Evaluation of the antidiarrhoeal activity of the hydroethanolic leafextract of Pupalia lappacea Linn. Juss. (Amaranthaceae)

A.J. Akindele n, O.A. Salako, U.V. OhonbamuDepartment of Pharmacology, Therapeutics & Toxicology (PTT), Faculty of Basic Medical Sciences, College of Medicine, University of Lagos,P.M.B. 12003 Lagos, Nigeria

a r t i c l e i n f o

Article history:Received 7 October 2013Received in revised form21 November 2013Accepted 7 December 2013Available online 14 December 2013

Keywords:Pupalia lappaceaAntidiarrhoeal activityCastor oilLoperamideIntestinal transit

a b s t r a c t

Ethnopharmacological Relevance: Pupalia lappacea is a medicinal plant found in savannah and woodlandlocalities and forest path sides from Senegal to Southern Nigeria. It has been used in the management ofdiarrhoea in Nigerian traditional medicine. This study was designed to evaluate the antidiarrhoealactivity of the hydroethanolic leaf extract of Pupalia lappacea (PL).Materials and methods: The antidiarrhoeal activity of PL was evaluated using the normal and castor oil-induced intestinal transit, castor oil-induced diarrhoea, gastric emptying and intestinal fluid accumula-tion tests in rodents.Results: PL (100–400 mg/kg, p.o.) produced a significant dose-dependent decrease in normal and castoroil-induced intestinal transit compared with the control group (distilled water 10 ml/kg, p.o.). This effectwas significantly (Po0.05) inhibited by pilocarpine (1 mg/kg, s.c.) but not by yohimbine (10 mg/kg, s.c.),prazosin (1 mg/kg, s.c.), or propranolol (1 mg/kg, i.p.). The extract produced a dose-dependent andsignificant increase in the onset of diarrhoea. PL (100–400 mg/kg) also reduced the diarrhoea score,number and weight of wet stools. The in-vivo antidiarrhoeal index (ADIin vivo) of 56.95% produced by theextract at the dose of 400 mg/kg was lower compared to that produced by loperamide 5 mg/kg (77.75%).However, PL (400 mg/kg) significantly increased gastric emptying in rats but significantly reduced thevolume of intestinal content in the intestinal fluid accumulation test. Phytochemical analysis of theextract revealed the presence of alkaloids, saponins, and fixed oils and fats. The acute toxicity studiesrevealed that the extract is relatively safe when given orally; no death was recorded at a dose of 10 g/kg.Conclusion: Results showed that the hydroethanolic leaf extract of Pupalia lappacea possesses antidiar-rhoeal activity possibly mediated by antimuscarinic receptor activity.

& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Diarrhoea is a symptom marked by rapid and frequent passageof semi-solid or liquid faecal material through the gastrointestinaltract. Various types of diarrhoea exist (based on their clinicalmanifestations), but it is broadly classified into acute and chronicfor diagnostic and therapeutic purposes. Acute diarrhoea is theprevalent form of the disease which has a major impact on themorbidity and mortality worldwide in all age groups, particularlyin infants and children under the age of three (Hirchhorn, 1980;Muriithi, 1996; Farthings, 2002). In less developed parts of theworld (such as Latin America, India and Africa), children mayexperience between 3 and 10 episodes of diarrhoea yearly(Farthings, 2002). In 2009, diarrhoea was estimated to have caused

1.1 million deaths in people aged 5 and 1.5 million deaths inchildren under the age of 5 (World Health Organization, 2009). Interms of etiology, diarrhoea can be classified as infectious andnon-infectious (de Hostos et al., 2011). Infectious diarrhoea iscaused by a virus, parasite or bacterium while non-infectiousdiarrhoea can be caused by toxins, chronic diseases or antibiotics(NHDHHS, 2009). Pharmacological models of non-infectious diar-rhoea were used in this study.

Pupalia lappacea Linn. Juss. (Amaranthaceae) is a plant found insavannah and woodland localities and forest path sides from Senegalto Southern Nigeria, but less common in the western part of theregion. Distribution is widespread elsewhere in tropical Africa and inAsia. Pupalia lappacea also commonly known as ram's bur and locallyas “Kaimin kadangari” (Hausa, Northern Nigeria), “Agbiriba” (Igbo,South-east Nigeria) or “Emo agbo” (Yoruba, South-west Nigeria) hasbeen used in traditional medicine for the treatment of cough, syphilis,skin diseases and diarrhoea (Odugbemi, 2008). The leaves mixed withpalm-oil, are used in Ghana to treat boils (Agyare et al., 2009). Theleaves are also used in topical applications to cuts, or used as enema or

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0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jep.2013.12.013

n Corresponding author. Tel.: þ234 8062359726.E-mail addresses: ajakindele@cmul.edu.ng,

jakindele@unilag.edu.ng (A.J. Akindele).

