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European Journal of Integrative Medicine 4 (2012) e113–e121

Original article

Cognition boosting effect of Canscora decussata (a South Indian

Shankhpushpi)
Neeraj K. Sethiya a,∗ , Alok Nahata b , V.K. Dixit b , S.H. Mishra a
a Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda, Vadodara 390002, Gujarat, India
b Department of Pharmaceutical Sciences, Doctor Hari Singh Gour Vishwavidyalaya, Sagar 470003, M.P., India

Received 27 September 2011; received in revised form 14 November 2011; accepted 14 November 2011

Abstract
Aim of the study: In southern India, ayurvedic practitioners traditionally employ the whole herb of Canscora decussata Schult. (CD) (Gentianaceae)
as a traditional ayruvedic medicine, Shankhpushpi for its memory potentiating, anxiolytic and tranquilizing properties. The present study investigated
the effect of CD, which is regarded as Shankhpushpi, for its effects on learning and memory in rodents. The extract was further studied for its
in vitro acetylcholinesterase (AChE) inhibitory potential which can correlate with its cognition boosting effect.
Materials and methods: Ethanol extract of CD was analyzed by high performance thin layer chromatography (HPTLC) and high performance
liquid chromatography (HPLC). Ethanol extract of CD was investigated for its AChE enzyme inhibitory activity. Nootropic activity using Elevated
plus maze apparatus, passive avoidance (Cook and Weidley’s pole climbing, step down) paradigms and active avoidance (two compartment shuttle
box) test were used to learning and memory.
Results: HPTLC and HPLC fingerprinting of ethanol extract revealed presence of mangiferin as its main constituent. It was found that CD
potentially inhibits AChE with 50% inhibitory concentration (IC50 ) of 165.667 ± 0.213 ␮g/ml. It was found that groups (n = 6), receiving ethanol
extract in doses of 200 and 400 mg/kg p.o. significantly reversed the amnesia induced by scopolamine (0.3 mg/kg i.p.). Nootropic activity was
compared using piracetam (100 mg/kg p.o.) as the standard.
Conclusion: Ethanol extract of CD showed significant effects on learning behavior and memory enhancement as evidenced from the experiments
performed. The activity may be attributed to the presence of various xanthones and mangiferin, a polyphenolic xanthone.
© 2011 Published by Elsevier GmbH.

Keywords: Xanthones; Amnesia; Memory; Cholinesterase inhibitor

Introduction of seven different plant species known as Shankhpushpi. The

Ethnopharmacological research into medicinal plants is
becoming an important part of advanced research during recent
years [1]. Shankhpushpi is an ayurvedic drug used for its action
on the central nervous system, especially for boosting mem-
ory and to improve intellect. Shankhpushpi is word of Sanskrit
which means ‘the plant with flowers shaped like a Shankha (a
musical instrument)’ [2]. Quantum of information gained from
ayurvedic and other Sanskrit literature revealed the existence
∗ Corresponding author at: Herbal Drug Technology Laboratory, Pharmacy
Department, The Maharaja Sayajirao University of Baroda, GH Patel Pharmacy
Building, Donor’s Plaza, Fatehgunj, Vadodara 390002, Gujarat, India.
Tel.: +91 265 2794051; fax: +91 265 2423898.
E-mail address: nscognosy2006@gmail.com (N.K. Sethiya).
sources comprise of entire herbs with following botanicals viz.,
Convolvulus pluricaulis Choisy. (Convolvulaceae), Convolvulus
microphyllus Sieber ex Spreng. (Convolvulaceae), Evolvu-
lus alsinoides Linn. (Convolvulaceae), Evolvulus nummularius
(Convolvulaceae), Clitorea ternatea Linn. (Papilionaceae),
Canscora diffusa R. Br. and Canscora decussata Schult (CD)
(Gentianaceae) [3–5].
In southern parts of India, CD is popularly known as “Shankh-
pushpi” and found above an altitude of 1300 m. It is also
grown in Sri Lanka and Myanmar. The entire plant, as well
as its fresh juice is used in medicine. It is used traditionally
for the treatment of insanity, epilepsy and nervous debility.
It contains xanthones, bitter substances and an oleoresin [6].
CD has proven its therapeutic potential in acetylcholinesterase
inhibition, CNS stimulation, hypertension, convulsions, tuber-
culosis, immunomodulation, inflammation, hepatoprotection,

