Pharmacological basis for the medicinal use of Viola odorata in diarrhea, bronchial asthma and hypertension

Abstract

Viola odorata is traditionally used in the management of gastrointestinal,
respiratory and vascular disorders. The present study was undertaken to validate
its folkloric uses. The application of V. odorata to spontaneous contractions
in isolated rabbit jejunum preparation exerted relaxant effect through
decrease in magnitude and frequency of contractions. Moreover, it also
caused relaxation of K+ (80 mM)-induced contractions and shifted the Ca2+
concentration response curves toward right in isolated jejunum similar to
verapamil (standard Ca2+ channel blocker), confirming Ca2+ channel blocking
activity. V. odorata also caused relaxation of carbachol (1 μM)- and K+ (80 mM)
-induced contractions in isolated rabbit tracheal preparations comparable to
verapamil, reflecting that observed relaxant effect may be the outcome of antimuscarinic
and/or Ca2+ channel blocking activities. It also exerted relaxant
effect on phenylephrine (1 μM)- and K+ (80 mM)-induced contractions in
isolated rabbit aortic preparations thus providing rationale for its folkloric
uses to treat diarrhea, asthma and hypertension.

Full text

Introduction
Viola odorata, Linn. (Violaceae), locally called banafsha,
is widely distributed throughout the world including
Pakistan (Said, 1972; Baquar, 1989). The plant has
traditionally been used to manage bronchial asthma,
cough, bronchitis (Nadkarni, 1976; Pullaiah, 2006),
anxiety (Keville, 1991) and hypertension (Duke et al.,
2002). It is also used as expectorant and laxative
(Ahmad et al., 2009).
The plant is reported to possess antioxidant (Ebrahim-
zadeh et al., 2010), diuretic, laxative (Vishal et al., 2009),
analgesic (Barkatullah et al., 2012), anti-inflammatory
(Koochek et al., 2003), antipyretic (Khattak et al., 1985),
sedative (Alireza and Ali, 2013), hypotensive and lipid
lowering effect (Siddiqi, 2012). Moreover, it has been
reported to possess antibacterial (Ramezani et al., 2012;
Khan et al., 2011), anthelmintic activity (Colgrave et al.,
2008), anti-fungal (Pawar and Thaker, 2006) and
mosquito repellant activity (Amer and Mehlhorn, 2006).
The qualitative investigation revealed presence of
violanthin, flavonoids, glycosides (Khare, 2007), stig-
masterol (Mittal, 2013), violaquercetin, saponins, alka-
loids, vitamins (Kathi, 1991), phenols (Ebrahimzadeh,
2010), glucosides, violin (Prajapati, 2004), violanthin
and violanin (Rastogi, 1979), vanillic acid (Evans, 1996),
benzofuranone (Akhbari et al., 2012), glucopyranosides,
(Karioti et al., 2011), shikimic acid (Anca et al., 2009),
cycloviolacin, (Rosengren et al., 2003; Craik et al., 1999),
dimethyldodecane (Cu et al., 1992), dimethylheptane
(Beierbeck and Saunders, 1980), glucopyranoside
(Henrick and Jefferies, 1964), violacin (Ireland et al.,
2006) and peptide (Svangård et al., 2003).
V. odorata has the folkoric repute of providing relief in
ailments pertaining to gastrointestinal, respiratory and
cardiovascular system. The present study was under-
taken to validate its folkloric uses in native systems of
medicine.
A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2015; 10: 836-843
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Abstract
Viola odorata is traditionally used in the management of gastrointestinal,
respiratory and vascular disorders. The present study was undertaken to vali-
date its folkloric uses. The application of V. odorata to spontaneous contrac-
tions in isolated rabbit jejunum preparation exerted relaxant effect through
decrease in magnitude and frequency of contractions. Moreover, it also
caused relaxation of K+ (80 mM)-induced contractions and shifted the Ca2+
concentration response curves toward right in isolated jejunum similar to
verapamil (standard Ca2+ channel blocker), confirming Ca2+ channel blocking
activity. V. odorata also caused relaxation of carbachol (1 µM)- and K+ (80 mM)
-induced contractions in isolated rabbit tracheal preparations comparable to
verapamil, reflecting that observed relaxant effect may be the outcome of anti-
muscarinic and/or Ca2+ channel blocking activities. It also exerted relaxant
effect on phenylephrine (1 µM)- and K+ (80 mM)-induced contractions in
isolated rabbit aortic preparations thus providing rationale for its folkloric
uses to treat diarrhea, asthma and hypertension.
