Keeping in view the medicinal use of
Viola betonicifolia in gut disorders, such as indigestion and constipation, its crude extract (VBME) was tested in mice, where it propelled charcoal meal through the small intestine and increased the production of wet feces, hence showing prokinetic and laxative activities, similar to the effect of carbachol, a standard cholinergic agonist and accelerator of intestinal contents (Brown and Taylor, 2006). These gut stimulatory actions of the extract were found partially sensitive to atropine, a muscarinic receptor blocker[
14], indicating the presence of some ACh-like component(s) in addition to other gut stimulant constituent(s). ACh is a neurotransmitter of the parasympathetic nervous system and is known to cause gastrointestinal stimulation through the activation of muscarinic receptors[
15], hence, the presence of ACh-like constituents partly explains its medicinal use in constipation and as digestive aid. To further study the possible mode of the observed prokinetic and laxative properties of the extract, isolated ileum preparations of mouse was used. But VBME did not show any stimulant effect on the baseline contraction (data not shown) of this tiny preparation. Hence, whentested in isolated rabbit jejunum and guinea-pig ileum, considered suitable preparations to study effects on gut motility[
16], VBME showed a dose-dependent gut stimulant effect, partially sensitive to atropine in both preparations, whereas, the comparable stimulatory effect of acetylcholine was completely inhibited in the atropine pretreated preparations. Partial atropine-sensitive gut stimulatory effect of VBME was also supported by acetylcholinesterase inhibition assay, thus, showing a common mechanism of muscarinic receptor activation along with some additional spasmodic component(s). However, the efficacy of gut stimulant effect was high in guinea-pig ileum than that found in rabbit jejunum. To further study the nature of the unknown additional spasmodic components, other than cholinergic, the stimulant effect was restudied in the presence of pyrilamine, a histaminic type-1 (H
1) receptor blocker[
17], hexamethonium, a ganglion blocker[
18] or SB203186, a serotonergic (5-HT) receptor antagonist[
19]. But VBME showed complete resistance to these antagonists, clearly suggesting some additional mechanism(s), independent of histamine, nicotine or 5-Hydroxytryptamine (5-HT, serotonin) receptors activation. Other mechanisms known for their gut stimulant property, which have not been ruled out in this study include certain prostaglandins[
20], platelet activating factor[
21], nitric-oxide-donating or releasing compounds[
22], dopaminergic antagonists[
23], cholecystokinin[
23] and tachykinins[
24].
Recently we have reported the anthelmintic and nematicidal properties of the VBME and its subsequent solvent fractions[
4,
25]. It is very interesting to note that VBME possesses combination of anthelmintic and laxative activities which has the merit because the laxative and prokinetic properties of the tested extract will be helpful in the expulsion of worms or nematodes from gut. Regarding the preliminary phytochemical study, the VBME is known to be a rich source of alkaloids, flavonoids, phenolic compounds and saponins[
5], while in case of quantitative phytochemical profile of the plant, it is clear from results of this study that VBME is a rich source of alkaloids and saponins. We have recently reported the quantification of flavonoids and phenolic contents in various solvent fractions of this plant. The total polyphenols were of higher in quantity in methanolic extract as compared to water fraction[
4]. The presence of alkaloids and saponins as the plant constituents, which are known to possess gut stimulatory properties[
26,
27], may explain the gut stimulant actions of the plant extract, though further studies are required to know the specific chemical(s) responsible for the tested biological activities.
The screening of methanolic extract instead of aqueous fraction for this study was due to the presence of polyphenols in the methanolic extract which are mostly responsible for such type of activities. More useful explanation for not using the aqueous extract can be ascribed to the presence of enzyme polyphenol oxidase, which degrades polyphenols in water extracts, whereas in methanol they are inactive. Moreover, water is a better medium for the occurrence of the micro-organisms as compared to methanol[
28]. Our research group is working on isolation of active constituents from this plant through column chromatography and we intend to make use of HPLC at later stage and hopefully our next publication will contain HPLC data for the isolation of active principles from this plant.