1876-3820/$ – see front matter © 2011 Published by Elsevier GmbH.

doi:10.1016/j.eujim.2011.11.003
e114 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121
spermatogenesis and postmenopausal osteoporosis [4,5]. It is
reported to contain several types of xanthones, triterpenoids, loli-
olide, sterols and flavanoids [7,8]. Mangiferin is one of the major
xanthones present in the CD, ameliorates scopolamine induced
learning deficit and acetylcholinesterase inhibition [9]. Kapadia
et al., also develop a HPTLC method in order to establish a dis-
tinct chemical profile for Shankhpushpi and for quantification
of scopoletein in Convolvulus pluricaulis Choisy and in com-
mercial formulations of Shankhpushpi [10]. The present study
investigated the effect of CD, which is regarded as Shankh-
pushpi, for its effects on learning and memory in rodents. The
extract was further studied for its in vitro acetylcholinesterase
(AChE) inhibitory potential which can correlate with its cogni-
tion boosting effect.
Methods
Plant material and preparation of extract
The whole CD plant was collected from the outskirts of
Raipur (Chattisgarh, India) in January, 2008. The plant was
identified by Dr. S.C. Agrawal (Department of Botany, CDRI,
Lucknow, India). A voucher specimen has been deposited in the
herbarium of the institute (Voucher Specimen no. NS/AN/Cd-
2409). Extraction was done according to the method followed in
our previous studies [11,12]. Plant material was shade dried and
ground into a coarse powder. Powdered material (120 g) was first
defatted with petroleum ether (500 ml). The marc was extracted
with ethanol to obtain the ethanol extract (yield 4.47%, w/w).
The extract was suspended in Tween-80 (0.1%, v/v) to get the
suspension for pharmacological investigations.
Drugs and chemicals
Acetylthiocholine iodide (ATCI), acetylcholinesterase
enzyme (AChE) from bovine erythrocytes, 5,5 -dithiobis[2-
nitrobenzoic acid] (DTNB) and galanthamine were obtained
from Sigma Ltd. (Mumbai, India). Scopolamine hydrobromide
(SHB) (99.98% pure) was obtained as a gift from Cadila
Healthcare Pvt. Ltd., Goa, India. SHB was dissolved in normal
saline for i.p. injection. TLC plates were purchased from
Merck, Darmstadt (Germany).
Characterization of extract and selection of marker
Thin layer chromatographic (TLC) studies were per-
formed using various solvent systems, and finally chloroform:
ethanol:toluene (7:2:1) was found to be suitable solvent sys-
tem for the proper separation of phytoconstituents (number of
spots = 12). The detection was carried out at 254 nm. Thin layer
chromatographic profile of the ethanol extract in n-butanol:
acetic acid: water (4:1:2), detected at 366 nm, revealed the pres-
ence of mangiferin (Rf 0.72), which is a key feature of ethanolic
extract [1,13].
TLC fingerprinting
Precoated and preactivated TLC plates of silica gel 60
F254 with the support of aluminum sheets 0.1 mm thick and
10 × 10 cm were used. Ethanol extract (10 mg) was weighed
accurately and dissolved in 10 mL of ethanol. Mangiferin
(10 mg) was dissolved in methanol (10 ml). The quantity of
standard applied was 5 ␮L and quantity of sample applied was
10 ␮L. The extract samples were applied in the form of a band
using CAMAG LINOMAT V, an automatic sample application
device, maintaining a band width 6 mm, space 10 mm, 250 nL/s
[14,15]. The solvent system used was chloroform: ethanol:
toluene (7:2:1) and n-butanol: acetic acid: water (4:1:2). The
plates were developed by placing in presaturated tanks (8 cm
height) with the respective solvent system. After drying the
plates, area under curve (AUC) was measured which was 7903.8
(Rf 0.72) for mangiferin sample. The AUC for ethanol extract
was found to be 4143.6. The mangiferin content of ethanol
extract calculated on the basis of AUC was found to be 52.43%.
HPLC
HPLC was done on Shimadzu Prominence UFLC (Pump:
LC-20 AD; Detector: SPD-20 AV; Column: Phenomenex 5 ␮,
C-18, 4.6 × 250 mm). The solvent system optimized for the fin-
gerprinting was methanol: water: acetonitrile (40:45:15) with
a flow rate of 1 ml/min (number of peaks = 9) and detection at
254 nm [15].