Article Info
Received: 26 June 2015
Accepted: 15 July 2015
Available Online: 19 October 2015
DOI: 10.3329/bjp.v10i4.23889
Cite this article:
Janbaz KH, Khan WU, Saqib F, Khalid
M. Pharmacological basis for the me-
dicinal use of Viola odorata in diarrhea,
asthma and hypertension. Bangladesh
J Pharmacol. 2015; 10: 836-43.
Pharmacological basis for the medicinal use of
Viola odorata
in diarrhea,
bronchial asthma and hypertension
Khalid Hussain Janbaz, Waseem-Ullah Khan, Fatima Saqib and Mamoona Khalid
Department of Pharmacy, Faculty of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan.
This article was downloaded by you on: Oct 19, 2015

Materials and Methods
Plant material and preparation of extract
The aerial parts of V. odorata were collected in May,
2012 from the botanical garden of Pakistan Institute of
Forestry, University of Peshawar and were identified
by the kind cooperation of an expert taxonomist (Prof.
Altaf Ahmad Dasti), at the Institute of Pure and
Applied Biology, Bahauddin Zakariya University,
Multan.
The plant material was shade dried and rendered free
of adulterants by manual picking and was grinded to
coarse powder with special herbal grinder. The pow-
dered material (1 kg) was macerated in 70% aqueous-
methanol for 2 weeks with occasional shaking. The
soaked material was passed through a muslin cloth to
remove the vegetative debris and the fluid obtained
was subsequently filtered through a Whatman No. 1
filter paper. The filtrate was evaporated on a rotary
evaporator (Rotavapor, BUCHI labrotechnik AG, Model
9230, Switzerland) at 40ºC under reduced pressure to
dark brown thick paste like semisolid material. The
percentage yield of crude V. odorata was calculated to
be 7.3% approximately. The extract obtained was stored
in amber colored air tight jars at -40ºC.
Chemicals
Acetylcholine chloride, atropine sulfate, carbachol, his-
tamine, potassium chloride, verapamil hydrochloride
and phenylephrine, magnesium chloride, ethylenetetra-
acetic acid (EDTA) were purchased form Sigma
Chemicals Co. (USA). Calcium chloride, glucose,
magnesium sulfate, potassium dihydrogen phosphate,
sodium bicarbonate, sodium dihydrogen phosphate,
and methanol were obtained from Merck, Darmstadt,
Germany. Ammonium hydroxide, sodium chloride,
and sodium hydroxide were purchased from BDH
Laboratory supplies, Poole, England.
The chemicals used in these experiments were of
highest purity and analytical research grade. Stock
solutions and subsequent dilutions were made fresh in
distilled water on the day of experiment. The drugs
were solubilized in vehicles which were without any
effect on tissue contractility in control and experiments.
Animals and housing conditions
Animals (♂/♀) used in this study were local strain
rabbits (1.0-1.8 kg). These were housed under control-
led environmental condition (23-25ºC) at the animal
house of Faculty of Pharmacy, Bahauddin Zakariya
University, Multan. Rabbits were provided with refresh
green fodder and tap water ad libitum. The animals
were deprived of food 24 hours prior to the
experiments but were given free access to water.
Rabbits were sacrificed following a blow on the back of
head to use for in vitro studies.
Preliminary phytochemical analysis
The crude extract of V. odorata was subjected to qualita-
tive phytochemical analysis for the presence of alka-
loids, saponins, anthraquinones, coumarins, sterols,
terpenes, flavonoids and phenols (Janbaz and Saqib,
2015).
In vitro experiment
Isolated tissues experiments were performed as descri-
bed previously (Janbaz et al., 2013).
