1. Phytomedicine 18 (2011) 219–223
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Phytomedicine
journal homepage: www.elsevier.de/phymed
Isolation of anxiolytic principle from ethanolic root extract of Cardiospermum
halicacabum
Rajesh Kumara
, G. Murugananthana
, K. Nandakumarb,∗
, Sahil Talwarb
a
Department of Pharmacognosy, PES College of Pharmacy, 50 Feet Road, Hanumanthnagar, Bangalore, Karnataka, India
b
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka 576104, India
a r t i c l e i n f o
Keywords:
Cardiospermum halicacabum
Cardiospermin
Anxiolytic effect
Elevated plus-maze test
Light dark model
a b s t r a c t
Cardiospermum halicacabum roots have been used traditionally for the treatment of epilepsy and anxiety
disorders. The purpose of this study was to characterize the putative phytoconstituents present in the
ethanolic root extract having anxiolytic activity using an elevated plus-maze (EPM) and light dark transi-
tion model. Control mice were orally treated with an equal volume of vehicle (4% gum acacia), and positive
control mice were treated with diazepam (1 mg/kg). In the EPM test, out of pool of 19 master fractions
(MF) only MF-14, 16 and 17 significantly (30 mg/kg, p < 0.05, p < 0.01 and p < 0.001) increased the number
of entries in the open arm. MF-14, 16 and 17 (10, 20 and 30 mg/kg) had also increased the time spent
by mice in illuminated part of the box significantly (p < 0.05, p < 0.01 and p < 0.001), as compared to con-
trol. However, significant changes (p < 0.05, p < 0.01 and p < 0.001) were recorded in other parameters, e.g.,
rearing, time spent in the closed arm and dark zone in both the models. These results suggested that C. hal-
icacabum root is an effective anxiolytic agent. The phytoconstituent responsible for the observed central
effects was isolated from MF-14 and identified as well-known compound, Cardiospermin, a cyanogenic
glucoside.
© 2010 Elsevier GmbH. All rights reserved.
Introduction
Anxiety disorders in a modern society have relatively high
prevalence affecting between 10 and 30% of the general popula-
tion with considerable financial resources (Rice and Miller 1998;
Greenberg et al. 1999; Wittchen and Hoyer 2001). Excessive anxi-
ety can debilitate and damage the quality of life (Seo et al. 2007). In
the clinical treatment of anxiety benzodiazepines, GABAA recep-
tor agonist and buspirone, 5-HT1A receptor agonist, are mainly
prescribed as first choice treatment. Chronic administration of
benzodiazepines, however result in physical dependence such as
sedation, myelorelaxation, ataxia, amnesia and pharmacological
dependence (Lader and Morton 1991). Moreover buspirone also
results in dizziness, headache, nervousness, paresthesia, diarrhea,
excitation and sweating as adverse effects (Jordan et al. 1996;
Rickels and Schweizer 1997). Therefore, research has been con-
ducted to identify safer, more specific medications possessing
anxiolytic effect without the complications. In past few years,
several herbal medicines have been used for the management of
anxiety in the world (Rex et al. 2002).
∗ Corresponding author. Tel.: +91 820 2922482x189; fax: +91 820 2571998.
E-mail address: nandakumar77@rediffmail.com (K. Nandakumar).
In India, Cardiospermum halicacabum (CH) has been used for sev-
eral centuries in the treatment of rheumatism, stiffness of limbs
and snake bite whereas the roots alone have been used for curing
diseases related to the nervous system (Muthu et al. 2006). Sim-
ilar uses of CH roots have been indicated in the folklore system
for anxiety and epilepsy as well (Venkteshbabu and Krishnakumari
2006). Initially study was conducted with ethanolic root extract
of CH and found to possess anxiolytic activity. The present study
was designed to fractionate the ethanolic extract and to isolate the
active constituent responsible for anxiolytic activity.
