Achyranthes aspera Attenuates epilepsy in

experimental animals: possible involvement

of GABAergic mechanism

Gollapalle Lakshminarayanashastry
Viswanatha, Marikunte
V. Venkataranganna, Nunna Bheema
Lingeswara Prasad & Ashok Godavarthi
Metabolic Brain Disease

ISSN 0885-7490

Metab Brain Dis

DOI 10.1007/s11011-017-9981-8

1 23
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 Author's personal copy
Metab Brain Dis
DOI 10.1007/s11011-017-9981-8

ORIGINAL ARTICLE

Achyranthes aspera Attenuates epilepsy in experimental animals:

possible involvement of GABAergic mechanism
Gollapalle Lakshminarayanashastry Viswanatha 1,2 & Marikunte V. Venkataranganna 3 &
Nunna Bheema Lingeswara Prasad 4 & Ashok Godavarthi 1

Received: 30 August 2016 / Accepted: 23 February 2017

# Springer Science+Business Media New York 2017

Abstract The present study was aimed to examine the possi-
ble anticonvulsant property of aerial parts of Achyranthes
aspera using various experimental models of epilepsy in mice.
Petroleum ether extract of aerial parts of A. aspera (PeAA),
methanolic eAA (MeAA) and aqueous eAA (AeAA) was ini-
tially evaluated against six-hertz seizure model in mice, based
on the outcomes the effective extract was further evaluated
against maximal electroshock (MES) and pentylenetetrazole
(PTZ) models in mice. In addition, the potent extract was
evaluated against the PTZ model by co-administering with
flumazenil (FMZ), and also evaluated for its effect on
GABA levels in brain and NMDA-induced lethality in mice.
Furthermore, the probable locomotor deficit-inducing prop-
erty of the extract was evaluated by actophotometer test in
mice. In results, only MeAA showed protection against six-
hertz-induced seizures in mice, based on these outcomes on-
ly MeAA was evaluated in MES and PTZ models. Notably,
the MeAA (200, 400 and 800 mg/kg) has offered mild and
dose dependent protection against MES and PTZ-induced
seizures in mice. Alongside, the MeAA (400 mg/kg) showed
a significant increase in GABA levels in the brain compared
to control, and in line with these findings the anti-PTZ effect
of MeAA (400 mg/kg, p.o.) was blocked when co-
administered with flumazenil (5 mg/kg, i.p.). However, the
MeAA has not shown significant protection against NMDA-
induced mortality and also did not cause significant change
in locomotor activity compared to before treatment. These
findings suggest that MeAA possess mild anticonvulsant ac-
tivity and the outcomes further confirmed the involvement of
GABAergic mechanism behind the anticonvulsant activity
of MeAA.
Keywords Achyranthes aspera . Anticonvulsant . Epilepsy .
Herbal medicine . Six-hertz seizures . MES –induced
seizures . PTZ-induced seizures . Gamma-amino butyric acid .
Diazepam