Microplate assay
AChE activity was determined in 96-well microplate reader
using Ellman’s method [16]. The substrate acetylthiocholine
is hydrolyzed by the enzyme which results in the product
thiocholine which reacts with Ellman’s reagent (DTNB) to
produce 2-nitrobenzoate-5-mercaptothiocholine and 5-thio-2-
nitrobenzoate which can be detected at 405 nm. In the 96-well
plates, 125 ␮L of 3 mM DTNB, 25 ␮L of 15 mM ATCI and
50 ␮L of buffer and 25 ␮L of sample dissolved in buffer contain-
ing not more than 10% methanol were added. The absorbance
was measured at 405 nm every 13 s for 65 s. 25 ␮L of 0.22 U/ml
of AChE enzyme was added and the absorbance was again read
every 13 s for 104 s. The absorbance was read using 680 XR
Microplate Reader S/N 10,519 at 405 nm. Percentage of inhibi-
tion was calculated by comparing the rates for the sample to the
blank. A range of concentration was used so that the IC50 value
could be calculated [17].
Animals
Male Sprague–Dawley rats (180–200 g) (2–3 months old)
were used for the study. The animals were housed in groups of
six in polypropylene cages, under standard laboratory conditions
of temperature (25 ± 2 ◦ C), lighting (08:00–20:00 h) and relative
humidity (50 ± 5%). The animals had free access to standard pel-
let chow (Brooke Bond-Lipton, India) and water. The animals
were acclimatized for a period of minimum 7 days and daily
 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121
observed between 0900 and 1400 h [11]. All the experiments
were conducted between 0900 and 1400 h. The experimental
protocol was approved by Institutional Animal Ethics Commit-
tee (IAEC) of B.R. Nahata College of Pharmacy, Mandsaur (Reg.
No. 918/ac/05/CPCSEA).
Acute toxicity studies with pharmacological behavioral
screening
Ethanol extract (2000 mg/kg) was administered as per OECD
guidelines per orally to 7 rats. Effects in behavior were observed
for 72 h. Rats were observed individually after dosing at least
once during the first 30 min, periodically during the first 24 h,
with special attention given during the first 4 h, and thereafter,
for a total of 72 h. Observations included changes in skin and fur,
eyes and mucous membranes, and also respiratory, circulatory,
autonomic and central nervous systems, and somatomotor activ-
ity and behavior pattern. Observations of tremors, convulsions,
salivation, diarrhea, lethargy, sleep and coma were recorded
[18].
Doses
Doses selected for studies were 100, 200 and 400 mg/kg
p.o. for ethanol extract, calculated after acute toxicity studies
(1/5th, 1/10th and 1/20th of the original dose, i.e. 2000 mg/kg
was selected as per OECD guidelines).
Assessment of nootropic activity
The activity was assessed using elevated plus maze as
described below:
Elevated plus maze
The elevated plus maze (EPM) consisted of two open arms
(50 × 10 cm) crossed with two closed arms (50 × 10 × 40 cm).
The arms were connected together with a central square
(10 × 10 cm) (Medicraft Electromedicals Pvt Ltd., India). The
apparatus was elevated to a height of 70 cm in a dimly illumi-
nated room. From day 1 to 6, each rat was placed at the end
of an open arm, facing away from the central platform. Transfer
latency (TL) was considered as the time taken by the rat to move
into any one of the covered arms with all its four legs. TL was
recorded for six successive days as training module. If the rat did
not enter into one of the covered arms within 90 s, it was gently
pushed into one of the two covered arms and TL was assigned as
90 s. The rat was allowed to explore the maze for 10 s and then
was returned to its home cage. Memory retention was exam-
ined 24 h and 48 h after the sixth day [19,20]. The animals were
divided into six groups containing six animals in each group.
Group I received the vehicle only. Group II received a single
dose of SHB on day 6. Group III received piracetam (100 mg/kg
p.o.) as positive control. Group IV–VI received ethanol extracts
(100, 200, and 400 mg/kg p.o.). All treatments were adminis-
tered daily 45 min prior to experiments. After attaining complete
training, except group I remaining five groups were given a
e115