Materials and methods
Animals
Adult male Swiss albino mice (18–24 g) were procured from
Bioneeds, Tumkur, India. They were housed in groups (n = 6) in
polypropylene cages (11 cm × 17 cm × 28 cm) with wood shavings
as bedding, under controlled conditions of light (12 h light dark
cycle, light on at 7 a.m.) and temperature (25 ± 2 ◦C). The animals
had free access to water and food except 1 h before and during
the experiments. All protocols and experiments were conducted
in strict compliance according to the ethical principles and guide-
lines provided by the Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA).
0944-7113/$ – see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.phymed.2010.07.002

2. 220 R. Kumar et al. / Phytomedicine 18 (2011) 219–223
Drugs
Diazepam was obtained from Ranbaxy Laboratories Limited,
Thane, India. Other chemicals petroleum ether, methanol and ethyl
acetate used for extraction, fractionation and phytochemical inves-
tigation were of analytical grade from SD Fine Chemicals, Mumbai,
India.
Plant material
The roots used for this study were collected from wild area of
Tiruchengodu, Tamil Nadu and was authenticated by Dr. K. Lak-
shman, Head of Department (Botany), Bangalore University, India.
Roots collected were sun dried and powdered coarsely.
Isolation and identification of the compound
Dried powdered roots (sieved # 40–60) were extracted with
absolute alcohol by soxhlation for 48 h. The extract was concen-
trated under vacuum. The major phytochemical constituents were
identified by optimization of thin layer chromatography (TLC).
The plates developed and scanned at 264 and 366 nm showed
prominent band separation in TEF (toluene:ethylactate:formic acid
4.5:4.5:1). Based on the results of preliminary screening, 30 g of
active crude ethanolic extract was chromatographed on silica gel
column (Merk 60–120 mesh, 600 g) and successively eluted with
stepwise gradient of petroleum, then ethyl acetate and finally
methanol. A total of 384 master fractions were collected and
each fraction was spotted on pre-coated silica gel (Merk-60 F254,
0.25 mm thick) plate and eluted in petroleum ether, ethyl acetate
and methanol in varying proportions. Fractions with similar Rf val-
ues in TLC were pooled together to get 19 similar fractions. Fractions
with sufficient yield were selected for further studies. Fraction 14
showed a single spot on the TLC. The structure of the isolated com-
pound was elucidated on the basis of 1H NMR, MS and FT-IR at the
Indian Institute of Sciences, Bangalore, India (Fig. 1).
Pharmacological evaluation
Elevated plus-maze test (EPM)
According to Pellow and File (1986) elevated plus-maze appara-
tus comprised of two perpendicular open (16 cm × 5 cm) and two
closed arms (16 cm × 5 cm × 12 cm) having an open roof, was ele-
vated (25 cm) from the floor to observe anxiolytic behaviour in mice
from the central platform (5 cm × 5 cm). The room was illuminated
with four 25 W red bulbs giving a light intensity 12 lx on the arms.
Mice (n = 6 per group) were randomly assigned to 13 experimental
groups (vehicle control 4% gum acacia p.o., 1 mg/kg diazepam p.o.
and 30 mg/kg of MF-2, 7, 10, 11, 13, 14, 15, 16, 17, 19). Drug admin-
istration was oral and 60 min prior to the test. The number of entries
into and the time spent on each of the two types of arms and the
latency to enter open arms were recorded during the 5 min trial.
During the 5 min session the following parameters were noted.
Fig. 1. Molecular formula of Cardiospermin with structure.
• Number of entries into open arm.
• Number of entries into closed arm.
• Time spent in the open arm.
• Time spent in the closed arm.
Every time before placing the animal, the arena was cleaned
with 5% alcohol to eliminate the possible bias due to the odour left
by the previous animal.
Light dark transition model
The method of Costall et al. (1989) was adopted and slightly
modified. The apparatus used was a box made of wood with overall
dimensions of 40 cm × 60 cm × 20 cm (length, width, height) and a
grid floor composed by bars spaced 5 cm apart. The box was further
divided by a barrier possessing a doorway (7 cm round hole), which
mice could cross in two chambers of measures (40 cm × 20 cm):
painted black, not illuminated, and (40 cm × 40 cm) painted white
and illuminated with a 60 lx light source. On the test day, mice were
administered orally diazepam (1 mg/kg, n = 6 for standard treated
group) and three doses (10, 20 and 30 mg/kg) active fractions of
extract (MF-14, 16 and 17) to other groups. MF-14, 16 and 17 were
selected due their significant result in EPM as comparing to other
MFs. Control group was administered vehicle (4% gum acacia, p.o.).
