stress marker studies revealed that administration of Rotenone increased the levels of
LPO compared to the Vehicle group. The EEAS (200mg/kg) and syndopa treated groups reduced the lipid
peroxidative tissue damage as revealed by the significant (P<0.001) reduction in the cellular malonaldehyde
levels. The ASAgNPs showed a moderate effect on LPO. (Figure 6a).
Reduced glutathione assay
Rotenone administration induced a significant (P<0.001) decrease in the tissue GSH content as compared to
vehicle group. Syndopa and EEAS showed an increase in GSH level as compared to Rotenone treated group
(Figure 6b).

V. Discussion And Conclusion

In the present investigation the preliminary phytochemical tests on EEAS and ASAgNPs gave positive
results for flavonoids, steroids glycosides, carbohydrates, terpenoids and sterols, and that may be responsible for
its biological activity. A. sessilis (Linn.) is a commonly known as sessile joy weed, found in humid and warm
regions of the world 18. The use of nanoparticles in drug delivery therapy holds much promise in targeting
remote tissue. Silver nanoparticles used as alternative strategies for drug delivery to alzhemier brain are able to
cross the blood brain barrier and penetrate into the cell cytoplasm and induce underlying cellular change 19.
Increased generation of oxidative free radicals (OFR) or impaired antioxidant defence mechanism have been
implicated in neurodegenerative diseases and in chronic stress induced perturbed homeostasis including
immunosuppression, inflammation, diabetes mellitus, peptic ulceration, depression and other stress related
disease 20. The extracts of A. sessilis (Linn.) have shown to exert significant antioxidant activity as in ferric
reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging assay 21 and
it has shown to improve the levels of superoxide dismutase and catalase in the livers of ovariectomized mice 22.
Hence in the present study an attempt was made to explore the possible neuroprotective activity of A. sessilis
(Linn.), keeping in mind its anti oxidant potential.
Rotenone administration to rats caused a significant increase in duration of catalepsy, decrease in
locomotor and muscle activity. The current data thus suggested damage to the motor control system (DAergic
neurons) and development of Parknsons disease like behavioural symptoms in rats exposed to Rotenone. Open
field test and catalepsy tests were used to asses the behavioural alterations in rats. The anti-parkinsons effect
was evaluated on the 6th and 12th day after the EEAS and ASAgNPs treatment in Rotenone induced
neurotoxicity. The duration of catalepsy was significantly decreased following EEAS (P<0.001) treatment
compared to Rotenone group. (Figure 4). Locomotor parameters were significantly enhanced on 12th day
followed by EEAS treatment and this was evidenced from the results of open field test confirming antiParkinsons activity (Figure 5a to 5c). Enhancement in Locomotor parameters and decrease in duration of
catalepsy was also seen in ASAgNPs treated group but non significant.
Rotenone exposure in rodents also provides a valuble model for studying mechanism of oxidative
stress induced dopaminergic damage 23. Oxidative stress generated as a result of mitochondrial dysfunction,
particularly mitochondrial complex-1 imparment plays an important role in PD pathogenesis. Rotenone leads to
depolymerization of microtubules causing rupture of transport vesicles, which then leads to release of dopamine
in or near DA-ergic neurons, oxidation of which further damages DA-ergic neurons 24. Chronic complex I
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inhibition by Rotenone in rats caused oxidative damage to proteins, induced a decrease in GSH level and rise in
enzyme level.
The extent of lipid peroxidase was estimated by measuring the levels of malonaldehyde, a product of
lipid peroxidation. Polyunsaturated fatty acid peroxides generate malonaldehyde (MDA) upon decomposition.
Malonaldehyde (MDA) formed from the breakdown of polyunsaturated fatty acids serves as a convenient index
for the determination of the extent of peroxidation reaction. There is substantial evidence of oxidative damage in
the brains of Parkinson disease patients. Increased levels of the lipid peroxidation have been found in the
substantia nigra of Parkinson disease patients 25. Similar results were observed in the brain homogenates of
Rotenone-treated animals. EEAS showed significant (P< 0.001) decrease in lipid peroxidase as compared to
Rotenone group (Figure 6a).
A defect in one or more of the naturally occurring antioxidant defenses particularly GSH is an
important factor in etiology of Parkinson disease. A similar defect in GSH has been observed in the nigra of
Parkinson disease patients who have been diagnosed to have incidental Lewy bodies which has been established