single dose of SHB on day 7 of the experiment so as to cre-
ate a temporary memory deficit. TL was measured on day 7 and
day 8 (24 h and 48 h after the first observations).
Inhibitory (passive) avoidance tests
The following parameters were used to assess effects on
learning and memory:
Scopolamine induced amnesia
This test was performed using the Cook and Weidley’s pole
climbing apparatus [21]. It consisted of a soundproof experi-
mental chamber with a grid floor which could be electrified and
equipped with a provision for buzzer tone. The enclosure had
a covering lid at the top, through which the animal could be
introduced into the chamber. A wooden pole, screwed onto the
inner surface of the lid of the chamber acted as the shock-free
zone (Medicraft Electromedicals Pvt Ltd., India). The stimu-
lus provided was a foot shock of 0.75 mA given for a period of
2 s from the electrified grid floor. Rats were initially trained to
escape the foot shock by climbing onto the pole, i.e. the shock
free zone. This initial trail was carried out by having three trials
sessions interspersed with an interval of 10 s. During each of the
initial trials, the rats were allowed to explore the apparatus for
10 s. Sensitivity of the rats towards foot shock was measured and
only those rats, which were sensitive to the foot shock and could
climb the pole, were included in the study [14,22]. The animals
were divided into six groups with six animals each. Group I
received the vehicle only. Group II received a single dose of
SHB on day 9. Group III received piracetam (100 mg/kg p.o.)
as positive control. Group IV–VI received ethanol extracts (100,
200, and 400 mg/kg p.o.). Training trial (TT) was conducted after
7 days treatment. Twenty-four hours later, on day 8, a retention
trial (RT) was conducted and the number of avoidance responses
(ARs) in the 10 trial sessions was noted. On day 9 of the exper-
iment, after attaining complete training, all the animals except
the control, were treated with a single dose of SHB (0.3 mg/kg
i.p.), 30 min before the administration of the extracts. Training
schedule was continued further with daily dosing of the extracts
till 15 days.
Step down test
The test apparatus was a rectangular box (45 × 30 × 40 cm)
with an electrified grid floor. It was made of transparent plex-
iglass to permit observations. An 8 cm high wooden platform
(17 × 12 cm) was fixed to the grid floor at the center of the
apparatus (Medicraft Electromedicals Pvt Ltd., India). A rat was
placed on the platform and allowed to step down. Twenty-four
hours later, on day 1 of the experiment, the rat was again placed
on the platform and foot shock (0.75 mA, 2 s) was delivered
through the grid floor as soon as it stepped down. The rat was
given foot shock only when all the four paws were touching the
grid floor. The rat was given three more training trials until the
latency of step down had stabilized. The test was repeated on
e116 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121
day 15. Memory retention score for each animal was calculated
by determining “Inflexion ratio” by the formula:
L15 − L1
Inflexion ratio =
L1
where L1 is the step down latency on day 1 in seconds and L15
is the step down latency on day 15 in seconds [23,24]. For this
experiment, the animals were divided in to five groups contain-
ing six animals in each group. Group I received the vehicle only.
Group II received piracetam (100 mg/kg p.o.) as positive control.
Group III–V received ethanol extracts (100, 200, and 400 mg/kg
p.o.). Test extracts and vehicle were administered once daily for
15 days.
Active avoidance test
Two compartment shuttle box
The animals were trained in an active avoidance paradigm
in a (25 × 25 × 40 cm) shuttle box with a buzzer tone located
at the midline on the lid of the box and floor consisting of the
stainless steel grid bars. The box was divided into two equal
compartments by a wooden panel with an (8 × 7 cm) hole in the
middle. The conditioned stimulus (CS) was a light source and
the buzzer for 10 s, followed by an unconditioned stimulus (US),
i.e. an electric shock (0.75 mA, 2 s). An avoidance response was
