Journal of

Pharmaceutical and
Allied Sciences

JOPHAS

A STUDY ON ANTINOCICEPTIVE, ANTIINFLAMMATORY AND

ANTIDIARRHOEAL ACTIVITIES OF VALLARIS SOLANACEA (Roth)
KUNTZE BARK EXTRACTS

RAHMAN MD. ASHIKUR1*, HAQUE EKRAMUL1, MD. HASANUZZAMAN1,

MUHURI SR2, SHAHID IZ1
1
PHARMACY DISCIPLINE, KHULNA UNIVERSITY, KHULNA-9208,
BANGLADESH.
2
UNIVERSITY OF SCIENCE AND TECHNOLOGY, CHITTOGONG-4202,
BANGLADESH.

*Correspondence author’s Email address: ashik031123@gmail.com

ABSTRACT
The present study investigated pharmacological activities to provide scientific basis for traditional
usage of the plant, Vallaris solanacea (Roth). Phytochemical analysis of the dried roots indicated the
presence of reducing sugars, tannins, saponins, gums, steroids, alkaloids and glycosides. The
pharmacological interest of these compounds, coupled with the use of this plant in traditional medicine
prompted the authors to check for its possible antinociceptive, anti-inflammatory and antidiarrhoeal
activities. The ethanolic extract showed statistically significant analgesic activity (p<0.01) in acetic
acid induced writhing inhibition in mice at the dose of 500 mg/kg body weight and also showed mild
effect at the dose of 250 mg/kg body weight. When given orally to rats at doses of 200 and 400 mg/kg,
the extract showed a significant (P<0.001) anti-inflammatory activity against carrageenan induced paw
edema in rats comparable to the standard drug phenyl butazone. The crude extract produced significant
antidiarrhoeal effect at the dose of 500 mg/kg of body weight comparable to that produced by
loperamide, used as standard drug. The results tend to suggest that the extract might possess some
chemical constituents that are responsible for analgesic, antiinflammatory and antidiarrhoeal activities.
Key words: Vallaris solanacea, Phytochemical tests, Analgesic, Antiinflammatory and
Antidiarrhoeal.