One hour later, each animal was placed in the middle of the light
compartment, facing the doorway separating the two compart-
ments. The behaviour of animal was noted for 5 min and following
parameters were recorded.
• Latency to the first crossing of the dark compartment.
• Number of crossings between light and dark area.
• Total time spent in the illuminated part of the cage.
• Total locomotion.
• Rearing.
Statistical analysis
Results are expressed as mean ± S.E.M. from (n = 6) animals per
group. Statistical differences in the mean were collected were cal-
culated/performed using One-way ANOVA followed by Dunnett’s
post hoc test for a significance level of p < 0.05.
Results
Identification of active compound
Fraction 14 of ethanolic extract of Cardiospermum halicacabum
yielded a dark brown coloured active compound which was
confirmed based on its FT-IR, MS and 1H NMR values. These data
led to the identification of a well-known compound, Cardiosper-
min, i.e. (2S)-6-hydroxy-4-methylene-2-[{3,4,5-trihydroxy-6-
(hydroxymethyl)-tetrahydro-2H-pyran2yl}oxy] hexanenitrile
(Fig. 1), with a molecular formula C13H21NO7 and a molecular
weight of 303. Molecular mass peak at 303 m/z and base peak at
141 m/z justifies that the base peak was formed by the aglycone
part fragmented from the parent compound.
Assessment of anxiolytic activity
Elevated plus-maze test
Diazepam has long been reported for its anxiolytic activity in
mice with EPM. In present study also, a pronounced anxiolytic affect
was observed on mice after administration of diazepam (1 mg/kg)
with significant increase in number of entries in the open arm
(p < 0.01). The results are shown in Table 1. 3 out of 10 MFs (mas-
ter fractions selected for activity), i.e. MF-14, 16 and 17 (30 mg/kg)

3. R. Kumar et al. / Phytomedicine 18 (2011) 219–223 221
Table 1
Effect of various master fractions of ethanolic extract of C. halicacabum on following parameters in EPM test.
Treatment No. of entries (counts/5 min) Time spent (s/5 min)
Open arm Closed arm Open arm Closed arm
Control 3.000 ± 1.265 17.167 ± 1.701 30.333 ± 12.041 231.17 ± 10.790
Diazepam (1 mg/kg) 13.500 ± 0.9220***
7.667 ± 0.6667**
127.17 ± 17.029***
102.00 ± 21.793***
MF-2 4.500 ± 1.668 13.667 ± 1.406 50.500 ± 13.537 209.33 ± 16.976
MF-7 2.667 ± 0.6667 11.833 ± 1.662 23.667 ± 7.027 221.50 ± 9.646
MF-10 3.333 ± 1.145 11.667 ± 2.076 21.000 ± 5.972 210.67 ± 10.775
MF-11 2.833 ± 1.195 14.167 ± 2.088 23.500 ± 8.217 217.50 ± 16.382
MF-12 5.667 ± 1.476 16.000 ± 1.653 28.833 ± 10.922 226.17 ± 14.786
MF-13 6.500 ± 1.285 12.500 ± 3.063 44.500 ± 9.197 196.50 ± 10.844
MF-14 9.167 ± 1.195*
10.000 ± 1.065*
79.833 ± 8.670 144.03 ± 24.002**
MF-15 5.833 ± 1.470 11.500 ± 1.285 61.500 ± 16.530 185.657 ± 13.781
MF-16 (A) 6.167 ± 1.493 10.667 ± 1.647 51.500 ± 9.243 166.67 ± 16.137
MF-16 (B) 12.000 ± 1.732***
8.667 ± 0.6667**
120.83 ± 19.017***
129.17 ± 20.756***
MF-17 13.333 ± 1.520***
8.500 ± 0.6191**
143.00 ± 13.967***
106.67 ± 21.239***
MF-19 6.66 ± 1.382 12.167 ± 1.470 60.000 ± 11.475 193.33 ± 12.085
Values are expressed as mean ± S.E.M. from six animals.