recorded when animal avoided the US by crossing to another
unelectrified dark compartment within 10 s after the onset of
CS. The experiment was continued for 14 days, which included
6 initial training days followed by treatment. Rats were divided
into five groups of six rats per group. Group I received the vehicle
only. Group II received piracetam (100 mg/kg p.o.) as positive
control. Group III–V received ethanol extracts (100, 200, and
400 mg/kg p.o.). During first six days of the experiment, the rats
were allowed to explore the apparatus and 30 CS were applied
without electric shock. Thus, each rat got adapted to the shut-
tle box. On day 7 of the experiment, 1 h post administration of
the extracts and the vehicle, each rat was subjected to initial
trial with electric shock. During this avoidance training, the rats
were placed in the electrified chamber and allowed to acclima-
tize for 30 s before being subjected to 30 avoidance trials, with
an interval of 18 s. During the first 10 s the CS was provided,
which was followed by the electric shock of 8 s. The avoidance
response (AR), characterized by escape to the adjoining “safe”
compartment during conditioned stimulus, was noted. % AR for
30 trials was computed in terms of absolute number for each ani-
mal. Retention trials were conducted 24 h (RT-1; on day 8), 48 h
(RT-2; on day 9) and 7 days (RT-3; on day 14) after the initial
trials [22,24].
Statistical analysis
The data are expressed as mean ± SEM and analyzed by One-
way ANOVA followed by Dunnett’s Multiple Comparison post
test. P and F values and degrees of freedom were calculated
for each experiment. The level of significance was P < 0.05. The
data was analyzed using Graph Pad-Prism-5 software (GraphPad
Software Inc., La Jolla, California).
Table 1
Acetylcholinesterase inhibitory activity of ethanol extract of CD.
Sr. No. Sample IC50 value (␮g/ml)
1 Ethanol extract of CD 165.667 ± 1.202
2 Galanthamine 5.543 ± 0.213
All values are mean ± SEM (n = 3).
Results
HPTLC and HPLC analysis
HPTLC and HPLC analysis confirmed the presence of
mangiferin in the tested extracts, a characteristic feature of stan-
dardized extract of CD as reported by Chaudhuri and Ghosal
earlier (Figs. 1a, b and 2a, b) [13].
Microplate assay
The ethanol extract of CD inhibited AChE moderately with
the IC50 value of 165.667 ± 0.213 ␮g/ml (Table 1). Galan-
thamine was used as a positive control, showed inhibition of
acetylcholinesterase with an IC50 value of 5.543 ± 0.213 ␮g/ml.
Acute toxicity studies with pharmacological behavioral
screening
The ethanol extract of CD did not show any toxicity at a dose
of 2000 mg/kg as evidenced by our observations. No signs of
any abnormal behavior or any mortality were observed during
the study period. Then 1/5, 1/10, 1/20th doses were selected for
further studies as per OECD guidelines.
Elevated plus maze
The elevated plus maze studies for nootropic activity showed
a significant decrease in the transfer latency in animals treated
with ethanol extract as compared to scopolamine treated control
group. The effect of ethanol extract increased with increase in
dose. A One-way ANOVA followed by Dunnett’s Multiple Com-
parison post test, conducted on the mean time of rats on day 6,
day 7 and day 8 [**P < 0.05; F = 6.548; df(6, 14) = 20] exhibited
that animals of extract treated group had significantly less trans-
fer latency (time taken to reach the safe compartment) on day 8
as compared to scopolamine treated control group (Fig. 3).
Scopolamine induced amnesia
Inhibitory avoidance test conducted by administration of
scopolamine produced amnesia as seen from the reduction in the
number of avoidance responses. However, continued treatment
of ethanol extract of CD produced better retention and recovery
in a dose dependent manner. Animals receiving 100 mg/kg of
the ethanol extract of CD have taken five to seven days whereas
groups treated with 200 and 400 mg/kg doses of the same
extracts required three to four or lesser days to reach the point
of reversal suggesting better retention and recovery. Animals
 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121 e117

