Evaluation of antimotility effect of Lantana camara L. var. acuelata constituents on neostigmine induced gastrointestinal transit in mice

Diarrhea is one of the most prevalent human disorders and understandably its remedy occupies a special place in the annals of medicine [1]. Neurohormonal mechanisms, pathogens, malnutrition, chronic diseases and drugs can alter gastrointestinal physiology resulting in changes in either secretion or absorption of fluid by the intestinal epithelium. Altered motility contributes in a general way to this process, as the extent of absorption, by and large, parallels transit time. Prokinetic agents, organophosphate pesticides, nerve gases, surgery, irritation bowel syndrome, collagen vascular disease and diabetes are some of the pathophysiological conditions that may alter intestinal motility and transit time. Antimotility compounds such as diphenoxylate, loperamide, opium alkaloids, anticholinergics etc. have been tried against diarrheal disorders but often with side effects after prolonged use [2].

Acetylcholine, the vagal neurotransmitter, enhances and atropine, a known anticholinergic agent decreases intestinal motility and secretion. Although various derivatives and congeners of atropine (such as propantheline, isopropamide and glycopyrrolate) have been advocated in patients in peptic ulcer or with non-specific diarrhea, the prolonged use of such agents is limited by other manifestations of parasympathetic inhibition such as dry mouth and urinary retention [3]. There is, thus, a need for identifying new compounds and evaluating their antimotility activity and developing these as selective inhibitors that decrease gastric secretion and intestinal motility at doses that have minimal anti-cholinergic effects at other sites and are completely free from other adverse effects [4].

Lantana camara L. (Verbenaceae) is one of the most prevalent and noxious weeds causing hepatotoxicity in grazing animals [5, 6]. Lantana poisoning causes obstructive jaundice and within a few hours of browsing upon its foliage, animals go off-feed and become severely constipated within 48 h [7]. On the contrary, Lantana plant has been reported to possess a number of medicinal properties [8, 9]. Some metabolites isolated from their leaves possess antitumor activity [10], antithrombin activity [11], anti-inflammatory, antinociceptive and antipyretic activity [12].

Present investigations were planned to study the effect of Lantana camara leaf powder, Lantana camara methanolic extract (LCME) and lantadene A administration on mice intestinal motility using neostigmine as a promotility agent. Antidiarrheal effect of LCME was studied using castor oil induced diarrhea in mice.

The percent intestinal transit was increased significantly with neostigmine, but was decreased significantly by all concentrations of LCME and lantadene A. The intraperitoneal administration of the LCME (125, 250 & 500 mg/kg) alone decreased the percent intestinal transit significantly. However, with a LCME dose of 1000 mg/kg, intestinal transit was nearly abolished. Leaf powder (1% of feed) feeding for 10 days reduced the % intestinal transit by 34.78. Inhibition in % intestinal transit by 85 and 170 mg/kg lantadene A was 39.47 and 27.34 respectively. The prokinetic effect of neostigmine was opposed by both the doses (1.0 g/kg and 500 mg/kg) of LCME and % intestinal transit was reduced to 11 and 24 respectively (Table 1).

Diarrhea was apparent in all the animals of control group 45 minutes after the administration of castor oil and fecal count was taken for 4 h. A marked reduction in the number of defecations over 4 h was observed with the i.p. administration of all doses of LCME. The animals of group 4 and 5 appeared to be completely constipated, where as those of group 2 and 3 showed a significant reduction in defecations as compared to control group (Table 2).

The pathophysiological mechanisms underlying the loss of intestinal fluid in diarrhea have been the subject of much debate for decades [17]. Diarrhea may be caused by an increase in osmotic load within the intestine, excessive secretion of electrolytes and water into the intestinal lumen, exudation of protein and fluid from the mucosa, infection and inflammation; and altered intestinal motility, resulting in rapid transit [18]. In most instances, multiple processes are simultaneously affected involving several factors, a particular factor becoming a dominant player in a given environment, however, motility and/or secretory disturbances usually remain a common denominator in most cases [2]. The mucosal lining of the gastrointestinal tract is provided with an extensive nerve supply from the enteric nervous system [19]. Neurotransmitters such as acetylcholine and noradrenaline and neurotransmitter candidates such as ATP, CGRP, CCK-8, ENK, GAL, GABA, serotonin, NO, somatostatin, SP, VIP etc have been implicated to different extents in normal and pathophysiological situations. Based on the knowledge gained about the divergent factors controlling the processes of secretion of electrolytes and motility, many interventional strategies have been adopted by researchers and numerous antidiarrheal compounds have been developed but not many compounds are without side effects and therefore there has always been a need for finding new ones.