Electronic supplementary material The online version of this article

(doi:10.1007/s11011-017-9981-8) contains supplementary material, Abbreviations
which is available to authorized users. PeAA Petroleum ether extract of Achyranthes aspera ae-
rial parts
* Gollapalle Lakshminarayanashastry Viswanatha MeAA Methanolic extract of Achyranthes aspera aerial
glv_000@yahoo.com parts
AeAA Aqueous extract of Achyranthes aspera aerial parts
1
Radiant Research Services Pvt Ltd, Peenya Industrial Area, MES Maximal Electroshock
Bangalore 560058, India PTZ Pentylenetetrazole
2
No.387/511/A, Megalabeedi, Kengeri, Bangalore, Karnataka 560 HLTE Hind limb Tonic extensor
060, India HLTF Hind limb Tonic flexion
3
Connexios Life Sciences Pvt Ltd, Basavanagudi, Bangalore 560 004, GABA Gamma amino butyric acid
India NMDA N-Methyl-D-aspartic acid
4
Oil Technological and Pharmaceutical Research Institute (OTPRI), LD lethal dose
JNT University Anantapur, Ananthapuramu 515 002, India ED Effective dose
 Author's personal copy
Introduction
Epilepsy is a serious neurological disease affecting more than
50 million population worldwide (Behr et al. 2016), the inci-
dences of epilepsy are significantly higher in the elderly pop-
ulation than other age groups (Hauser et al. 1993). Currently,
many synthetic classes of anticonvulsant drugs are being used
either alone or in combination for the treatment of epilepsy
(Hernan and Holmes 2016). Moreover, recently many newer
drugs like vigabatrin, levetiracetam, topiramate, lamotrigine,
zonisamide, lacosamide, rufinamide, stiripentol have been de-
veloped for epilepsy treatment (Satinder and Suvasini 2013;
Mula 2016; Marvin 2010). Nonetheless, all the currently
available synthetic anticonvulsant drugs are prone to cause
one or more side/adverse effects such as impotence, dizziness,
mental slowing, ataxia, impaired concentration, mental confu-
sion, sleep disturbance, anorexia, somnolence, aggression and
so on (Mula 2016; Hamed 2016; Yang and Wang 2016). In
this scenario, there is a tremendous scope for development of
safe and potent drug for the management of epilepsy, in these
lines plant based remedies are well known and got better edge
over synthetic drugs, thus many researchers are extensively
working on herbal remedies to discover better and safe med-
icine for epilepsy. In this perspective, many plant based med-
icines such as Carum carvi L. (Showraki et al. 2016),
Kalanchoe pinnata Lam. (Mora and Hernandez 2016),
Citrus sinensis (Citraro et al. 2016), Abies webbiana
Lindl.(Parkash et al. 2015),Crocus sativus (Khazdair et al.
2015), Laggera aurita (Linn. F) (Malami et al. 2016), and
Dodonaea viscosa (Linn) (Karim et al. 2015) have been sci-
entifically proved to possess potent anticonvulsantactivity
against experimental models of epilepsy. In view of this,
Achyranthes aspera Linn., belongs to family
Amaranthaceae, has been extensively used in the traditional
system of medicine for the treatment of various ailments
(Talukder et al. 2012). In line with its potential usage in folk
medicine, various parts of the plant has been scientifically
proved to possess anti-hypergycemic (Talukder et al. 2012),
anticancer (Shagun 2014), diuretic (Saurabh et al. 2011), an-
tiobesity (Rani et al. 2012), antimicrobial (Ndhlala et al.
2015), antineoplatic and immunomodulatory (Narayan and
Kumar 2014), antiherpes (Mukherjee et al. 2013), antiarthritic
(Kothavade et al. 2015), antioxidant and hepatoprotective ac-
tivities (Khan et al. 2015), furthermore, Achyranthes aspera is
scientifically proved to possess various biological activities
related to central nervous system such as nootropic
(Gawande and Goel 2015), antianxiety (Chandana et al.
2012), antidepressant (Barua et al. 2009a, b), CNS depressant
(Bhosale et al. 2011a, b), and evidently various parts of the
plant has been effectively used in the management of epilepsy
by various folk medicine practicing communities in India
(Sharma et al. 2013). Based on the available literature on
antioxidant and neuroprotective properties of Achyranthes
Metab Brain Dis
aspera and its current use in folk medicine for the manage-
ment of epilepsy, the present study was undertaken to evaluate
the anticonvulsant activity of various extracts of Achyranthes
aspera aerial parts against experimental models of epilepsy.
Materials and methods
Drugs and chemicals
Phenytoin was purchased from Sun Pharma, (Mumbai, India),
diazepam and flumazenil was procured from Ranbaxy (New
Delhi, India), and pentylenetetrazole was purchased from
Sigma-Aldrich (Bangalore, India). All the liquid
chromatography-mass spectrometry (LCMS/MS) grade sol-
vents used in this study were procured from Merck Ltd.
(Mumbai, India). All other chemicals and reagents were of
analytical grade and were purchased from HiMedia
Laboratories Pvt. Ltd. (Mumbai, India).
Collection of plant material
The aerial parts of Achyranthes aspera was collected from
Kengeri, Bangalore (Karnataka, India), in the month of
June–July 2015, and it was identified and authenticated by
D r. K . M a d h a v a c h e t t y, P r o f e s s o r o f B o t a n y, S r i
Venkateshwara University, Tirupati. A voucher specimen of
the plant material is preserved in the department with Voucher
specimen number 2184.
Plant material preparation
The aerial parts of Achyranthes aspera was shade dried, pow-
dered, and subjected to extraction process with various sol-