Fig. 1. HPTLC chromatogram: (A) ethanol extract of CD (254 nm); (B) ethanol extract of CD and mangiferin (366 nm).

receiving only SHB on day 9 showed a considerable loss of mem-
ory and amnesia produced was persistent. In lower doses, the
retention of memory and retrieval as seen in the ethanol extract
treated groups was weak (5–7 days). In higher doses, there was
a significant increase in retention suggesting the effect of CD
on memory and its retrieval in a dose dependent manner. Thus,
antiamnesic effects of ethanol extract of CD on scopolamine-
induced amnesia were successfully demonstrated through the
study. A One-way ANOVA followed by Dunnett’s Multiple
Comparison post test, conducted on the avoidance response of
rats when they were administered a single dose of scopolamine
on 9th day followed by trials on 10th, 11th, 12th, 13th, 14th and

Fig. 2. HPLC chromatogram in methanol:water:acetonitrile (40:45:15) at 254 nm: (A) standard mangiferin; (B) ethanol extract of CD.
e118 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121

Fig. 3. Effect of ethanol extract of CD and piracetam on nootropic activity by scopolamine induced amnesia in rats using elevated plus maze apparatus. EE100 –
ethanol extract of CD (100 mg/kg p.o.), EE200 – ethanol extract of CD (200 mg/kg p.o.), EE400 – ethanol extract of CD (400 mg/kg p.o.), scopolamine 0.3 mg/kg
i.p, piracetam 100 mg/kg p.o. One-way ANOVA followed by Dunnett’s test. All values are mean ± SEM (n = 6) [** P < 0.05; F = 6.548; df(6, 14) = 20]. Before the
commencement of the experiment, the animals were provided prior training (6 days) on the apparatus for acclimatization.

Fig. 4. Effect of ethanol extract of CD on scopolamine-induced amnesia using Avoidance Responses (ARs) to analyze the effects in rats. EE 100 – ethanol extract of
CD (100 mg/kg), EE 200 – ethanol extract of CD (200 mg/kg p.o.), EE 400 – ethanol extract of CD (400 mg/kg p.o.). The test drugs and the vehicle were administered
per orally once daily for 15 days. On the ninth day, a single dose of SHB (0.3 mg/kg i.p.) was administered 30 min before the daily dosing of the extracts. One-way
ANOVA followed by Dunnett’s test. All values are mean ± SEM (n = 6) [*** P < 0.001; F = 95.48; df(6, 56) = 62].

15th day [***P < 0.001; F = 95.48; df(6, 56) = 62] demonstrated
that the extract treated groups exhibited a significant increase in
the number of ARs as compared to the scopolamine treated and
vehicle treated groups (Fig. 4).