Isolated rabbit jejunum preparations
Plant extract was tested on isolated rabbit jejunum
preparations for possible presence of spasmogenic and/
or spasmolytic activity. Isolated rabbit jejunum seg-
ments of approximately 2 cm in length were suspended
in isolated tissue baths containing Tyrode’s solution, at
37ºC, aerated with carbogen (95%O2 and 5%CO2). The
composition of the Tyrode’s solution (mM) was: KCl
(2.68), NaC1 (136.9), MgC12 (1.05), NaHCO3 (11.9),
NaH2PO4 (0.42), CaC12 (1.8) and glucose (5.55). A
preload of 1 g was applied and intestinal responses
were recorded isotonically through Power Lab data
acquisition system (AD Instruments, Sydney, Australia)
attached to a computer installed with lab chart software
(version 7.1). The tissues were allowed to equilibrate for
at least 30 min prior to the addition of any drug.
Isolated rabbit jejunum preparations exhibit sponta-
neous rhythmic contractions and allowed testing of the
antispasmodic (relaxant) effect without application of
an agonist (Janbaz et al., 2015a; Saqib et al., 2012). The
observed response of the test material was quantified
by the application of doses in a cumulative fashion. The
relaxant effects on the part of test substance were taken
as the percent change in spontaneous contractions of
the preparation recorded immediately before the addi-
tion of test substances.
The possible mechanism of the relaxant activity of the
test material was investigated through the relaxation of
the observed sustained spasmodic contractions follow-
ing exposure to high concentration of K+ (80 mM) (Farre
et al, 1991). The test material was applied in a
cumulative manner to the sustained contractions to
achieve concentration dependent inhibitory responses
(vanRossum, 1963). The observed relaxant effect of the
test material on K+ (80 mM)-induced contraction was
expressed as percent of the control contractile response.
Calcium channel blocking effect of the test substance
was confirmed by the method described previously
(Janbaz et al., 2015b). The isolated rabbit jejunum
preparation was allowed to stabilize in normal Tyrode’s
solution, which was subsequently replaced for 30 min
with Ca2+-free Tyrode’s solution to which EDTA (0.1
mM) was added in order to remove calcium from the
tissue. This bath solution was further replaced with K+-
rich and Ca2+-free Tyrode’s solution, having the
Bangladesh J Pharmacol 2015; 10: 836-843 837

following composition (mM): KC1 (50), NaCl (91.04),
MgCl2 (1.05), NaHCO3 (11.9), NaH2PO4 (0.42), glucose
(5.55) and EDTA (0.1). Subsequent to an incubation
period of 30 min, cumulative Ca2+ concentrations were
applied to the tissue bath to obtain control calcium dose
-response curves. On achievement of the super-
imposable control calcium dose-response curves
(usually after two cycles), the tissues were then washed
and allowed to equilibrated with the plant extract for 1
hour and then the concentration response curves of
Ca2+ were recorded and compared to the control
curves. The dose-response curves of Ca2+ were recor-
ded in the presence of different concentrations of the
plant extract in tissue bath (Janbaz et al., 2015b)
Isolated rabbit tracheal preparations
Rabbit tracheas were dissected out and kept in Kreb's
solution having the following composition (mM): NaCl
(118.2), NaHCO3 (25), CaCl2 (2.5), KCl (4.7), KH2PO4
(1.3) MgSO4 (1.2) and glucose (11.7). The trachea was
cleaned free from the surrounding fatty tissues and
rings of 2-3 mm width containing 2-3 cartilages were
prepared. Each ring was opened by a longitudinal
incision on the ventral side opposite to the smooth
muscles layer to form a strip with smooth muscles layer
in middle and cartilages on both sides. These tracheal
preparations were mounted in 20 mL organ bath
containing Krebs solution being maintained at 37ºC and
aerated with carbogen. A preload tension of 1 g was
applied and tissue preparations were allowed to be
equilibrated for 1 hour prior to any challenge by the