Acetylcholine is the endogenous neurotransmitter at cholinergic synapses in the central and peripheral nervous system. The stimulation of vagal input to the gastrointestinal tract increases tone, amplitude of contraction and secretory activity of the stomach and intestine. Since such responses are inconsistently seen with administered acetylcholine, possibly because of poor perfusion and rapid hydrolysis by plasma butryl cholinesterase, use of neostigmine was made in the present investigation. Neostigmine is an inhibitor of acetylcholinesterase and increases the amount of acetylcholine at the synapse [3] and thus exerts a pro-kinetic effect. The results show that the Lantana camara leaf powder and LCME significantly reduced the % intestinal transit in a dose dependent manner. Lantadene A also produced a statistically significant reduction in % intestinal transit.

The induction of diarrhea with castor oil results from the action of ricinoleic acid formed from hydrolysis of its triglyceride in the oil [20, 21]. The released ricinoleic acid produces changes in the transport of water and electrolytes resulting in a hypersecretory response and speeds intestinal transit [3]. The involvement of nitric oxide from neurons in the diarrhea induced by the castor oil has also been proposed [22]. Castor oil increases the induction of prostaglandins [23], causes changes in the permeability and mucosal injuries and stimulates PAF [24] biosynthesis which may result in inflammation of intestinal mucosa. The preventive administration of LCME was associated with significant protection against diarrhea induced by castor oil in mice. Lantana camara might possess some compounds with antisecretory properties which may account for its efficacy against diarrhea induced by castor oil in mice.

Lantana camara has been reported to be toxic to grazing animals such as cow, buffaloes, sheep and goats [7, 25] and laboratory animals such as guinea pigs [8] and female rats [26]. In spite of its widespread toxicity in the Lantana affected animals, various parts of this plant have been used in the traditional medicines for treating cuts, ulcers, swelling, eczema, inflammation, fever etc [8]. Gastrointestinal stasis, ruminal stasis, constipation, discolorization of conjunctiva, photosensitization, decreased bile flow and urinary retention in the Lantana poisoned animals has been noticed [27‐29]. These symptoms resembled those due to atropine toxicity i.e., anticholinergic excess [30, 31].

Anti-dysenteric and anti-diarrheal properties of medicinal plants have been suggested to be due to tannins, alkaloids, saponins, flavonoids, sterols and triterpenes and reducing sugars [32]. The sesquiterpene lactones have been reported to have the ability to relax smooth muscles and thereby relieve gastrointestinal disorders [33]. The phytochemical analysis of the Lantana camara leaf extract has earlier been shown to contain flavonoids [34], terpenes [35] and their derivatives and pentacyclic triterpenoids [36]. These constituents may mediate the anti-diarrheal action of the Lantana camara extract. A verbascoside [37] isolated from Lantana camara has been shown to be an inhibitor of protein kinase C. The role of this enzyme has been demonstrated in signal transduction, inflammation and smooth muscle contraction [38] and an inhibition of its activity by a constituent of Lantana camara shall result in decrease in motility. Although the anti-diarrheal properties of the reported active terpenoids are well established, aspects of their mechanism of action remain poorly understood. Terpenes, flavonoids and terpenoid derivatives may act by inhibiting release of autocoids and prostaglandins [39, 4] thereby inhibit the motility and secretion induced by neostigmine. Intestinal motility alterations in Lantana camara foliage poisoned sheep has been described by Pass et al. [40] but no mechanism has been suggested.

The remarkable antimotility effect of Lantana camara methanolic extract against neostigmine as promotility agent points towards an anticholinergic effects due to Lantana camara constituents and attest to its wide range of utility in secretory and functional diarrheas and other gastrointestinal disorders in the folklore. This effect was further confirmed by significant inhibition of castor oil induced diarrhea in mice by various doses of LCME. Whatever may be the mechanism of action, LCME may be useful in a wide range of diarrheal states due to disorders of intestinal transit and secretion. Further studies with purified constituents are needed to completely understand the mechanism of anti-diarrheal action of Lantana camara.