as a preclinical symptom of Parkinson disease. 26
A reduction in GSH levels may impair H2O2 clearance and promote hydroxyl radical formation leading
to the generation of pro-oxidant milieu. A reduction in GSH levels was evident in Rotenone-treated animals.
EEAS showed a significant (P< 0.001) increase in level of GSH as compared to Rotenone group (Figure
6b).Increase in levels of GSH and decrease in levels of lipid peroixidase were also seen in ASAgNPs treated
group but non significant.
Thus, the Rotenone treated group showed a significant increase in the levels of MDA which is an
indication of extent of lipid peroxidation, decrease in the levels of GSH in the brain as compared to the control
animals. All these indicate an increase in the oxidative stress in the brain of animals treated with Rotenone.
Pretreatment with standard and EEAS resulted in a decrease in lipid peroixidase level and increase in the levels
of GSH, indicating its antioxidant effect in the brain of Rotenone treated animals.
A possible underlying mechanism of this protection can be associated with the presence of alkaloids,
poly phenols and flavonoids in the extracts, which are an important source of antioxidants 27. Oxidative
processes are an important factor in the pathogenesis of several disorders, and postmortem studies have
consistently implicated
oxidative damage in Parkinson disease pathogenesis 28. Thus, compounds with
potential antioxidant activity are notable candidates to become new therapeutic agents, since perspectives for
treatment of Parkinson disease in the future could include antioxidant therapies. Therefore, the results are
consistent with others studies which showed protective activity by substances such as alkaloids, poly phenols
and flavonoids, known by their antioxidant power in the same experimental model 29-30. Finally, the data
revealed that EEAS could be a potential therapeutic tool for neurodegenerative diseases. Active components of
this extracts have to be determined. The research for substance with neuroprotective activity has increased in
recent years. Since, oxidative stress produced in brain due to Rotenone toxicity seems to be important in
producing motor defects; therefore use of antioxidants could prove beneficial 31. The present study which thus
explored the potential of EEAS, proving to be an antioxidant, showed a promising effect in animals with
Parkinsonism disease. Rotenone can cause selective dopaminergic neurodegeneration in vitro and in vivo32.
Behaviour symptoms on Rotenone administration are due to the depletion of dopamine in the striatum, which is
caused by the dopaminergic neurodegeneration in substantia nigra. The change in dopamine level cause
neuronal dysfunction, leads to Parkinsonism symptoms. The restoration of behavioural and locomotor alteration
in Parkinsonism rats administered with EEAS indicates its potent antioxidant activity and presence of higher
levels of flavonoids and phenolics in plant. From the study we found that that EEAS and ASAgNPs have shown
protective effect against Rotenone induced Parkinsonism. The neuroprotective and ex-vivo antioxidant activity
of plants may be due to the presence of flavonoids and phenolic compounds. Significant neuroprotection was
produced by EEAS. Further studies are required for screening and evaluation of the particular phytoconstituents
present in plant, which might exhibit a protective effect in this study. In addition, further studies are needed to
explore other possible mechanisms involved in the neuroprotective effect of A.sessilis and to prove the safety
and efficacy of long term administration of EEAS and ASAgNPs.

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FIGURES

Figure 1: UV absorbance spectra at 444nm.

Figure 2: SEM image of ASAgNPs

Figure 3: DLS (Size distribution by intensity)

Figure: 4. Effect of EEAS and ASAgNPs on Catalepsy

test in rotenone model.

Figure 5b. Effect of EEAS and ASAgNPs in rearing

frequency on Open field test in rotenone model.

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Figure 5a. Effect of EEAS and ASAgNPs in

ambulation frequency on Open field test in rotenone
model.

Figure 5c: Effect of EEAS and ASAgNPs in self

grooming on Open field test in rotenone model.

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Figure 6a: Effect of EEAS and ASAgNPs on Lipid

peroxidation

Figure 6b: Effect of EEAS and ASAgNPs on GSH

Values are expressed as Mean SEM (n=6), analyzed by one-way ANOVA followed by Tukeys multiple
comparisons test, * Represents P < 0.05, **P <0.01, ***P<.0.001 when compared to rotenone group. a
Represents P<.0.001 when compared to vehicle only group.

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DOI: 10.9790/3008-10512532

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