drug. Tissue preparations were stabilized by repeated
applications of carbachol (1 µM) until constant respon-
ses were recorded. The carbachol (1 µM)- and high K+
(80 mM)-induced sustained contractions were subse-
quently used for testing of different doses of the test
material in a cumulative fashions. The isometric
responses were recorded through Power Lab data
acquisition system (AD Instruments, Sydney, Australia)
attached to a computer installed with lab chart software
(Version 7.1). The standard drug with Ca2+ channel
blocking effect (verapamil) was tested on high K+ (80
mM)- and carbachol-induced spastic contractions in
order to confirm the possible mechanism of action
(Janbaz et al., 2014a)
Isolated rabbit aorta preparation
To see the effect of plant extract on systemic vascular
resistance, rabbits of either sex were sacrificed by a
blow on the back of head and descending thoracic aorta
was dissected out and kept in the normal Kreb’s
solution having composition as described earlier. It was
then cut into rings of about 2-3 mm width and each ring
was mounted in a tissue bath containing Kreb's
solution. Temperature was maintained at 37ºC and
tissue was continuously aerated with carbogel. A pre-
load of 2 g was applied to each preparation and allowd
to equilibrate for a period of 1 hour. After equilibrium,
tissue was stabilized by repeated exposure to K+ (80
mM) or phenylephrine (1 µM) depending upon the
protocol of the experiment. The vasorelaxant/ vaso-
constrictive effects of the test substances were studied
by addition in tissue organ baths containing pre-
stabilized tissue in a cumulative manner (Janbaz et al.,
2014b). Changes in isometric tension of aortic rings
were obtained via force-displacement transducer
(Model FORT100, WPI, USA) coupled to Power Lab
data acquisition system (AD Instruments, Sydney,
Australia) and computer running Lab Chart software
(version 7.1).
Statistical analysis
The data was expressed as mean ± standard error of
mean (S.E.M., n = 5) and median effective concentration
(EC50) with 95% confidence interval (CI). The statistics
applied was Student's t-test. The logarithmic, dose/
concentration-response curves of different treatments
were analyzed by non-linear regression using computer
software (Graph Pad Software, San Diego, CA, USA).
Results
Preliminary phytochemical analysis of V. odorata
revealed the presence of alkaloids, flavonoids,
glycosides, steroids, terpenes, saponins and tannins
among the methanol soluble extractable constituents of
V. odorata.
Effect on isolated rabbit jejunum preparations
The crude extract of V. odorata on application to the
spontaneously contracting isolated rabbit jejunum
preparations exerted relaxant effect in tissue bath
concentration dependent manner with EC5o value of
0.05 mg/mL (95% CI: 0.03-0.08 mg/mL; n=5). More-
over, it caused complete relaxation of K+ (80 mM)-
induced contraction with EC50 value of 0.41 mg/mL
(95% CI: 0.14-1.15 mg/mL; n=5). Verapamil (standard
Ca2+ channel blockers), relaxed the spontaneous and K+
(80 mM)-induced contractions with respective EC50
values of 0.49 µM (95% CI: 0.35- 0.73 µM; n=5) and 0.33
µM (95% CI: 0.16-0.66 µM; n=5) (Figure 1). Furthermore,
extract of plant also caused rightward shift of
concentration response curves for Ca2+ in a manner
comparable to verapamil in isolated rabbit jejunum
preparations (Figure 2).
Effect on isolated rabbit tracheal preparations
The crude extract of V. odorata exerted relaxant effect
on application to carbachol (1 µM) and K+ (80 mM)-
induced contractions in isolated rabbit tracheal
838 Bangladesh J Pharmacol 2015; 10: 836-843

preparations with respective EC50 values of 0.54 mg/
mL (95% CI: 0.30-0.96 mg/mL; n=5) and 0.68 mg/mL
(95% CI: 0.37-1.24 mg/mL; n=5). Similarly, verapamil
also caused relaxation of carbachol (1 µM) and K+ (80
mM)-induced contractions with respective EC50 values
of 2.15 µM (95% CI: 0.03-4.26 µM; n=5) and 0.32 µM
(95% CI: 0.02-0.62 µM; n=5) (Figure 3).
