Journal of Ethnopharmacology 102 (2005) 298–301
Antinociceptive activity of alcoholic extract of
Hemidesmus indicus R.Br. in mice
Prashant R. Vermab,∗, Amit A. Joharapurkara, Vivekanand A. Chatpalliwara,
Alpana J. Asnanib
aDepartment of Pharmaceutical Sciences Nagpur, Nagpur University, Nagpur 440033, MH, India
bPharmacognosy, J.L. Chaturvedi College of Pharmacy, 846, New Nanandanvan, Nagpur 440009, MH, India
Received 12 February 2005; received in revised form 20 May 2005; accepted 26 May 2005
Available online 2 August 2005
The ethanolic extract of roots of Hemidesmus indicus R.Br. (family: Asclepiadaceae) was investigated for possible antinociceptive effect in
licking test) and hot plate test in mice. Hemidesmus indicus R.Br. extract was administered in the dose range of 25, 50 and 100mg/kg orally
1h prior to pain induction. The preliminary phytochemical screening of the extract showed the presence of triterpenes, flavonoids, pregnane
glycosides and steroids. Oral administration of Hemidesmus indicus extract revealed dose-dependent antinociceptive effect in all the models
for antinociception and it blocked both the neurogenic and inflammatory pain and the nociceptive activity was comparable with the reference
drug. The results indicate that alcoholic extract of Hemidesmus indicus R.Br. possesses a significant antinociceptive activity. The activity can
be related with the significant phytochemicals such as triterpenes, flavonoids, and sterols reported in the root extract.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antinociceptive; Hemidesmus indicus; Hot plate test; Writhing test; Paw licking test
Hemidesmus indicus R.Br. Indian sarsaparilla (Anan-
tamul) belonging to the family Asclepiadaceae, a twining
shrub commonly found in India. The roots of the plant
are woody and have a sweet taste, with cooling effect,
and used in various ailment of diseases, a well-known
drug in the Ayurveda system of medicine (Kirtikar and
Basu, 1991). Inflammation induced by Viper venom and
Propiono bacterium acne are reported to be treated by root
extract (Jain and Basal, 2003). A lep of root powder applied
topically is used to treat swellings, inflammation and chronic
rheumatism (Alam et al., 1994; Alam and Gomes, 1998a,b).
Many other reports suggest the activity against inflam-
Abbreviations: i.p., intra peritoneal; p.o., per oral; %MPE, maximum
possible antinociceptive effect; H. indicus, Hemidesmus indicus
E-mail address: firstname.lastname@example.org (P.R. Verma).
mation induced due to pathogenesis for example, asthma,
leprosy, swelling of joints, inflamed eyes and in chronic
These said uses of the drug emphasize on its anti-
inflammatory activity. But mechanisms are not clearly
reported, whether activity is due to suppression of central
and peripheral pathways of inflammation or both. In view of
this and on account of the reported usefulness of this plant
in traditional system of medicine and inflammatory condi-
tions, the present study was aimed at investigating possible
antinociceptive activities of ethanolic extract of the roots in
2. Materials and methods
2.1. Plant materials and extract
The roots of Hemidesmus indicus obtained from
Amarkantak forest was authenticated by Mr. S. Chilate, the
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
P.R. Verma et al. / Journal of Ethnopharmacology 102 (2005) 298–301
authorized plant identifier of Department of Botany, Nagpur
University Nagpur. A specimen is preserved in the college
pulverized into a dry powder and extracted with, ethanol for
dark brown colored semisolid mass (yield 9.2%, w/w) under
2.2. Phytochemical screening (Stahl, 1969; Harbone,
1976; Wagner et al., 1984)
The extracts were screened for the presence of tan-
nins, saponins, unsaturated sterols, triterpenes, alkaloids,
anthraquinones, flavonoids, lactones/esters, protein/amino
acids and carbohydrates and/or glycosides with thin layer
chromatography (TLC). Thin layer plates precoated with sil-
ica gel G (Merck, 0.25mm thickness) were used. Devel-
opment was carried out with different solvent systems ethyl
acetate:methanol:water (100:13.5:10, v/v/v), ethyl acetate:
formic acid:acetic acid:water (100:14:10:27, v/v/v/v), chlo-
roform:methanol:water (64:50:16, v/v/v), benzene:ethyl
acetate (80:20, v/v) and ethyl acetate:methanol:water:acetic
acid (65:15:15:10, v/v/v/v). After development in the sol-
vents the plates were dried and sprayed with Dragendorff’s,
AlCl3, hydroxylamine-ferric chloride, ninhydrin and anti-
mony trichloride for the discovery of alkaloids, flavonoids,
lactones/esters, protein/amino acids, unsaturated sterols, and
triterpenes. Detection of anthraquinones, saponins, tannins,
carbohydrate and/or glycosides is carried out using KOH,
anisaldehyde-sulphuric acid reagents, ferric chloride and
naphthoresorcinol reagents, respectively. Detection was car-
ried out by visualization in visible light and under UV light
Swiss male mice 25–28g, procured from animal house
of J.L. Chaturvedi College of Pharmacy, Nagpur were used.