Journal of Ethnopharmacology 151 (2014) 984–989

febrifuge (Agyare et al., 2009). In Ivory Coast, a decoction is taken indraught and applied in frictions for oedema of the legs and is used invarious remedies for dysentery and diarrhoea. It is used as an anti-emetic in the south-western region of Nigeria and the Republic ofBenin (Adjanohoun and Akjakidje, 1989).

Pupalia lappacea preparations have been reported to haveantioxidant activity (Aladedunye and Okorie, 2008), anti-canceractivity (Ravi et al., 2012), antinociceptive and antipyretic activities(Neeharika et al., 2013). However, no scientific report of theantidiarrhoeal activity of Pupalia lappacea is available. The aim ofthis study is to evaluate the antidiarrhoeal effects of the hydro-ethanolic leaf extract of Pupalia lappacea (PL) in search for safe,effective and cheap remedies for diarrhoea management.

2. Materials and methods

2.1. Reagents and drugs

Castor oil (Finest Cold Drawn Commercial Castor oil, UK),loperamide hydrochloride (Imodiums, Janssen PharmaceuticalN.V., Belgium), methylcellulose (Koch-light Laboratories Ltd.,England), pilocarpine hydrochloride, propranolol hydrochloride,prazosin hydrochloride and yohimbine (Sigma Chemical Company,St. Louis, USA) were used in this study.

2.2. Plant material

The fresh leaves of Pupalia lappacea were collected from thefarm of a traditional herbal practitioner in Ogun State, Nigeria, andthe plant was identified and authenticated at the herbarium of theDepartment of Botany, University of Lagos, Lagos, Nigeria, by Mr.T.K. Odewo. A voucher specimen with reference number LUH 5092was deposited in the herbarium of the department.

2.3. Experimental animals

Adult albino Wistar mice (15–30 g) and rats (120–220 g) (8weeks old) of either sex obtained from the Laboratory AnimalCentre of the College of Medicine, University of Lagos, Lagos,Nigeria, were used for the experiments. Animals were maintainedunder standard environmental conditions (12 h light and 12 h darkcycle; 23–25 1C) and had free access to standard pelleted feed(RAAF Animal Feeds Ltd., Akute, Ogun State, Nigeria) and water ad-libitum in accordance with the Guidelines for Care and Use ofLaboratory Animals in Biomedical Research (National Academy ofSciences, 2011).

2.4. Preparation of extract

The fresh leaves of the plant were collected and air-dried for aperiod of three weeks. The dried leaves were thereafter groundinto powder with an electric blender. The powdered leaves werethen macerated in hydroethanol (1:1; 50 g/L) for 48 h. Exhaustiveextraction was done.

Filtration was carried out after 48 h and the combined filtratewas evaporated to dryness under reduced pressure at 40 1C using aHeidolph rotavapor. The dried hydroethanolic extract had a stickyconsistency which was readily soluble in water. It had a darkbrown colour and pH of 5.2. The percentage yield of the extractwas 3.25%. The dried extract obtained was kept in the refrigeratorat 4 1C and reconstituted in distilled water prior to each experi-mental session.

2.5. Phytochemical analysis

A portion of the dried extract was used for phytochemicalscreening in order to determine the presence of pharmacologicallyactive constituents using the methods described by Trease andEvans (1989) and Edeoga et al. (2005).

2.5.1. Test for alkaloids0.5 g of PL was added to 5 ml of 1% HCl with stirring on a water

bath. Three portions of 1 ml each of the filtrate were then treatedwith few drops of Mayer's, Dragendorff's, and Wagner's reagent.Observation of turbidity of precipitation was taken as indicationfor the presence of alkaloids.

2.5.2. Test for saponins2 g of PL was boiled in 20 ml of distilled water on a water bath

and filtered. 10 ml of the filtrate was mixed with 5 ml of distilledwater and the mixture was vigorously agitated to obtain a stablefroth. The froth was mixed with three drops of olive oil and shakenvigorously. Observation was made for the formation of emulsion.In the haemolysis test, two test tubes were labelled A and B. 0.2 mlsolution of PL, prepared in 1% normal saline, and 0.2 ml of 10%(v/v) blood in normal saline was put into test tube A while Bcontained 0.2 ml of 10% blood in normal saline with 0.2 ml ofnormal saline. The observation of red supernatant in test tube A,absent in test tube B, confirmed the presence of saponins.

