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

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. 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 medicinal 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 work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. 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 powdered material (1 kg) was macerated in 70% aqueousmethanol 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.

V. odorata has the folkoric repute of providing relief in ailments pertaining to gastrointestinal, respiratory and cardiovascular system.The present study was undertaken to validate its folkloric uses in native systems of medicine.

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 powdered material (1 kg) was macerated in 70% aqueousmethanol 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.
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 controlled 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 qualitative phytochemical analysis for the presence of alkaloids, saponins, anthraquinones, coumarins, sterols, terpenes, flavonoids and phenols (Janbaz and Saqib, 2015).

In vitro experiment
Isolated tissues experiments were performed as described 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 segments 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 spontaneous 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 addition 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 following 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 (van Rossum, 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 Ca 2+ -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 Ca 2+ -free Tyrode's solution, having the 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 Ca 2+ concentrations were applied to the tissue bath to obtain control calcium dose -response curves.On achievement of the superimposable 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 Ca 2+ were recorded and compared to the control curves.The dose-response curves of Ca 2+ were recorded 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) MgSO 4 (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 responses were recorded.The carbachol (1 µM)-and high K + (80 mM)-induced sustained contractions were subsequently 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 Ca 2+ 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 preload 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/ vasoconstrictive effects of the test substances were studied by addition in tissue organ baths containing prestabilized 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 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: 3.97-6.65mg/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: 0.34-6.66mg/mL; n=5).

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 Ca 2+ 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 Ca 2+ concentration (Karaki et al., 1997).The intracellular Ca 2+ concen-tration is known to be increased either influx through voltage dependent Ca +2 channels or Ca 2+ released from sarcoplasmic stores (Godfraind et al., 1986).The spontaneous contractions in isolated rabbit jejunum preparations are manifestation of alternative depolarization and repolarization, where tissues at height of depolarization, permit fast influx of Ca 2+ 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 Ca 2+ 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 Ca 2+ 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 Ca 2+ channel blockade (van Rossum, 1963).The abovementioned findings were confirmed further as extract treatment in isolated rabbit jejunum preparation caused decreased response to Ca 2+ and rightward shift of the concentration response curves for Ca 2+ in a manner similar to verapamil as standard Ca 2+ channel blocker (Fleckenstein, 1977).The Ca 2+ channel blockers is an established class of therapeutic agents and are known to be effective in hyperactive diseases of the gut (Brunton et al., 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 Ca 2+ channels.The Ca 2+ 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 phenylephrine (1 µM)-and K + (80 mM)-induced contractions 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 Ca 2+ 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 Ca 2+ channels.The increase in intracellular Ca 2+ due to increased influx of Ca 2+ can cause further Ca 2+ release from sarcoplasmic reticulum (Gurney, 1994;Karaki et al., 1997).Similarly, phenylephrine-induced contraction in vascular smooth muscles is known to be mediated through increase in cytsoplasmic Ca 2+ through two possible means, i.e., Ca 2+ influx via receptor operated channels and subsequent release of Ca 2+ from intracellular stores (Graham et al., 1996).The relaxation of phenylephrine-induced contractions on the part of V. odorata at elevated tissue bath concentrations can be viewed on focusing the point that V. odorata like other Ca 2+ channel blockers can only block Ca 2+ influx through voltage dependent Ca 2+ channels and do nothing with Ca 2+ influx through receptor operated channels and subsequent increase in intracellular Ca 2+ due to release of Ca 2+ from intracellular 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.