Step down test

The learning behavior of the animals was studied by step

down apparatus. The animal learns that if it steps down, it
will have a foot shock and thus avoids stepping down. The
latency of stepping down reflects the retention of learned behav-
ior/memory. The step-down latency was significantly increased
on administration of ethanol extract of CD. A one-way ANOVA
conducted on the mean inflexion ratios of the animals on day 15 Fig. 5. Effects of ethanol extract of CD on CS induced learning retention
[***P < 0.001; F = 79.456; df(5, 35) = 40] suggest that extract (memory) deficit in the step-down test in rats. EE – ethanol extract of CD.
The test extracts and the vehicle were administered once daily for 15 days
significantly increased the inflexion ratio as compared to vehi- in the unstressed group or 45 min before stress. All values are mean ± SEM
cle in a dose dependent manner. A dose dependent action of the (n = 6). One-way ANOVA followed by Dunnett’s test [***P < 0.001; F = 79.456;
drug on memory retention was also evident (Fig. 5). df(5, 35) = 40].
 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121
Fig. 6. Effect of ethanol extract of CD on the avoidance responses in the two-compartment shuttle box paradigm. 0.0. – TT (training trial), 24.0 h; 48.0 h; 168.0 h
– RT (retention trial), EE – ethanol extract of CD. The test drugs and the vehicle were administered once daily for 7 days in the unstressed group or 45 min before
stress. All values are mean ± SEM (n = 6). One-way ANOVA followed by Dunnett’s test [*** P < 0.001; F = 50.51; df(5, 18) = 23].
Two compartment shuttle box
In the shuttle box avoidance paradigm, experiment was con-
ducted for active avoidance test. Significant increase in the
% ARs was observed in all the groups as compared to the
vehicle in a dose dependent manner indicating the memory
potentiating activity of ethanol extract of CD. The dose of
400 mg/kg p.o. of the extract was found to be more potent
in comparison to lower doses, indicating the dose dependent
action of the drugs on cognitive functions and memory. The
% ARs which was 46.10 ± 1.02, 56.77 ± 1.39 and 64.99 ± 0.74
in training trials was increased to 55.55 ± 0.70, 63.33 ± 0.85
and 72.77 ± 1.02 respectively in retention trials (day 7) for 100,
200 and 400 mg/kg p.o. dose of the ethanol extract. A One-way
ANOVA followed by Dunnett’s Multiple Comparison post test,
conducted on the % Avoidance Response (ARs) of rats when
they were placed in two compartment apparatus [***P < 0.001;
F = 50.51; df(5, 18) = 23] demonstrated that the extract treated
groups exhibited a significant increase in the number of % ARs
as compared to the vehicle treated and piracetam treated groups
(Fig. 6).
Discussion
Dysfunction of cholinergic neurotransmission in the brain
contributes to the salient cognitive decline [17]. It was found that
CD potentially inhibits AChE and protects the loss of neurotrans-
mitter acetylcholine, which is directly responsible for cognitive
function. The question of reliability and validity is of prime
importance in establishing experimental paradigms of practi-
cal predictable value. These factors assume further importance
when animal models of human behavior and its perturbations are
being used. The paradigms used in the present study have been
subjected to thorough critical appraisal and validated as animal
models of acquisition and retention of memory of the learned
task [14,25]. The findings of the present study clearly indicate
that the ethanol extract of CD at doses of 200 and 400 mg/kg
significantly improve the acquisition and retention of memory.
The elevated plus-maze paradigm has been used for
evaluating learning and memory in rodents [26]. It is an extero-
ceptive behavioral model for evaluating learning and memory.
e119
Scopolamine produces a temporary loss of memory. Simulta-
neous administration of scopolamine along with test extracts
depicts their antiamnesic potential. The increase in transfer
latency as exhibited by time taken by animal to reach safe com-
partment was observed during our experiment. Graded doses of
ethanol extract, i.e. 100, 200 and 400 mg/kg showed a significant
increase in transfer latency. The time required for reaching the
safe area was reduced as compared to the vehicle treated control
animals.
Stressful conditions adversely affect cholinergic system and
results in learning deficits, but the cholinergic response to the
stressful stimuli is variable and depends on the type and duration