2.5.3. Test for phlobatanninsObservation of the deposition of a red precipitate upon the

boiling of PL with 1% aqueous HCl was taken as indication for thepresence of phlobatannins.

2.5.4. Test for reducing sugars0.5 g of PL was diluted with 1 ml of distilled water and 1 ml of

Fehling's solution (A and B) was added. This was heated on a waterbath. A brown colouration indicates the presence of reducingsugars.

2.5.5. Test for fixed oils and fatsThe filter paper and saponification tests were used in this

respect. In the filter paper test, a small quantity of PL was pressedbetween two filter papers. The appearance of oil stain was taken asindication for the presence of fixed oils. In the saponification test,few drops of 0.5 M potassium hydroxide were added to a smallquantity of PL along with a drop of phenolphthalein. On a waterbath, the mixture was heated for 1–2 h. The presence of fixed oilsand fats was indicated by the formation of soap.

2.6. Acute toxicity studies

Albino mice of either sex were fasted for 12 h prior to testing.The animals were randomly allotted to groups of five animals each.A dose of 10,000 mg/kg of extract was administered, in divideddoses, by oral intubation to a group. The other groups of mice weregiven the extract intraperitoneally at the dose of 400 and 800 mg/kg respectively.

The general symptoms of toxicity such as restlessness, pantingand mortality in each group within 24 h were recorded. The LD50

was estimated using Miller and Tainter's log-probit analysismethod (Adeyemi et al., 2010). Animals were further observedfor one week for any delayed toxic effects.

A.J. Akindele et al. / Journal of Ethnopharmacology 151 (2014) 984–989 985

2.7. Normal intestinal transit

The method used was as described by Hsu (1982). Mice wereallotted to five groups containing six animals each. Three groupswere separately treated orally with the hydroethanolic extract(100, 200 and 400 mg/kg) while distilled water (10 ml/kg) andloperamide hydrochloride (5 mg/kg) were separately administeredorally to the control and standard groups respectively. One hourafter treatment, the mice were administered a standard charcoalmeal (0.2 ml/mouse, made up of 5% charcoal suspension in 5%methylcellulose) orally.

The mice were then sacrificed 30 min after administration ofcharcoal meal and the small intestine immediately isolated.Peristaltic index for each mouse was expressed as a percentageof the distance travelled by the charcoal meal relative to the totallength of the small intestine (Aye-Than et al., 1989).

2.8. Castor oil-induced intestinal transit

The animals were divided into five groups of six mice each.Three groups were given the hydroethanolic extract (100, 200 and400 mg/kg) via the oral route. Another group was given loper-amide hydrochloride (5 mg/kg) orally and the control group wasgiven distilled water (10 ml/kg) orally. Thirty minutes later, allgroups of mice were administered castor oil (0.2 ml/mouse).

In respect of mechanism of action elucidation, four groupscomprising six mice each were separately given yohimbine(10 mg/kg, s.c.), prazosin (1 mg/kg, s.c.), propranolol (1 mg/kg,i.p.) and pilocarpine (1 mg/kg, s.c.) 15 min before the administra-tion of hydroethanolic extract of PL at the dose of 400 mg/kg (mosteffective extract dose in this model).

Thirty minutes after the administration of castor oil, theanimals were given a standard charcoal meal (0.2 ml/mouse, madeup of 5% charcoal suspension in 5% methylcellulose) orally. All theanimals in each treatment group were sacrificed 30 min after theadministration of the charcoal meal and the small intestineimmediately isolated. The peristaltic index, which is the distancetravelled by the charcoal meal relative to the total length of smallintestine expressed in percentage, was determined for each mouse(Aye-Than et al., 1989; Adeyemi and Akindele, 2008).

2.9. Castor oil-induced diarrhoea

The animals were divided into five groups of six mice each.Four groups were treated with the extract (100, 200 and 400 mg/kg) and loperamide hydrochloride (5 mg/kg) orally. The controlgroup was given distilled water (10 ml/kg) orally. Pre-treatmentwas done one hour before the administration of castor oil (0.2 ml/mouse). Each mouse was kept for observation under a transparentfunnel, the floor of which was lined with paper and observed forfour hours (Izzo et al., 1992).