vents (petroleum ether, methanol and water) in the ascending
order of their polarity. In brief, in a round bottom flask 100 g
of the plant material was initially defatted with 1 l of petro-
leum ether for 24 h, at room temperature; the marc obtained
was completely dried and extracted with 1 l of methanol at 60o
c for 48 h in Soxhlet apparatus, subsequently the marc obtain-
ed was extracted with 1 l of double distilled water in a round
bottom flask at room temperature for 48 h. Extracts obtained
were concentrated at room temperature under reduced pres-
sure using Rota-evaporator (Rotavap-Remi instruments).
LC-MS/MS analysis of Achyranthes aspera methanol
extract
The LC-MS/MS instrument used in our study comprised
an high-performance liquid chromatograph (HPLC)
coupled with an API 4000 mass spectrometer (Applied
Biosystem/MDS SCIEX, Canada) coupled with an
electron-spray ionization source with an electron
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Metab Brain Dis
multiplier detector and a chromatographic system. Batch
acquisition and data processing were controlled by using
Analyst® Software Version 1.5 (Applied Biosystems/
MDS Analytical Technologies, CA, USA).
The parameters of the mass spectrometer were opti-
mized by using a 1-mg mL-1 sample solution of
Achyranthes aspera methanol extract (MeAA) prepared
in Double distilled mineral (DM) water. The sample was
directly and continuously infused (flow injection analy-
sis) into the electron spray-MS (ES-MS) by using a
syringe pump. The intensity of the response was
checked in both the positive and the negative ionization
modes. A response with good intensity was obtained in
the positive mode. Other parameters, such as the de-
clustering potential (DP: 60–100 V), ion spray voltage
(IS: 4500 V), pressures of the nebulizing gas (GS1
45 pounds per square inch (psi)) and the heater gas
(GS2 = 55 psi), focusing potential (FP = − 400 V),
entrance potential (EP = 10–15 V), source temperature
(TEM = 450–500 °C) and EP 10–15 V, were optimized
with respect to the ionization-intensity response.
The mobile phase consisting of water and 0.2% formic acid
in pump A, and acetonitrile in pump B. The system was op-
erated at 0.7 ml/min flow rate, injection volume 2–14 μL (lin-
ear gradient of 2 microleters) with total runtime of 4.5 min.
The outcomes of LC-MS/MS analysis showed presence of
gallic acid, kaempferol, quercetin and rutin as chief active
ingredients, the LC-MS/MS chromatogram and compounds
identified from methanolic extract of Achyranthes aspera ae-
rial parts (MeAA) were given in Fig. 1.
Formulation of extracts for administration
Various concentrations of PeAA, MeAA, and AeAA were
prepared in 3% tween 80 in distilled water. In short, required
quantity of the extract was weighed and suspended in tween
80, and distilled water was slowly added with occasional stir-
ring, and made up to the volume with distilled water, the
concentration of tween 80 in the final formulation would be
3%. All the preparations were freshly prepared and adminis-
tered by oral route at 10 ml/kg dose volume.
Experimental animals
Inbred Swiss albino mice (25–30 g) were used for the study.
The animals were maintained in polypropylene cages at a
temperature of 25 °C ± 1 °C and relative humidity of 45%
to 55% in a clean environment under 12-h light/dark cycle.
The animals had free access to standard rat/mice pellets
(Pranav Agro Industry, Bangalore, India) and purified water
ad libitum.
All the experimental protocols were approved by
Institutional Animal Ethics Committee (IAEC) and were
conducted according to the principles and guidelines of the
Committee for the Purpose of Control and Supervision of
Experimentation on Animals (CPCSEA), India.
Experimental protocol
Acute oral toxicity studies
Acute oral toxicity of extracts of Achyranthes aspera aerial
parts were determined as per OECD guideline no. 425. In brief,
nulliparous and non-pregnant female mice weighing between
25 and 30 g were fasted for 3 h and single dose of extract was
administered by oral route, and the animals were observed for
the toxic signs and mortality, initially for 48 h (short term tox-
icity), and the survived animals were further observed for
14 days (long term outcomes). LD50 was calculated using
AOT425 stat program (OECD guidelines no.425, 2001).
Six-hertz seizure test
This test was performed as per Brown et al., (Brown
et al. 1953; Kaminski et al. 2004). In short, Swiss albi-
no mice were randomized based on the body weight
and they were pretreated with extracts (100 to
800 mg/kg, p.o.) for 3 days. On third day, sixty minutes
after the last dose, tetracaine ophthalmic solution (0.5%
tetracaine hydrochloride) was applied; after thirty mi-
nutes electrical stimulation was given to all the animals
(3 s, 32-mA monopolar rectangular pulses at 6 Hz)
through corneal electrodes using ECT Unit 5780 (Ugo
Basile, Comerio, Italy) constant current device. In gen-
eral, the electrical stimulation will induce a stunned
posture, associated with rearing and automatic move-
ments, which lasts from 60 to 120 s in control animals.
After the seizures, animals will regain their normal ex-
ploratory behavior.
The end point is considered as protection against the sei-
zures, and the animal gain its normal exploratory behavior
within 10 s of electrical stimulation is considered as being
protected (Magdalena et al. 2014). The percentage protection
is calculated using the equation given below.
  