The animals received a standard pelleted diet (M/s Hindus-
tan Lever Foods, Calcutta, India) and water ad libitum, and
were maintained under standard environmental conditions
(22±5◦C with 12h of light/dark cycle). The animals were
divided into a group of six mice each. The Institutional Ani-
mal Ethical Committee (JLC/IAEC/2000/10) approved the
Pentazocine was obtained from Ranbaxy Laboratories
Ltd., New Delhi. Acetic acid and formalin used are of ana-
lytical grade obtained from Loba chemicals. The root extract
was suspended with the help of gum acacia in distilled water
at the time of oral administration in the doses of 25, 50 and
100mg/kg body weight as per acute toxicity studies carried
2.5. Acetic acid-induced writhing in mice (Koster et al.,
1959; Amos et al., 2002)
Mice were divided into five groups, of six mice each and
pre-treated as follows: group I received only vehicle, group
II received a standard drug pentazocine (2mg/kg, i.p.) and
served as control and positive control, respectively. Groups
III, IV and V, received 25, 50, and 100mg/kg extract p.o.,
with pre-treatment time of 1h. Each group was administered
10ml/kg body weight (i.p.) of an aqueous solution of
acetic acid (0.6%). The mice were then observed for the
number of abdominal constrictions and stretching, counted
over a period of 0–20min. The percentage inhibition was
determined for each experimental group as follows:
= 100 ×1 − number of writhing in experimental group
number of writhing in control group
2.6. Formalin test in mice (Dubuisson and Dennis,
1977; Tjolsen et al., 1992)
Groups of six Swiss male mice were pre-treated p.o., with
Hemidesmus indicus extract (25, 50, and 100mg/kg), stan-
dard biphasic drug pentazocin (2mg/kg i.p.) and vehicle.
lin, subcutaneously under the sub-plantar region of the left
They were then placed in an observation chamber and
monitored for 1h, recording severity of pain responses based
on the following scale: (0) mice walked or stood firmly on
injected paw; (1) the injected paw was favored or partially
elevated; (2) the injected paw was clearly lifted off the floor;
(3) the mice licked, chewed or shook the injected paw. This
thus showing finer degrees of antinociception as opposed to
the method in which only the time the animal spent lick-
ing the injected paw is recorded. Antinociceptive effect was
determined in two phases, the early phase (I) being recorded
during the first 5min while the late phase (II) was recorded
during the last 45min with a 10min lag period in between
2.7. Hot-plate test in mice (Turner, 1965)
This test was carried out on a group of six Swiss male
mice using a hot-plate apparatus (Lyabco model D-S37),
maintained at 55±1◦C and the method described earlier
was employed. Only mice that showed initial nociceptive
responses within 20s were selected for the experiment. The
latency to first sign of hind paw licking or jump response to
avoid heat nociception was taken as an index of nociceptive
threshold with cut off time of 30s. The nociceptive threshold
was observed before and after at 30 and 60min, after admin-
istration of the extract at doses of 25, 50 and 100mg/kg p.o.
P.R. Verma et al. / Journal of Ethnopharmacology 102 (2005) 298–301
The control group was administered distilled water at simi-
lar volumes to the test group and positive control group with
drug pentazocine (2mg/kg i.p.). The result was expressed as
%MPE (maximum possible antinociceptive effect).