*
p < 0.05 statistically significant as compared to control.
**
p < 0.01 statistically significant as compared to control.
***
p < 0.001 statistically significant as compared to control.
resulted in a significant increase in the entries into open arms
(p < 0.05, p < 0.01and p < 0.001). However, some differences were
observed between the two drugs, e.g., diazepam increased total
time spent in the open arm and total arm entries at the same time
only MF-16(B) and 17 had shown such effect.
Light dark transition model
The results of the light/dark test are shown in Figs. 2–4. MF-
14, 16 and 17 at the doses of 10, 20 and 30 mg/kg and diazepam
(1 mg/kg) induced a significant increase in the time spent in the
light zone (p < 0.05 and p < 0.01) without affecting other parame-
ters. The significant increase (p < 0.05, p < 0.01 and p < 0.001) in time
of locomotion compared to control group showed decrease in fear
and anxiety in animal which indicates anxiolytic activity of MFs
fractionated from C. halicacabum roots (Table 2).
Discussion
There has been a considerable popular interest in the use of
the so-called natural remedies, or herbal products, to treat anxi-
ety and depression. St. John’s wort is the most well-known herbal
product available over the counter. Recently, several plants have
been reported to possess anxiolytic effects through animal models
of anxiety (Beaubrum and Gray 2000).
The EPM is currently the first choice test for anxiolytic drugs
and has been validated for both rats and mice (Pellow et al. 1985;
Lister 1987). It is based on the natural conflict between the drive to
Fig. 2. Effect of single treatment of MF-14 on the time spent in the illuminated
part of light dark box over a 5 min period of time. Mice were administered MF-14
(10, 20 and 30 mg/kg, p.o.). Bar represents mean ± S.E.M. (n = 6). p values for group
comparisons were made using One-way ANOVA followed by post hoc Dunnett’s test
(*p < 0.05, and ***p < 0.001 versus the vehicle treated control group).
explore a new environment and the tendency to avoid potentially
dangerous area. More recently, it has been argued that the incorpo-
ration of a range of ethological parameters may enhance the utility
of this paradigm (Rodgers and Johnson 1995). Therefore, we chose
Table 2
Effect of MF-14, 16 and 17 from ethanolic extract of C. halicacabum on various parameters in light dark transition model.
Treatment Latency Time spent in dark zone (s/5 min) Rearing Total locomotion
Control 6.617 ± 0.9098 215.33 ± 12.743 4.333 ± 0.988 86.500 ± 15.684
Diazepam (1 mg/kg) 13.667 ± 1.820**
105.83 ± 6.969***
9.833 ± 1.537*
239.83 ± 15.230***
MF-14 (10 mg/kg) 9.667 ± 1.746 196.83 ± 10.326 5.167 ± 1.600 161.83 ± 14.178*
MF-14 (20 mg/kg) 9.333 ± 0.8819 177.83 ± 15.611 8.500 ± 1.803 193.83 ± 13.004***
MF-14 (30 mg/kg) 8.000 ± 1.506 159.67 ± 12.945 7.833 ± 1.400 174.67 ± 22.435**
MF-16 (10 mg/kg) 9.333 ± 1.453 149.33 ± 13.880*
10.667 ± 0.5578*
177.33 ± 19.243**
MF-16 (20 mg/kg) 10.167 ± 1.701 113.17 ± 17.884***
8.833 ± 0.9098 208.33 ± 13.213***
MF-16 (30 mg/kg) 12.167 ± 0.9098*
95.667 ± 11.445***
9.500 ± 0.7683 203.33 ± 20.704***
MF-17 (10 mg/kg) 8.500 ± 1.565 141.00 ± 22.710**
9.333 ± 1.174 190.33 ± 14.099***
MF-17 (20 mg/kg) 11.833 ± 1.078 96.167 ± 17.945***
9.000 ± 1.844 217.67 ± 7.911***
MF-17 (30 mg/kg) 11.667 ± 1.308 85.000 ± 12.359***
11.000 ± 1.317**
210.17 ± 20.355***
Values are expressed as mean ± S.E.M. from six animals.