No:of animal showed seizures
%Protection ¼ 100−  100
Total no:of animals used
Maximal electroshock-induced convulsions
In this method, in bred male Swiss albino mice (25–30 g
weight) were randomized and assigned in to six groups,
each group consisting of eight animals (n = 8). Group 1
and group 2 were served as control and standard groups,
received 3% tween 80 (10 ml/kg) and Phenytoin (25 mg/
 Author's personal copy
Compound Molecular formula
1 Gallic acid C7H6O5
2 Kaempferol C15H10O6
3 Quercetin C15H10O7
4 Rutin C27H30O16
Fig. 1 LC-MS/MS spectra of methanol extract of Achyrathes aspera aerial parts
kg) respectively. While, group 3, 4, 5, and 6 received 100,
200, 400 and 800 mg/kg doses of MeAA respectively, all
the treatments were given orally for three days. On day-3,
sixty minutes after the drug administration, tetracaine oph-
thalmic solution (0.5% tetracaine hydrochloride) was ap-
plied to the cornea and corneal electrodes were applied to
the cornea and electrical stimulation (60 mA of current for
0.2 s) was given for all the animals. Subsequently, all the
animals were individually observed in a rectangular
open top plastic cage for 30 min. The parameters like
period of hind limb tonic flexion (HLTF), hind limb
tonic extensor (HLTE), stupor, and death/survival were
recorded, and the percentage protection was calculated
using the survival rate (Chrościńska et al. 2016; Joshi
et al. 2015; Mishra et al. 2015).
Metab Brain Dis
Actual mass (g/moL) Observed m/z value in
LC-MS/MS spectra
170.12 171.9
286.23 287.1
302.23 303.1
610.52 611.1
Pentylenetetrazole-induced convulsions
Similar to MES model, male Swiss albino mice (25–30 g)
were divided into six (G-1 to G-6) groups (n = 8). Group
1 and group 2 served as control and standard groups,
received 3% tween 80 (10 ml/kg) and diazepam (5 mg/
kg) respectively. While, group 3, 4, 5, and 6 received 100,
200, 400 and 800 mg/kg doses of MeAA respectively, all
the treatments were given orally for a three days. On day-
3, one hour after the assigned treatments, PTZ (80 mg/kg,
i.p.) was administered intraperitoneally to all the animals,
and they were individually observed in an open top plastic
cage initially for 30 min, and later up to 24 h. During the
observation period, the parameters such as onset of clonus,
onset of tonic convulsions, and survival/death were
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Metab Brain Dis
recorded and expressed as percentage protection (Showraki
et al. 2016).
Effect of MeAA on GABA levels in the brain
This test was performed to evaluate the effect of single dose
(1 day) and multiple dose (3 days) administration of MeAA on
GABA levels in the brain. In this model, male Swiss albino
mice (25–30 g) were grouped and treated similar to PTZ mod-
el, in two different sets of experiments.
The first set (1 day treatment) and second set (3 days treat-
ment) experimental animals were sacrificed on day-1 and day-
3 respectively, 1 h after the last dose of MeAA. The brain
tissue was collected and frozen, and the cerebellum and whole
brain were separated using microtome blade. The whole brain
was weighed and transferred to 10 ml of ice-cold 0.1 M
Perchloric acid containing 15 μg/ml of Valine (internal stan-
dard). Under the ice-cold conditions, the contents were ho-
mogenized and centrifuged at 5000 rpm for 10 min at 4 °C;
the supernatant was separated and subjected for GABA esti-
mation as per Suher et al. (Suher et al. 2000).
Effect of flumazenil on anti-PTZ action of MeAA – to explore
the GABAergic mechanism
Similar to PTZ model, male Swiss albino mice (25–
30 g) were divided into seven groups (G-1 to G-7,
n = 8). Group 1, group 2 and group 3 served as control,
standard and MeAA alone treated groups, received 3%
tween 80 (10 ml/kg), diazepam (5 mg/kg, p.o.) and
MeAA (400 mg/kg, p.o.) respectively; while group 4,
group 5, and group 6 received vehicle (3% tween 80)
(10 ml/kg), diazepam (5 mg/kg, p.o.) and MeAA
(400 mg/kg, p.o.,) respectively, in combination with
the GABAA receptor antagonist flumazenil (3 mg/kg,
i.p.); group 7 received diazepam (5 mg/kg, p.o.) along
with MeAA (400 mg/kg, p.o.). Everyday, 15 min after
the assigned treatments the animals of the group 4, 5,
and 6 animals received flumazenil (3 mg/kg) intraperi-
toneally and other groups (group 1, 2, 3 and 7) received
1% dimethyl sulfoxide (DMSO) in sterile water (5 ml/
kg) intraperitoneally; all the treatments were given for
three days. On day-3, one hour after the assigned treat-
ments, PTZ (80 mg/kg, i.p.) was administered intraper-
itoneally to all the animals and observed similar to the
PTZ model (Showraki et al. 2016; Ishola et al. 2013;
Nogueira and Vassilieff 2000).
N-methyl-D-aspartic acid (NMDA) -induced lethality test
Swiss albino mice were divided into six groups of 10
animals each, Group 1 and group 2 served as control
and standard groups, received 3% tween 80 (10 ml/kg)
and Promethazine (80 mg/kg) respectively. While, group
3, 4, 5, and 6 received 100, 200, 400 and 800 mg/kg
doses of MeAA respectively, all the treatments were
given orally for three days. On day-3, one hour after
the last dose, all the animals were challenged with a
lethal intraperitoneal dose (200 mg/kg) of NMDA.
Subsequently, all the animals were observed for a period
of 3 h for their survival, and the results were expressed
as percentage protection against lethality (Moreau et al.
1989; Yamaguchi et al. 1993).
Number of animals survived
Percentage protection ¼ 100
Total number of animals used
Actophotometer test
The actophotometer test was performed to evaluate the effect
of MeAA on locomotor activity. In short, forty male swiss
albino mice (25–30 g) were assigned into five groups
(n = 8), and they were treated similar to PTZ model.
To measure locomotor activity, all the animals were
individually placed into actophotometer and photocell
counts were recorded for 10 min, the locomotor activity
was measured before and after the treatment
(Viswanatha et al. 2013). The change in the locomotor
activity was calculated using the following equation.
  