INTRODUCTION with a new glycoside, benzyl 2-O-β-

Vallaris solanacea (Roth) Kuntze (Family:
Apocynaceae), local name: Agarmoni,
Bread flower, is a tall climbing shrub
which is locally used as medicinal plant. It
is native in India and Burma and also
found in Sylhlet in Bangladesh. It is
traditionally used for sores and wounds.
Barks are chewed for fixing teeth.
Previous research revealed that it contains
a mixture of glycosides vallarisoside and a
known one, 3β-O-(α-acofriosyl) along
apiofuranosyl-(1→2)-β-D-glucopyranosyl-
2 (1) and also O- acetyl-solanoside (O-
acetyl-acofreosyl digitoxigenin) (2). In this
project work an attempt was made to
justify scientifically, the traditional uses of
the plant. Moreover by using various
standard qualitative chemical tests the
Journal of Pharmaceutical and Allied Sciences
Vol. 8 No. 1 (2011)
ISSN: 1596-8499
Website: http://ajol.info/index.php/jophas
 Rahman Ashikur et al/Journal of Pharmaceutical and Allied Sciences 8 (1) (2011) 1268 - 1275
presence of reported compounds were
detected. In the present study, we
evaluated the antinociceptive, anti-
inflammatory and antidiarrhoeal activity of
the ethanolic leaf extract.
MATERIALS AND METHODS
Plant material collection and
extraction
The barks of Vallaris solanacea (Roth)
Kuntze were collected from Jessore,
Bangladesh in August 2008 and identified
by the experts at Bangladesh National
Herbarium, Mirpur, Dhaka (Accession No.
DACB- 44320). The identified plant bark
was dried under shade. After complete
drying, the sample was cut into small
pieces and then reduced to coarse powder
with the help of a mechanical grinder and
the powder was stored in a suitable
container. About 500 mg of the powder
was extracted by maceration over 20 days
with 1200 ml of 80% ethanol. The extract
was filtered with a piece of cotton wool,
followed by filtration through Whatmann
filter paper grade 1. The filtrate thus
obtained was concentrated using a rotary
evaporator (Bibby RE200, Sterilin Ltd.,
U.K.) to get the crude extract.
Animal samples
Swiss-Albino mice of either sex (20-25 gm
body weight) were collected from animal
resources branch of the International
Center for Diarrhoeal Disease Research,
Bangladesh (ICDDR, B) and were used for
the experiments. The animals were kept in
the standard polypropylene cages and
provided with standard diets (ICDDR, B
formulated). The animals were
acclimatized in animal house, Pharmacy
Discipline, Khulna University, Khulna
under standard laboratory conditions
(relative humidity 55-60%, room
0
temperature 25±2 C and 12 hours light:
dark cycle) for a period of 14 days prior to
performing the experiments.
Drug samples
Diclofenac sodium and phenyl butazone
were sourced from Beximco Pharmaceuticals
Ltd., Bangladesh.
Preliminary phytochemical analysis
The crude extracts were subjected to
preliminary phytochemical screening for
the detection of major chemical groups. In
each test, 10% (w/v) solution of the extract
in methanol was used unless otherwise
mentioned in individual tests (3, 4).
Tests for reducing sugar
Benedict’s Test: A 0.5 ml volume of the
extract was placed in a test tube and then 5
ml Benedict’s solution was added to it.
This was boiled for 5 min and allowed to
cool spontaneously.
Fehling’s Test (Standard Test): A 2 ml
volume of the extract was added in 1 ml of
a mixture of equal volumes of Fehling’s
solutions A and B, and was boiled for few
min.
Combined Reducing Sugar test: A 1 ml
volume of the extract was boiled with 2 ml
of dilute hydrochloric acid for 5 min. After
cooling, the mixture was neutralized with
sodium hydroxide solution and then
Fehling’s test was performed as earlier
described.
Tests for tannins
Ferric Chloride Test: A 5 ml volume of the
extract was placed in a test tube and then 1
ml of 5% ferric chloride solution added to
it.
Potassium dichromate test: A 5 ml volume
of the extract was placed in a test tube and
then 1 ml of 10% potassium dichromate
solution added.
Test for flavonoids
A few drops of concentrated hydrochloric
acid were added to 5 ml of the extract.
Test for saponins
A 1 ml volume of the extract was placed in
a graduated cylinder, diluted to 20 ml with
distilled water and shaken gently for 15
min.
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Test for gums
A 5 ml volume of the extract was placed in
a test tube and then Molisch’s reagent and
sulphuric acid were added to it.
Tests for steroids
Libermann-Burchard test: A 1 ml volume
of the extract was placed in a test tube and
2 ml of Libermann-Burchard reagent
added.
Sulphuric acid test: A 1 ml volume of the
extract was placed in a test tube and 1 ml
sulphuric acid added.
Tests for alkaloids
Mayer’s test: A 2 ml volume of the extract
and 0.2 ml of dilute hydrochloric acid
were placed in a test tube and 1 ml of
Mayer’s reagent added.
Dragendorff’s test: A 2 ml volume of the
extract and 0.2 ml of dilute hydrochloric
acid were placed in a test tube and1 ml
Dragendorff’s reagent added.
Wagner’s test: The extract (2 ml) and 0.2
ml of dilute hydrochloric acid were placed
in a test tube. Then 1 ml of Wagner’s
reagent was added.
Hager’s test: A 2 ml volume of the extract
and 0.2 ml of dilute hydrochloric acid
were placed in a test tube. Then 1 ml of
picric acid solution (Hager’s reagent) was
added.
PHARMACOLOGICAL STUDIES
Determination of antinociceptive
activity
The analgesic activity of the sample was
studied using acetic acid induced writhing
model in mice. Experimental animals were
randomly selected and divided into four
groups denoted as Control group, Positive
control group, Test group I and Test group
II consisting of 5 mice in each group.
Control group received orally, 1% Tween-
80 at the dose of 10 mg/kg body weight
and Positive control group received orally,