3. Statistical analysis
All data were expressed as the mean±S.E.M. data was
subjected to ANOVA followed by Dunnett’s multiple com-
parison test. P≤0.05 was considered significant.
The phytochemical screening of ethanolic extract of
Hemidesmus indicus root shows the presence of flavonoids,
terpenoids, tannins, coumarins and glycoside. Test for alka-
loids, anthraquinones, lactones/ester, protein/amino acids
and saponins showed negative responses.
The extract of Hemidesmus indicus at doses of 25, 50
and 100mg/kg, showed antinociceptive activity in all the
models for nociception and the effect was found to be dose-
dependent. The extract decreased the number of acetic acid-
induced abdominal constrictions in mice and the values were
imum percentage inhibition of constrictions of 46.8% was
observed at 100mg/kg for the extract.
The formalin test in mice revealed a similar pattern
of antinociceptive effect, which was also dose-dependent
(Fig. 2). There was a significant reduction in responses to
nociception during both phases I and II for the extract at
100mg/kg, when compared to the control and standard drug
the animal’s reaction time to the heat stimulus. Values were
found to be significant (P<0.05) at 60min after treatment
with 100mg/kg. While values for 50mg/kg were significant
Fig. 1. Antinociceptive activity of Hemidesmus indicus in acetic acid
induced writhing test. Bar represents mean±S.D. (n=6). P<0.05 compare
to other group.
licking test. Bar represents mean±S.D. (n=6).*P<0.05 as compared to
Fig. 3. Antinociceptive activity of Hemidesmus indicus in hot plate reac-
tion time. Bar represents mean±S.D. (n=6). %MPE calculated from basal
latency in each test.
at 60min after treatment with extract. Maximal percentage
inhibition of nociception was 60.12% observed during phase
I, at 100mg/kg for the extract (Fig. 3).
5. Discussion and conclusion
Three different animal models were employed to inves-
tigate the potential antinociceptive activity of the ethanolic
extract of Hemidesmus indicus in this study. The methods
methods elucidated peripheral and central activity, respec-
tively, while the formalin test investigated both.
The extract at the doses tested was shown to possess
antinociceptive activity evident in all the nociceptive models
signifying it possess both the central and peripherally
P.R. Verma et al. / Journal of Ethnopharmacology 102 (2005) 298–301
mediated activities. The extracts at the doses tested was
shown to possess dose-dependent antinociceptive activity.
Results indicated that the extract exhibited significant
antinociceptive activity against all the three models the mod-
els of pain, and it blocked both the neurogenic and inflam-
matory pain. The activity was dose-dependent that reached
et al., 1998). It is also called as the abdominal constriction
response, it is very sensitive and able to detect antinocicep-
tive effects of compounds and dose levels that may appear
inactive in other methods like tail-flick test (Bentley et al.,
1981). Local peritoneal receptors are postulated to be partly
involved in the abdominal constriction response (Bentley et
al., 1983). The method has been associated with prostanoids
in general, e.g. increased levels of PGE2 and PGF2? in
peritoneal fluids as well as lipooxygenase products (Derardt
et al., 1980). Therefore, the result of the acetic acid induced
writhing strongly suggests that the mechanism of this
extract may be linked partly to lipooxygenase and/or cyclo-
In the formalin test there is a distinctive biphasic noci-
ceptive response termed early and late phase. Drugs that
act primarily on the central nervous system inhibit both
phases equally while peripherally acting drugs inhibit the
late phase (Chen et al., 1995). Inhibition of the late phase is
due to inflammation with a release of serotonin, histamine,
sensitization of central nociceptive neurons. Suppression of
both phases of pain as observed with the extract (50mg/kg)
in this study also lends strong credence to the presence of
both central and peripheral effects.
In conclusion, this study has shown that the ethano-
lic extract of Hemidesmus indicus possess significant
antinociceptive effects in laboratory animals at the doses
investigated. The results support the traditional use of this
plant in some painful and inflammatory conditions, and
activity can be related with the significant phytochemicals
ther studies are in fact currently underway to isolate and
characterize the active principle(s) of the crude extract.
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