Photocell counts after treatment
%Reduction in locomotor activity ¼ 100−  100
Photocell counts before treatment
Statistical analysis
In Siz-hertz-seizure test and NMDA - induced lethality
test, the results were expressed as percentage protection.
All other experimental findings were expressed as
mean ± SEM. The outcomes of locomotor activity were
statistically analyzed by paired t-test and the data of the
remaining experiments were statistically analyzed by
one way ANOVA followed by Dunnett’s multiple com-
parison test using GraphPad Prism version 5.0 for
Windows, GraphPad Software, San Diego California
USA. The minimum level of significance was fixed at
p < 0.05.
Results
Extraction of plant material
The extractive values of petroleum ether (PeAA), methanol
(MeAA) and aqueous (AeAA) extracts were found to be 0.33,
8.75, and 10.24% w/w (gram by gram) respectively.
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Metab Brain Dis

Table 1 Anticonvulsant activity of Achyranthes aspera on 6-Hz Anticonvulsant activity

induced seizures in mice

Sl.no Treatment No. of animals % Protection Six-hertz (6 Hz) seizure test

showed
seizures/Total In this test, various doses of PeAA (100, 200 and
animals used
400 mg/kg), MeAA (100, 200, 400 and 800 mg/kg)
1 Control (3% tween 80) 10/10 0% and AeAA (100, 200, 400 and 800 mg/kg) were evalu-
2 Diazepam 5 mg/kg, p.o. 0/10 100% ated for the possible anticonvulsant property. In the out-
3 PeAA 100 mg/kg, p.o. 10/10 0% comes, except MeAA none of the extracts showed pro-
4 PeAA 200 mg/kg, p.o. 10/10 0% tection against six-hertz-induced seizures. Further, the
5 PeAA 400 mg/kg, p.o. 10/10 0% effect of MeAA was found to be mild and dose depen-
6 MeAA 100 mg/kg, p.o. 1/10 10% dent, and attained saturation effect after 400 mg/kg
7 MeAA 200 mg/kg, p.o. 3/10 30% dose. Notably, the reference standard diazepam (5 mg/
8 MeAA 400 mg/kg, p.o. 4/10 40% kg) has completely abolished six-hertz-induced Seizures.
9 MeAA 800 mg/kg, p.o. 4/10 40% The results are given in Table 1.
10 AeAA 100 mg/kg, p.o. 10/10 0%
11 AeAA 200 mg/kg, p.o. 10/10 0%
12 AeAA 400 mg/kg, p.o. 10/10 0%
Maximal electroshock-induced convulsions
13 AeAA 800 mg/kg, p.o. 10/10 0%
In six-hertz-seizure test, except MeAA none of the extracts
PeAA Petroleum ether extract of Achyranthes aspera aerial parts, MeAA showed protection, therefore only MeAA was shortlisted for
Methanol extract of Achyranthes aspera aerial parts, AeAA,Aqueous ex- further evaluation in MES and PTZ models. In this model, the
tract of Achyranthes aspera aerial parts
MeAA (200, 400 and 800 mg/kg) has decreased the duration
of MES-induced HLTE (P < 0.05 and P < 0.01), HLTF
(P < 0.05 and P < 0.01) and stupor (P > 0.05 and P < 0.01),
Acute oral toxicity study and also increased the survival rate (25% and 50%) compared
to control group. Further, the protective effect of MeAA was
Acute oral toxicity study for the plant extracts were dose dependent in 200 and 400 mg/kg doses, however there
performed as per OECD guidelines no. 425 up and was no significant difference between 400 and 800 mg doses
down procedure. The outcome of the study showed that in terms of anticonvulsant activity, it may be due to saturation/
the PeAA, MeAA and AeAA were safe up to 5000 mg/ sealing effect. Noteworthy, the reference standard phenytoin
kg, p.o. Further, no signs of toxicity were observed (25 mg/kg) has completely abolished the MES-induced con-
during short term (48 h) and long term (14 days) ob- vulsions and mortality. The ED50 value of MeAA was found
servation period. to be 585.36 mg/kg (Fig. 2).

Fig. 2 Effect of MeAA on MES-

induced convulsions in mice.
Note- MES, Maximal
electroshock, MeAA, Methanolic
extract of Achyranthes aspera
aerial parts, HLTE, hind limb
tonic extensor, HLTF, hind limb
tonic flexion, ns, Not significant.
All the values are expressed as
mean ± SEM (n = 8); means of
various groups were statistically
compared by one way ANOVA
followed by Dunnett’s multiple
comparison test using Graph Pad
version 5.0. *P < 0.05, **P < 0.01
compared with MES control
 Author's personal copy
Metab Brain Dis

Pentylenetetrazole-induced convulsions
In continuation with MES model, MeAA (100, 200, 400 and
800 mg/kg) was evaluated against PTZ-induced seizures in
mice. The animals of control group exhibited clonic and tonic
convulsions, and all animals were found dead as a conse-
quence of intraperitoneal PTZ (80 mg/kg) administration.
Remarkably, MeAA at 200 and 400 mg/kg doses has dose
dependently prolonged the onset of clonus (P < 0.05 and
P < 0.01), onset of tonus (P > 0.05 and P < 0.05), and in-
creased the survival rate (25% and 62.5%) compared to con-
trol group, further the anticonvulsant effect of MeAA at
800 mg was almost similar to 400 mg, which implies the
sealing effect and there was no significant change in anticon-
vulsant activity upon increasing the dose beyond 400 mg,
therefore for further studies 400 mg/kg dose of MeAA was
used. Exceptionally, the reference standard diazepam (5 mg/
kg) has ameliorated the PTZ-induced convulsions and
completely inhibited the mortality rate. The ED50 value of
MeAA was found to be 428.65 mg/kg (Fig. 3).
Effect of MeAA on GABA levels in the brain
The MeAA has evolved as a good anticonvulsant extract in all
the anticonvultant tests (Six-hertz-seizure test, MES and PTZ
models); Furthermore, to explore the mechanism behind the