diclofenac sodium at the dose of 25 mg/kg
body weight. Test groups I and II were
treated with the test sample orally, at the
dose of 250 and 500 gm/kg body weight.
A thirty minutes interval was given to
ensure proper absorption of the
administered substances. Then the
writhing inducing chemical, acetic acid
solution (0.7%) was administered intra-
peritoneally to each of the animals of a
group. An interval of 5 minutes was given
for absorption of acetic acid and number of
writhings in 15 minutes was noted. The
animals do not always perform full
writhing. The incomplete writhing was
taken as half-writhing, so two half-
writhings were taken as one full writhing.
This is why total writhing was halved to
convert all writhing to full writhing or real
writhing (5, 6).
Anti-inflammatory activity
Anti-inflammatory activity of Vallaris
solanacea was tested by using carrageenan
induced rat paw edema model (6, 7). Rats
were randomly divided into four groups,
each consisting of six animals. Group I
was kept as ‘control’ giving 1% (v/v)
Tween-80 solution in water; group II was
kept as ‘positive control’ and was given
the standard drug phenylbutazone at a dose
of 100 mg/kg of body weight; group III
and IV were test groups, treated with
extracts at the doses of 200 and 400 mg/kg
of body weight respectively. Control
vehicle, standard drug and the extracts
were given orally 1 h prior to the injection
of 0.1 ml of 1% freshly prepared
suspension of carrageenan. The paw
volume was measured by using a
plethysmometer just before and 1, 2, 3, 4,
5 h after the carrageenan injection.
Antidiarrhoeal activity
Antidiarrhoeal screening of the Vallaris
solanacea extract was carried out by the
method described by Shoba (8) and
Thomas (9). Animals were screened for
diarrhoeal activity by administering 0.3
mL castor oil (Hospital pharmacy, Khulna
Medical College) and those showing
diarrhoeal activity were selected for the
experiment. The animals were divided into
groups of five mice per group for
experimental control (1% Tween 80, 10
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mL/kg), positive control (3 mg/kg body
weight of loperamide) and for test group
(500 mg/kg body weight of bark extract).
Forty minutes after treatment, the mice
were administered with 0.3 mL castor oil
to induce diarrhoea. The mice were placed
in individual cages with floor lined with
blotting paper. The floor lining was
changed every hour for the next 4 hours of
the observation period. Non-infective
diarrhoea is characterized by looseness of
bowel (10). Hence, we count the total
number of diarrheic faecal output and
latent time for each of sample set. A
numerical score based on stool consistency
was assigned as follows: normal stool=1
and watery stool=2.
Statistical analysis
Student’s t-test was used to determine
significant differences between the control
group and test group.
RESULTS
Preliminary phytochemical analysis
Results of different chemical tests on the
methanol extract showed the presence of
Reducing sugar, Tannins, Saponins, Gums,
Steroids, Alkaloid, Glycosides (Table1)
Antinociceptive activity
Analgesic activity of the ethanolic extract
was tested by acetic acid induced writhing
model in mice. The extract produced
51.68% (p<0.01) acetic acid induced
writhing inhibition in mice at the dose of
500 mg/kg body weight, which is
comparable to diclofenac sodium 71.42%
(p<0.001) at the dose of 25 mg/kg body
weight (Table 2).
Anti-inflammatory activity
The experimental findings from the
carrageenin-induced rat paw edema model
showed that the ethanolic extract of
Vallaris solanacea reduced the paw
volume significantly (P<0.001) from 1h to
5h. The extract showed highest effects at
the third hour where the inhibition was
about 29% and 41% at dose of 200 and
400 mg/kg respectively, which were
comparable to the standard drug
phenylbutazone, where the inhibition was
about 51% at the dose of 100 mg/kg of
body weight (Table 3).
Antidiarrhoeal activity
Antidiarrhoeal activity of the methanol
extract of Vallaris solanacea extract was
tested by castor oil-induced diarrhea in
mice. Diarrheal initiation time and the
number of stools excreted by the animals
in 4 hours were collected. The extract
caused an increase in latent period (1.1h)
i.e. delayed the onset of diarrhoeal episode
of 500 mg/kg body of weight significantly
(P<0.01) which was comparable to the
standard drug loperamide at the dose of 50
mg/kg body weight in which the resulted
value was 1.5h (P<0.01) (Table 4). The
selected concentration of the extract also
showed a good diarrheal inhibition with
65.36%. Loperamide, standard
antidiarrheal agent showed an inhibition of
72.22%. The latent period for the initiation
of stool excretion was noted. This was
1.114 hrs, which is 0.432 hrs earlier than
loperamide treated mice but, 0.43 hrs latter
than experimental control mice.
DISCUSSION
To get preliminary idea about the active
constituents present in the plant extracts
different chemical tests were performed
and found the presence of Reducing Sugar,
Tannins, Saponins, Gums, Steroids,
Alkaloids, and Glycosides.
Analgesic activity of the ethanol extract of
V. solanacea was tested by acetic acid
induced writhing model in mice. Acetic
acid induced writhing model represents
pain sensation by triggering localized
inflammatory response. Acetic acid, which
is used to induce writhing, causes algesia
by liberation of endogenous substances,
which in turn excite the pain nerve endings
(11). Increased levels of PGE2 and PGF2α
in the peritoneal fluid have been reported
to be responsible for pain sensation caused
by intraperitoneal administration of acetic
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Table 1. Results of preliminary phytochemical analysis