Effect on isolated rabbit aortic preparations
The V. odorata crude extract on application to isolated
rabbit aortic preparation, exerted relaxant effect on
phenylephrine (1 µM)-induced contractions in isolated
rabbit aortic preparations up to the extent of 5 mg/mL
tissue bath concentrations with EC50 values of 5.37 mg/
mL (95% CI: 3.97-6.65 mg/mL; n=5), whereas K+ (80
mM)-induced contractions in isolated rabbit aorta were
relaxed at lower tissue bath concentrations with EC50
values of 1.5 mg/mL (95% CI: 0.34-6.66 mg/mL; n=5).
The standard Ca2+ channel blocker (verapamil), relaxed
the phenylephrine (1 µM) and K+ (80 mM)-induced
contractions with respective EC50 of 1.08 mg/mL (95%
CI: 0.08-2.52; n=5) and 0.55 mg/mL (95%CI: 0.04-2.10;
n=5) (Figure 4).
Figure 1: Effect of crude extract of V. odorata (Vo. Cr) (A) and verapamil (B) on spontaneous and K+ (80 mM)-induced contractions
in isolated rabbit jejunum preparations (values are expressed as mean ± S.E.M.; n=5)
A B
A B
Figure 2: Effect of crude extract of V. odorata (Vo. Cr) (A) and verapamil (B) on concentration-response curves for Ca2+ in isolat-
ed rabbit jejunum preparations (values are expressed as mean ± S.E.M.; n=5)
0.03 0.3 3
0
25
50
75
100
Spontaneous
K+ (80mM)- induced
[Vo.Cr] mg/ml
-4.5 -3.5 -2.5 -1.5
0
25
50
75
100 Control
Vo.Cr mg/ml
Vo.Cr mg/ml
Log [Ca++] M
% Of Control Max
-4.5 -3.5 -2.5 -1.5
0
25
50
75
100 Control
Verapamil 0. M
Verapamil 0. M
Log [Ca++] M
% Of Control Max
Bangladesh J Pharmacol 2015; 10: 836-843 839
% of Control

Discussion
Preliminary phytochemical analysis of V. odorata
revealed the presence of alkaloids, flavonoids,
glycosides, steroids, terpenes, saponins and tannins
among the constituents of V. odorata.
The V. odorata exerted relaxant effect on spontaneous
contractions in isolated rabbit jejunum preparation, i.e.,
exhibiting antispasmodic activity. Studies reported on
plant materials reflected anti-spasmodic activity may
possibly be mediated through blockade of Ca2+ channels
(Janbaz et al., 2015b). The contractile activities of
smooth muscle preparations, i.e., isolated rabbit
jejunum preparations is mediated through increase/
decrease of cytoplasmic free Ca2+ concentration
(Karakai et al., 1997). The intracellular Ca2+ concen-
tration is known to be increased either influx through
voltage dependent Ca+2 channels or Ca2+ released from
sarcoplasmic stores (Godfraind et al., 1986). The
spontaneous contractions in isolated rabbit jejunum
preparations are manifestation of alternative depolari-
zation and repolarization, where tissues at height of
depolarization, permit fast influx of Ca2+ through
voltage dependent Ca+2 channels (Brading, 1981). Thus,
the spasmolytic effect on the part of V. odorata may
possibly be mediated through either blockade of
voltage dependent Ca+2 channels or suppression of Ca2+
release from sarcoplasmic reticulum. The isolated
smooth muscle preparations on exposure to K+ (80
mM) exhibit sustained contractile activity due to rapid
influx of extracellular Ca2+ through opened voltage
dependent Ca+2 channels (Bolton, 1979; Godfrained et
al., 1986) and relaxant effect of extract on K+ (80 mM)-
induced contractions may possibly mediated through
Ca2+ channel blockade (van Rossum, 1963). The above-
mentioned findings were confirmed further as extract
treatment in isolated rabbit jejunum preparation caused
decreased response to Ca2+ and rightward shift of the
A
0.01 0.1 1
0
20
40
60
80
100
CCh (1µM)-induced
K+(80mM)-induced
5
[Vo.Cr] mg/ml
% Of Control
0.0001 0.001 0.01 0.1
0
20
40
60
80
100
CCh (1µM)-induced
K+(80mM)-induced
[Verapamil] 
% Of Control
Figure 3: Effect of crude extract of V. odorata (Vo. Cr) (A) and verapamil (B) on carbachol (1 µM) and K+ (80 mM)-induced contrac-
tions in isolated rabbit tracheal preparations (values are expressed as mean ± S.E.M.; n=5)
Figure 4: Effect of crude extract of V. odorata (Vo. Cr) (A) and verapamil (B) on phenylephrine (PE) (1 µM) and K+ (80 mM)-
induced contractions in isolated rabbit aortic preparations (values are expressed as mean ± S.E.M.; n=5)
0.03 0.3 3
0
25
50
75
100
PE(1  M)- induced
K+ (80mM)- induced
[Vo.Cr] mg/ml
% of control
0.03 0.3 3
0
25
50
75
100
PE(1  M)- induced
K+ (80mM)-induced
[Verapamil] M
% of control
840 Bangladesh J Pharmacol 2015; 10: 836-843
B
A B

concentration response curves for Ca2+ in a manner
similar to verapamil as standard Ca2+ channel blocker
(Fleckenstein, 1977). The Ca2+ channel blockers is an
established class of therapeutic agents and are known
to be effective in hyperactive diseases of the gut
(Brunton, 1996).