The following parameters were observed: time elapsed betweenthe administration of the castor oil and the excretion of the firstdiarrhoea faeces, the total amount of hard stool, semi-solid stooland watery stool. The weight of watery stool (which comprises ofthe semi-solid and watery stool) and the total weight of all stools(which comprises of hard stool, semi-solid stool and watery stool)were determined. A numerical score based on stool consistency wasassigned: 1 (hard stool), 2 (semi-solid stool), and 3 (watery stool).The in vivo antidiarrhoeal index (ADIin vivo) was expressed accordingto the formula: ADIin vivo¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiDfreq � Gmeq � Pfreq

3p

(Aye-Than et al.,1989; Adeyemi and Akindele, 2008), where Dfreq is the delay indefaecation time or diarrhoea onset (in % of control), Gmeq is the gutmeal travel reduction (in % of control), and Pfreq is the purgingfrequency, as number of stool reduction (in % of control).

2.10. Measurement of gastric emptying

The method used was that described by Droppleman et al.,1980. Albino rats of either sex (previously fasted for 24 h prior tothe experiment), were randomly allotted to two groups of sixanimals each. The test meal was prepared by mixing 10% charcoalsuspension in 5% methylcellulose. Group one received distilledwater (10 ml/kg, p.o.) while group two received hydroethanolicextract (400 mg/kg, p.o.). One hour later, each rat received 3 ml(3.05 g) of the test meal. One hour later, each rat was sacrificed,laparatomized and the stomach removed and weighed on ananalytical balance. The stomach was opened and the stomachcontents poured out and rinsed. Excess moisture was removedwith cotton wool, and the empty stomach was weighed.

The difference between full and empty stomach was subtractedfrom the weight of 3 ml of the test meal, indicating the quantityemptied from the stomach during the test period.

2.11. Intestinal fluid accumulation

Albino rats were divided into two groups of six rats each.Intestinal fluid accumulation was induced by oral administrationof castor oil (2 ml/rat) (Robert et al., 1976). The rats were fasted for24 h, but had access to water prior to the experiment.

The control group was administered distilled water (10 ml/kg)orally and the second group was administered the extract(400 mg/kg) orally. One hour later, castor oil (2 ml/rat) wasadministered intragastrically and 1 h after that, the rats weresacrificed. The rats were then dissected and their small intestineswere immediately isolated after ligation at the pyloric sphincterand at the ileo-caecal junction respectively. Each intestine wasweighed and the intestinal contents were expelled into measuringcylinder and the volume determined. The intestine was reweighedand the difference between the full and empty intestine wasdetermined.

2.12. Statistical analysis

Results were expressed as mean7SEM. Statistical analysis ofthe data was done using One-way ANOVA followed by the Tukey'smultiple comparison test or Unpaired Student's t-test, whereappropriate. Results were considered significant when Po0.05.

3. Results

3.1. Phytochemical analysis

Phytochemical analysis carried out revealed the presence ofalkaloids, saponins, and fixed oils and fats, and absence ofphlobatannins and reducing sugars.

3.2. Acute toxicity study in mice

The hydroethanolic extract administered to animals by the oralroute did not produce any visible signs of toxicity and no mortalitywas recorded at a dose of 10,000 mg/kg. The animals were keptunder observation for additional seven days and this showed nodelayed toxic effect.

Administration of the extract via the intraperitoneal routeproduced writhing and calming of the animals especially at thedose of 800 mg/kg. Mortality was 20% at the dose of 800 mg/kg.This implies that the LD50 for the intraperitoneal route is4800 mg/kg.

A.J. Akindele et al. / Journal of Ethnopharmacology 151 (2014) 984–989986

3.3. Normal intestinal transit

In control animals, the charcoal meal traversed 72.5770.63% ofthe total length of the small intestine. The hydroethanolic extract(100–400 mg/kg) produced a dose-dependent decrease in theintestinal transit with the peak effect of 34.78% inhibition at400 mg/kg compared to loperamide (5 mg/kg) with inhibitionvalue of 66.93% (Table 1).