Fig. 3 Effect of MeAA on PTZ-

induced convulsions in mice.
Note- MeAA, Methanolic extract
of Achyranthes aspera aerial
parts, ns, Not significant. All of
the values are expressed as
mean ± SEM (n = 8); means of
various groups were statistically
compared by one way ANOVA
followed by Dunnett’s multiple
comparison test using Graph Pad
version 5.0. *P < 0.01,
**P < 0.001 compared with PTZ
control
 Author's personal copy
Fig. 4 Effect of MeAA on GABA levels in brain. Note- MeAA,
Methanolic extract of Achyranthes aspera aerial parts; ns, Not
significant. The photocell counts are expressed as mean ± SEM (n = 8).
The basal values of the respective treatments were compared with post
treatment values by paired t-test using Graph Pad version 5.0. *P < 0.05,
**P < 0.01, ***P < 0.001 compared with control
Anticonvulsant activity, 200 and 400 mg/kg doses of MeAA
were evaluated for its effect on GABA levels in whole brain
other than cerebellum. The results showed significant eleva-
tion of GABA levels in whole brain, upon multiple dose
(3 days) (P < 0.01) administration of MeAA compared to
control. In similar lines, the reference standard diazepam
(5 mg/kg) also showed significant elevation of GABA com-
pared to control (Fig. 4).
Effect of flumazenil on anti-PTZ action of MeAA
In this test, MeAA (400 mg/kg) per se treatment showed sig-
nificant anticonvulsant effect against PTZ-induced convul-
sions (anti-PTZ effect). In contrary, when MeAA (400 mg/
kg, p.o.) was co-administered with flumazenil (5 mg/kg,
i.p.), the anticonvulsant effect of MeAA was completely
abolished, due to GABAA receptor antagonistic action of
flumazenil. In support of this, the anticonvulsant effect of
reference standard diazepam (5 mg/kg, p.o.) was also blocked
when administered along with flumazenil, which further val-
idated the method to confirm the GABAergic mechanism of
MeAA. The results are given in Fig. 5.
N-methyl-D-aspartic acid (NMDA) -induced lethality test
N-Methyl-D-aspartic acid-induced lethality test is a most
commonly used test to explore the possible role of
glutaminergic pathway in the anticonvulsant effect of the test
drug. In the present study, none of the tested doses of MeAA
(100, 200, 400 and 800 mg/kg) showed significant protection
against NMDA-induced behavioral changes, and lethality, at
high dose (800 mg/kg) MeAA showed 30% inhibition.
However, the reference standard, promethazine (80 mg/kg)
Metab Brain Dis
showed significant (90%) protection against NMDA-induced
mortality in mice. The results are given in Tables 2.
Actophotometer test
Since most of the currently available antiepileptic drugs pos-
sess the inherent character of inducing locomotor deficits, we
thought to evaluate the effect of MeAA on normal locomotor
activity of mice by actophotometer test; diazepam (5 mg/kg)
was used as a reference standard. Interestingly, MeAA did not
induce significant changes in locomotor activity in any of
tested doses (100, 200 and 400 mg/kg) compared to basal
locomotor activity. In contrary, the reference standard diaze-
pam (5 mg/kg) has significantly decreased the photocell
counts and thereby showed significant reduction in locomotor
activity (78.34%) compared to its basal values recorded before
treatment (P < 0.001). Furthermore, the percentage reduction
in locomotor activity in MeAA treatment was significantly
less compared to diazepam. The results are illustrated in
Fig. 6.
Discussion
In present study various extracts of Achyranthes aspera aerial
parts were evaluated for anticonvulsant activity in three dif-
ferent experimental models in Swiss albino mice.
Based on the recent recommendations of anticonvulsant
discovery program, all the extracts (PeAA, MeAA and
AeAA) were initially evaluated against six-hertz-seizure test
in mice (Wojda et al. 2009). The six-hertz-seizure test mimics
the drug-resistant epilepsy observed in humans, and it is con-
sidered as mild, effective, and economical model for the early
identification of anticonvulsant property of test samples
(Wolfgang 2011), and as per the guidelines, test drugs which
fail in conventional models (MES, PTZ etc.) are strongly
recommended to screen against Six-hertz- seizure test
(Wojda et al. 2009;Jarogniew; Jarogniew et al. 2012).
Indeed, six-hertz-seizure test resembles the clinical condition
of partial or limbic epilepsy associated with a minimal clonic
phase, followed by stereotyped and automatistic behaviors, in
this model most of the currently available anticonvulsant
drugs will fail to show protection, and hence called as drug-
resistant epilepsies (Wolfgang 2011). Hence, this model is
considered as highly useful in identifying even a very mild
anticonvulsant drug, and thus valuable in avoiding false neg-
atives. With the objective of identifying even a very mild
anticonvulsant sample, all the extracts were initially evaluated
against six-hertz-seizure test. However, in this test only
MeAA (100, 200, 400 and 800 mg/kg) has shown potent
anticonvulsant effect. Conversely, PeAA and AeAA were fu-
tile and only high dose of AeAA (400 and 800 mg/kg, p.o.)
showed very minimal inhibition. Based on these observations,
 Author's personal copy
Metab Brain Dis

Fig. 5 Effect of Flumazenil on

anti-PTZ action of MeAA in
mice. Note- FMZ, Flumazenil,
MeAA, Methanolic extract of
Achyranthes aspera aerial parts,
ns, Not significant. All of the
values are expressed as
mean ± SEM (n = 8); means of
various groups were statistically
compared by one way ANOVA
followed by Dunnett’s multiple
comparison test using Graph Pad
version 5.0. *P < 0.05,
**P < 0.01, **P < 0.001
compared with PTZ control