The V. odorata caused relaxation of carbachol (1 µM)-
and K+ (80 mM)-induced contractions in isolated rabbit
tracheal preparations in a manner comparable to
verapamil and is possibly mediated through blockade
of Ca2+ channels. The Ca2+ channel blockers are useful
bronchodilator in conditions of increased sensitivity of
the airway (Ahmed, 1992), hence this study provided a
scientific basis to validate traditional uses of V. odorata,
Linn. in the management of respiratory disorders
including asthma, cough and bronchitis.
The V. odorata caused complete relaxation of the phe-
nylephrine (1 µM)- and K+ (80 mM)-induced contrac-
tions in isolated rabbit aorta preparations, however,
phenylephrine-induced contractions were found to be
relaxed at elevated tissue bath concentrations. The
isolated rabbit aorta preparations have been used for
characterization of Ca2+ channel blocking activities
(Janbaz et al., 2014b), which on exposed to K+ (80 mM),
resulted in contraction of smooth muscles via opening
of voltage dependent Ca2+ channels. The increase in
intracellular Ca2+ due to increased influx of Ca2+ can
cause further Ca2+ release from sarcoplasmic reticulum
(Gurney, 1994; Karaki et al., 1997). Similarly, phenyl-
ephrine-induced contraction in vascular smooth mus-
cles is known to be mediated through increase in
cytsoplasmic Ca2+through two possible means, i.e., Ca2+
influx via receptor operated channels and subsequent
release of Ca2+ from intracellular stores (Graham et al.,
1996). The relaxation of phenylephrine-induced contrac-
tions on the part of V. odorata at elevated tissue bath
concentrations can be viewed on focusing the point that
V. odorata like other Ca2 + channel blockers can only
block Ca2+ influx through voltage dependent Ca2+
channels and do nothing with Ca2+ influx through
receptor operated channels and subsequent increase in
intracellular Ca2+ due to release of Ca2+ from intra-
cellular stores (Graham et al., 1996). The observed
relaxant effect of V. odorata on aorta may provide a
scientific basis for the folkloric use of V. odorata in the
management of hypertension.
The V. odorata exhibited Ca2+ channel blocking activity
in isolated rabbit tissue preparations (i.e., jejunum,
trachea and aorta) which can be attributed to the
presence of alkaloids (Khalid et al., 2004; Gilani et al.,
2005, ), flavonoids (Revuelta et al., 1997; Di-Carlo, 1993)
and tannins (Azhar et al., 1997) among the constituents
of V. odorata detected in the preliminary phytochemical
screening.
Ethical Issue
All the experiments performed were complied with the
rulings of Institute of Laboratory Animal Resources,
Commission on Life Sciences (NRC, 1996), approved by
the Ethical Committee of Bahauddin Zakariya
University, Multan.
Conflict of Interest
The author(s) declare that there is no conflict of interests
regarding the publication of this article.
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Author Info
Khalid Hussain Janbaz (Principal contact)
e-mail: khjanbaz@hotmail.com; Phone: +923067388951, +9261921055; Fax: +92619210129
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