Reducing
Alkaloids Glycosides Steroids Gums Flavonoids Saponins Tannins
Compound sugars

Observation -ve +ve +ve +ve - ve + ve + ve +ve

Key: +ve = Presence -ve = Absence

Table 2: Effect of Vallaris solanacea on acetic acid induced writhing in mice

Writhing Count %Writhing
Animal Group Treatment (%Writhing) Inhibition

Control (n=5) 1% tween-80 16 ± 1.415 (100)

solution in water 0

Positive Control Diclofenac sodium 4.4 ±

(n=5) (25mg/kg) 0.51***(28.58) 71.42

Test group I Et. Extract 12.4 ±3.015*

(n=5) (250mg/kg) (77.5) 22.5

Test group I Et. Extract 7.4 ±

(n=5) (500mg/kg) 1.355**(48.32) 51.68

Values are expressed as mean ± SEM, SEM=Standard error of Mean, n=No. of mice, Et.= Ethanolic, *P<0.05;
**
P<0.01; ***P<0.001 vs. control.

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Table 3. Effect of ethanolic extract of Vallaris solanacea on carrageenin induced rat paw
edema
Time after carrageenin injection

Animal group / 1 hr 2 hr 3 hr 4 hr 5 hr

Treatment Increase in Paw Edema volume (ml)×1000 ± S.E.M.

Control
(1% Tween 80) 137.00±0.70 164.50±1.02 244.50±0.85 272.00±0.65 231.00±0.97
10 ml/kg; p.o.

Positive control 96.30±0.78** 100.50±0.99** 119.2±1.25*** 183.00±1.06** 146.75±0.88**

Phenylbutazone *
100 mg/kg; p.o.
Test group-1 114.20±1.25* 128.70±1.57* 174.40±0.88** 222.00±0.68* 183.8±0.86*
Ethanol extract
200 mg/kg; p.o.

Test group-2 100.40±1.06* 113.3±0.89** 144.3±0.97*** 200.80±1.65** 163.60±1.05**

Ethanol extract
400 mg/kg; p.o.

Values are expressed as mean ± S.E.M, SEM=Standard error of Mean; (n=6) ; *P<0.05; **
P<0.01; ***
P<0.001 vs.

control; p. o., per oral

Table 4. Effects of Vallaris solanacea ethanolic extract on inhibition of castor oil diarrhoea
Latent Mean
Dose P value (One
Treatment Period number of % Inhibition SD
(mg/kg) way Anova)*
1
(Hrs) stools*

Experimental
0.684 +
control (1% 10 3.6 - 0.19
0.19
Tween80)

Positive control 1.546 +

25 1 72.22 0.57 P < 0.01
(Loperamide) 0.57

Vallaris 1.114 +
500 1.25 65.36 0.41 P < 0.01
solanacea 0.41

Key: *- (http://faculty.vassar.edu/lowry/VassarStats.html) Vallaris solanacea Crude Extract. 40 minutes after

treatment, 0.3mL castor oil was administered orally. Latent period of castor oil induced diarrhea was noted. Number of
stools excreted for the next 4 hours were noted. *1 – Mean number of stools was an average number of stools for 4
hours for each treatment. % inhibition, SD and P value was also calculated with respect to the number of stools. N=5.

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acid (12). The ethanolic extract of V. Gums, Steroids, Alkaloids, and

solanacea produced significant writhing Glycosides, which have potential role in its
inhibition comparable to the standard drug Antinociceptive, Anti-inflammatory and
diclofenac sodium. On the basis of this Antidiarrhoeal activity. This could provide
result it can be concluded that the ethanol a rationale for traditional uses of this plant
extract of V. solanacea possesses analgesic and suggests for further investigation and
activity. isolation of biologically active constituents
Preliminary phytochemical screening responsible for the activity.
showed the presence of various classes of
constituents, such as alkaloids, glycoside, ACKNOWLEDGEMENT
tannins and saponins. Since several tannins The authors are grateful to the authority of
isolated from medicinal plants have been International Centre for Diarrhoeal
discovered for their significant Disease and Research, Bangladesh
antinociceptive and/or anti-inflammatory (ICDDR, B) for providing the
activity (13-15), it is, therefore, possible experimental mice. The authors are also
that the antinociceptive effects observed grateful to the authority of Beximco
with this extract in the present study may Pharmaceuticals Ltd. for providing
be attributing to its flavonoids and tannins Diclofenac sodium and Phenyl butazone.
component.
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