we thought to evaluate only MeAA against MES and PTZ-

Table 2 N-Methyl-D-aspartic acid (NMDA) -induced lethality test
induced convulsion models.
Sl.no Treatment No. of animals % Protection Furthermore, MES-induced epilepsy is a conventional and
survived/Total most widely accepted model for screening the drugs against
animals used
epilepsy, this model mimics the petit mal epilepsy observed in
1 Control (3% tween 80) 10/10 0% clinic (Carter et al. 2000). In principle, the electroshock deliv-
2 Promethazine 80 mg/kg, p.o. 9/10 90% ered in MES model is well known to potentiate the sodium
6 MeAA 100 mg/kg, p.o. 0/10 0% influx by opening of sodium channels, and also increases glu-
7 MeAA 200 mg/kg, p.o. 1/10 10% tamate levels, glutamate is an excitatory neurotransmitter, and
8 MeAA 400 mg/kg, p.o. 2/10 20%
it binds to NMDA receptors and induces the symptoms that
9 MeAA 800 mg/kg, p.o. 3/10 30%
exactly mimic the petit mal epilepsy in humans (Carter et al.
2000). With the detailed understanding of underlying mecha-
MeAA Methanol extract of Achyranthes aspera aerial parts, nism, it is stated and scientifically proved that, the agents
 Author's personal copy
Fig. 6 Effect of MeAA on
locomotor activity evaluated by
Actophotometer test. Note-
MeAA, Methanolic extract aerial
parts of Achyranthes aspera, ns,
Not significant. The photocell
counts are expressed as
mean ± SEM (n = 8). The basal
values of the respective
treatments were compared with
post treatment values by paired t-
test using Graph Pad version 5.0.
*P < 0.001 compared with their
respective basal values
which shows protection in this model will act either by
blocking the voltage-dependent sodium channels (phenytoin,
sodium valproate,) and/or by decreasing excitatory amino acid
levels and/or by antagonizing their actions (e.g., felbamate)
(Viswanatha et al. 2013).
Besides, PTZ is a potent antagonist at GABA receptor site,
this model is equally old and widely used as MES model;
however, this model mimics grand mal epilepsy in humans
(Huang et al. 2001). In fact, PTZ binds to GABAA receptor
and closes the chloride channel and thereby prevents the entry
of chloride, and prevents the neurons from undergoing hyper-
polarization, further PTZ is also known to decrease the GABA
levels and density of GABAA receptors in various parts of the
brain (Psarropoulou et al. 1994), all these changes together
leads to continuous stimulation of cortical neurons, and results
in convulsions similar to absence seizures/grand mal epilepsy
observed in humans (Huang et al. 2001). Hence, it is thought
that the agents which shows protection in this model would act
either by acting as GABAA receptor agonists (like diazepam),
and/or by enhancing GABA levels in brain, and/or through
enhancing GABAA receptor density in the brain (Viswanatha
et al. 2013).
In both MES and PTZ models, the MeAA (200 and
400 mg/kg) showed significant and dose dependent protec-
tion. However, further increase in the dose of MeAA to
800 mg/kg did not show significant increase in anticonvulsant
activity, and it may be due to sealing effect, notably, in none of
the tested doses the MeAA has showed complete inhibition of
MES and PTZ-induced convulsions. Interestingly, the phenyt-
oin (25 mg/kg) and diazepam (5 mg/Kg) were used as refer-
ence standards in MES and PTZ models respectively, have
Metab Brain Dis
abolished the MES and PTZ-induced convulsions and mortal-
ity completely.
With the above finding it can be hypothesized that,
the MeAA is eliciting potent anticonvulsant activity by
increasing the inhibitory neurotransmitters (GABA) and/
or, decreasing the excitatory neurotransmission and/or,
by blocking the sodium channels and/or by neutralizing
the PTZ binding site. To explore the possible mecha-
nism of action, the effect of MeAA was evaluated for
its influence on GABA levels in brain. Interestingly,
pretreatment with MeAA (200 and 400 mg/kg) showed
significant increase in GABA levels in whole brain
other than cerebellum, upon multiple dose (3 days)
administration, compared to control. Thus we can
conclude that one of possible mechanism behind the
anticonvulsant effect of MeAA may be through GABA
mediated inhibition in brain. To confirm this hypothesis
further, the anticonvulsant effect of MeAA was exam-
ined in presence of flumazenil, which is a GABA A
receptor antagonist; in literature many researchers have
used this test to confirm the GABA mediated mecha-
nism behind the anticonvulsant, sedative and hypnotic
activities (Shannon et al. 1997; Rundfeldt et al. 1995;
John 2013; Li et al. 2006; Bruno et al. 2016). In these
studies, researchers have used GABAA receptor agonist
like benzodiazepine drugs (diazepam) along with
GABAA receptor antagonists such as flumazenil, and
reported that the GABAA receptor mediated activities
such as anti-seizure, sedative, hypnotic and addiction
properties of benzodiazepines were reversed in presence
of flumazenil, and these results were further confirmed
 Author's personal copy
Metab Brain Dis
in receptor binding studies (Li et al. 2006; Sean et al.
2012;Christiaan et al. 2012). In addition, flumazenil is
also used as a tool to identify the putative endogenous
and exogenous ligands at benzodiazepine receptor sites
(Uhlirova et al. 2004). Keeping this strong scientific
literature, in present study we have evaluated the anti-
convulsant effect of MeAA (400 mg/kg, p.o.) and diaz-
epam (5 mg/kg, p.o.) in presence of flumazenil (5 mg/
kg, i.p.) against PTZ-induced convulsions. Interestingly,
the diazepam (5 mg/kg, p.o.) and MeAA (400 mg/kg,
p.o.) alone treated groups showed significant anticonvul-