*
p < 0.05 statistically significant as compared to control.
**
p < 0.01 statistically significant as compared to control.
***
p < 0.001 statistically significant as compared to control.

4. 222 R. Kumar et al. / Phytomedicine 18 (2011) 219–223
Fig. 3. Effect of single treatment of MF-16 on the time spent in the illuminated
part of light dark box over a 5 min period of time. Mice were administered MF-16
(10, 20 and 30 mg/kg, p.o.). Bar represents mean ± S.E.M. (n = 6). p values for group
comparisons were made using One-way ANOVA followed by post hoc Dunnett’s test
(**p < 0.01 and ***p < 0.001 versus the vehicle treated control group).
Fig. 4. Effect of single treatment of MF-17 on the time spent in the illuminated
part of light dark box over a 5 min period of time. Mice were administered MF-17
(10, 20 and 30 mg/kg, p.o.). Bar represents mean ± S.E.M. (n = 6). p values for group
comparisons were made using One-way ANOVA followed by post hoc Dunnett’s test
(**p < 0.01 and ***p < 0.001 versus the vehicle treated control group).
this test to investigate the anxiolytic potential of the various frac-
tions of ethanolic root extract of Cardiospermum halicacabum. The
indices of anxiety in this test, percent of open arm entries and time
spent in the open arm are sensitive to agents thought to act via the
GABAA receptor complex, justifying the use of diazepam as a posi-
tive control in this study. In agreement with previously published
reports, diazepam increased the number of open arm entries and
the time spent in the open arms (Moser 1989; Helton et al. 1996;
Eguchi et al. 2001), confirming its anxiolytic effects. The 3 MFs (14,
16 and 17) had similar effects on these parameters. The effect of
MF-14, 16 and 17 (30 mg/kg) on the elevated plus-maze test was
significant (p < 0.05, p < 0.01 and p < 0.001), as compared to control.
These observations clearly indicate these MFs exert an anxiolytic
activity.
The light dark transition model box is also widely used for
rodents as a model for screening anxiolytic or anxiogenic drugs.
A good agreement has been observed between relative potency
of drugs clinically used in the treatment of anxiety in humans
and their ability to facilitate exploratory activity in the light/dark
paradigm in mice (Crawley and Goodwin 1980). Transitions have
been reported to be an index of activity exploration because of
habituation over time, and the time spent in each compartment to
be a reflection of aversion (Belzung et al. 1987). Young and Johnson
(1991) concluded that simply the measurement of the time spent in
the light area, but not the number of transfers, was the most con-
sistent and useful parameter for assessing anxiolytic-like action.
Furthermore, Lepicard et al. (2000) reported that the time spent in
the light was a stronger indication in the study of anxiety, whereas
the number transfers reflected both anxiety and exploration. These
observations seem to be in good agreement with our results. The
present data showed that MF-14, 16 and 17 (10, 20 and 30 mg/kg)
could increase the time spent in the light area, suggesting again
these fractions possesses anxiolytic properties. In the above study,
time of locomotion was increased with MFs and diazepam treat-
ment demonstrated that animals were more active in the light
box.
Results of this study indicated that the ethanolic root extracts
of CH had central anxiolytic effects. The phytoconstituent respon-
sible for the observed central effects has been isolated from MF-14
and identified as well-known compound, Cardiospermin (Fig. 1), a
cyanogenic glucoside (Hubel and Nahrstedt 1979). Future prospects
of this work include the identification of the active constituents
present in MF-16 and 17 along with the pharmacological mecha-
nisms underlying the activity of Cardiospermin.
Conclusion
In conclusion, bioactivity guided fractionation identified Car-
diospermin as major active compound in roots of CH. Since there
is a need for new anxiolytic compounds with fewer side effects
compared to synthetic medication, natural products could be an
inspiration of new prototypes for drug development.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors like to thank PES College of Pharmacy and Manipal
University for their support for this research work.
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