of the stressor [27]. Biochemically, the cholinergic system plays
an important role in memory. Anticholinergic agents such as
atropine and scopolamine interfere with memory. Choline acetyl
transferase (the enzyme catalyzing the formation of acetyl-
choline) and nicotinic cholinergic receptors are known to be
deficient in the cortex of patients with Alzheimer’s disease.
There is substantial evidence that muscarinic receptor blockade
by drugs like scopolamine results into disruptions of behavioral
inhibition, working (short-term) memory, retrieval from refer-
ence (long-term memory), attention and decisional processes,
movement and strategy selection and altered sensory processing
[14,28]. In the present study, ethanol extract has shown better
retention and recovery in a dose dependent manner than the
vehicle treated group. Animals receiving only SHB on day 9
showed a considerable loss of memory and amnesia produced
was also persistent. The dose administered to animals appears
to influence recovery as high dose has greater effect on mem-
ory and its retrieval. The retrieval of memory as evidenced by an
increase in the number of avoidance responses seen in the groups
receiving 400 mg/kg of the extract was stronger than the 100
and 200 mg/kg treated groups (five to seven days). Thus, anti-
amnesic effects of CD on scopolamine induced amnesia were
successfully demonstrated throughout the study.
An animal in an open field spends most of the time close to the
walls and in the corners. When placed on an elevated platform
in the center of a rectangular compartment, it steps down almost
immediately to the floor to explore the enclosure and to approach
the wall [14,22]. In our experiments of step-down apparatus, the
latency, i.e. the time taken by the animal to step down, was
e120 N.K. Sethiya et al. / European Journal of Integrative Medicine 4 (2012) e113–e121
significantly affected and the animal avoided stepping down for
a longer period of time suggesting that it remembers that it will
meet an electric shock on stepping down. The ethanol extract of
CD at doses of 100, 200 and 400 mg/kg significantly increased
the step-down latency, and hence the inflexion ratio, on day 15
of the experiment, indicating the memory enhancing activity of
the drug.
In the shuttle box avoidance paradigm of our study, a signifi-
cant increase in the % ARs was observed in groups receiving the
ethanol extract as compared to the vehicle treated control group.
The dose of 400 mg/kg was found to be more potent indicat-
ing the dose dependent action of CD on cognitive functions and
memory.
Preliminary phytochemical studies on CD showed the pres-
ence of mangiferin, a glucosylxanthone [13]. Mangiferin has
already been reported to have memory enhancing properties by
Andreu et al. [29]. They reported that mangiferin stimulated
cell proliferation and induced a significant increase in the super-
natant levels of nerve growth factor (NGF) and tumor necrosis
factor (TNF)-␣ in vitro in human U138-MG glioblastoma cells.
The results indicate that mangiferin enhances recognition mem-
ory through a mechanism that might involve an increase in
neurotrophin and cytokine levels. The presence of mangiferin
in CD can thus be correlated to the cognitive and memory
enhancing activity of CD. C. decussata also contains many more
xanthones other than mangiferin and may be responsible for the
synergistic activity [30]. Our findings strongly support this view.
Conclusion
Our studies thus support and validate the earlier findings for
the use of CD as Shankhpushpi (a nervine tonic in traditional
ayurvedic medicine) is not invalid. The pharmacological actions
of CD, justify the therapeutic uses of the plant in mental disorders
(e.g., melancholia). These findings validate the traditional claims
of CD as a memory improving herb.
Conflicts of interest
The authors report that they have no potential conflicts of
interest.
Acknowledgements
The authors would like to express their sincere thanks to the
Director, B.R. Nahata Smriti Sansthan, Contract Research Cen-
ter, Mandsaur (M.P.), India for granting permission to carry out
the in vivo studies. We would also like to thank Cadila Healthcare
Pvt. Ltd, Goa, India for providing a gift sample of SHB. Two
of the authors, Neeraj K. Sethiya and Alok Nahata are thankful
to the University Grants Commission, New Delhi for providing
junior research fellowship.
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