sant activity compared to control. However, the anticon-
vulsant effect of diazepam (5 mg/kg, p.o.) and MeAA
(400 mg/kg, p.o.) was completely abolished when co-
administered along with flumazenil. The blockade of
anticonvulsant action of diazepam validates the test pro-
cedure, while blockade of MeAA anticonvulsant activity
in presence of flumazenil (5 mg/kg, i.p.) confirmed the
GABAA mediated mechanism behind the anticonvulsant
effect of MeAA. Notably, the MeAA (400 mg/kg, p.o.)
and diazepam (5 mg/kg, p.o.) combination showed syn-
ergistic effect against PTZ-induced convulsion, the anti-
convulsant effect of the combination (p < 0.001) was
more compared to their per se treated groups (MeAA
400 mg/kg (p < 0.05), diazepam 5 mg/kg (p < 0.01)).
Thus we can conclude that anticonvulsant effect of
MeAA is mediating through GABAergic pathway.
In addition, to study the possible involvement of the excit-
atory neurotransmitter (glutamate), the MeAA (100, 200, 400
and 800 mg/kg) was evaluated against NMDA-induced lethal-
ity in mice, systemic administration of NMDA to mice
showed variable effects such as scratching behavior, tile bit-
ing, tonic-clonic seizures and lethality. Since the behavioral
effects and convulsions produced are not consistent with the
animals, lethality was selected as a reliable end point (Leander
et al. 1988). In this study, MeAA did not showed significant
protection against NMDA-induced lethality, and thus con-
firmed the negligible role of glutaminergic pathway in the
anticonvulsant effect of MeAA.
In addition, the effect of MeAA was evaluated for its pos-
sible locomotor deficits; however the MeAA did not show
significant influence on locomotor activity.
In conclusion, all the findings in the present study are in
mutual relation and supports that MeAA possess potent anti-
convulsant activity and it could be a useful agent in treating
both petit mal and grand mal epilepsy. Furthermore, In support
of all these findings, the LC-MS/MS analysis of MeAA con-
firmed the presence of kaempferol, gallic acid, quercetin and
rutin; and previously various phytoconstituents such as quer-
cetin. Triterpenoid saponins (A&B), achyranthine, betaine,
steroids and so on were isolated from Achyranthes aspera
(Bhosale et al. 2011b), and these phytoconstituents were
earlier reported to be responsible for CNS depressant and
anxiolytic activity of Achyranthes aspera (Bhosale et al.
2011a, b). In similar lines, the plant constituents identified
in MeAA such as kaempferol (Calderón-Montaño et al.
2011), gallic acid (Maria et al. 2016), quercetin (Formica
and Regelson 1995; Gregory 2011), rutin (Aditya and Ajay
2016 were scientifically well demonstrated to possess
neurological properties such as anticonvulsant, anxiolyt-
ic, sedative, and CNS depressant and so on (Anandhan
et al. 2010; Chakraborty et al. 2010; Barua et al. 2009a,
b; Sasso et al. 2010). Furthermore, triterpenoid saponins
are well known exhibit agonistic/facilitatory activities at
GABAA receptor complex, which led to the hypothesis
that they act similar to benzodiazepine-like molecules,
which is supported by their behavioral effects in animal
models of CNS depression and anxiety (Anandhan et al.
2010; Chakraborty et al. 2010; Barua et al. 2009a, ;
Gatta et al. 2016). In conclusion, based on these obser-
vations we can declare that, the MeAA contains mixture
of few neuroprotective phytoconstituents and the syner-
gistic interaction of the phytoconstituents present in
MeAA is eliciting anticonvulsant activity through
GABAA receptor agonistic action by enhancing GABA
levels in the brain.
Conclusion
These findings demonstrate that MeAA possess signifi-
cant anticonvulsant property, interestingly the MeAA
showed significant increase in brain GABA levels, and
notably the anticonvulsant action of MeAA was
abolished by flumazenil, thus confirmed the involve-
ment of GABAergic pathway, and thus confirmed that
one of the possible mechanism/s behind the anticonvul-
sant activity of MeAA may be mediating through
GABAergic pathway. Furthermore, the MeAA has failed
to inhibit the NMDA-induced lethality, and thus con-
firmed the negligible role of glutaminergic pathway.
All these findings together strongly confirmed the pos-
sible mechanism of GABAergic pathway behind the an-
ticonvulsant activity of MeAA. Indeed, there is scope
for supplementary studies to isolate the component re-
sponsible for anticonvulsant activity of MeAA.
Acknowledgements The authors acknowledge the technical help pro-
vided by Dr. Nandakumar K, Associate Professor, Department of
Pharmacology, Manipal college of Pharmaceutical sciences, Manipal.
The authors greatly acknowledge Ms.Radiant Research Services Pvt.
Ltd., Bangalore, Vitthartha Life Sciences, Bommasandra Industrial Area,
Bangalore, and Manipal College of Pharmaceutical Sciences, Manipal for
providing all the neccesary facilities and Technical help in completing the
research work.
Authors Contribution GLV, MVV, NBLP and AG have contributed
equally for designing, conducting the study, data collection, analysis
 Author's personal copy
and preparation of manuscript, MVV and NBLP involved in critical anal-
ysis and interpretation of findings, MVV, NBLP, AG have proof read and
approved the final version of manuscript.
Compliance with ethical standards
Conflicts of interest The authors declare there are no conflicts of in-
terest and they guarantee no further ethical conflict among the authors and
the experimental methodology. Also, no formal funding from any agen-
cies was used for this project.
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