3.4. Castor oil-induced intestinal transit

The hydroethanolic extract and loperamide (5 mg/kg) causedsignificant (Po0.001) reduction in the distance travelled by thecharcoal meal in the small intestine, relative to the total length of

the small intestine, compared to the control mice. The highestpercentage inhibition produced by the extract (44.60%) was seenat the dose of 400 mg/kg and this was significantly (Po0.01) lessthan the effect (63.84%) produced by loperamide (5 mg/kg). Also,there was no significant difference when the peristaltic index ofthe extract at 400 mg/kg was compared with that of yohimbi-neþextract (400 mg/kg), prazosinþextract (400 mg/kg) and pro-pranololþextract (400 mg/kg). However, there was a significant(Po0.05) difference when the peristaltic index of the extract(400 mg/kg) was compared with that of pilocarpineþextract400 mg/kg (Table 2).

3.5. Castor oil-induced diarrhoea

As shown in Table 3, 4 h after the administration of castor oil allgroups produced watery stool. However, there was a dose-dependent significant (Po0.001) delay in the onset of diarrhoeaproduced by the hydroethanolic extract with the peak effectproduced at the dose of 400 mg/kg (173.67 min) compared withcontrol (59.33 min.). Similarly, there was reduction in the numberof wet stools, total number of stools, total weight of wet stools andtotal weight of all stools and diarrhoea score for all doses of thehydroethanolic extract when compared with control. Also, thehydroethanolic extract produced its highest percentage protectionin terms of reduction in diarrhoea score and in vivo antidiarrhoealindex at the dose of 400 mg/kg (62.68% and 56.95% respectively),but this was less than that produced by loperamide (84.69% and77.75% respectively).

3.6. Gastric emptying

As shown in Table 4, the extract (400 mg/kg) significantly(Po0.01) increased the quantity of test meal emptied (2.0870.10 g) compared to that of control (1.5670.10 g).

3.7. Intestinal fluid accumulation

In the fluid accumulation test, the extract at 400 mg/kgsignificantly (Po0.01) reduced both the weight and volume of

Table 1Effect of Pupalia lappacea on normal intestinal transit.

Group Dose (mg/kg) Peristaltic index (%) Inhibition (%)

Control – 72.5770.63nnn –

PL 100 66.1770.53nnn 8.82PL 200 63.4870.46nnn 12.53PL 400 47.3370.38nnn 34.78Loperamide 5 24.0070.48nnn,a 66.93

Values are mean7SEM (n¼6).nnn Po0.001 vs. Control.a Po0.001 vs. PL (400 mg/kg) (One way ANOVA followed by the Tukey's

Multiple Comparison Test). PL¼Pupalia lappacea.

Table 2Effect of Pupalia lappacea on castor oil-induced intestinal transit.

Group Dose (mg/kg) Peristaltic index (%) Inhibition (%)

Control 82.06 –

PL 100 56.05nnn 31.70PL 200 53.36nnn 34.97PL 400 45.46nnn,a 44.60PLþ 400 44.05 46.32Yohimbine 10PLþ 400 34.00 58.57Prazosin 1PLþ 400 44.99 45.18Propranolol 1PLþ 400 60.28 26.54Pilocarpine 1Loperamide 5 29.67nnn,b 63.84

Values are mean7SEM (n¼6).a Po0.05 vs. PLþPilocarpine.b Po0.01 vs. PL (400 mg/kg).nnn Po0.001 vs. Control (One way ANOVA followed by the Tukey's Multiple

Comparison Test). PL¼Pupalia lappacea.

Table 3Effect of Pupalia lappacea in castor oil-induced diarrhoea in mice.

Group Dose(mg/kg)

Onset ofdiarrhoea(min)

Number ofwet stools

Total numberof stools

Total weight ofwet stools(g)

Total weight ofall stools(g)

DiarrhoeaScore

Percentageprotection

In vivoantidiarrhoealindex (%)

Control 59.3372.68 9.1770.83 13.8370.31 0.3470.03 0.3870.03 34.8371.40 – –

PL 100 129.5074.99nnn 7.5070.76 10.0070.86nn 0.2570.03 0.2870.02n 26.3372.39n 24.40 28.18PL 200 137.8379.16nnn 6.5070.67 9.1770.79nnn 0.2270.02nn 0.2570.02nn 23.6772.26nn 32.06 35.36PL 400 173.6776.17nnn 3.1770.48nnn 5.5070.76nnn 0.1570.01nnn 0.1770.01nnn 13.0072.01nnn 62.68 56.95Loperamide 5 218.3375.24nnn,a 1.5070.43nnn 2.1770.40nnn 0.1370.01nnn 0.1470.01nnn 5.3371.28nnn 84.69 77.75

Values are mean7SEM (n¼6).n Po0.05.nn Po0.01.nnn Po0.001 vs. Control.a Po0.001 vs. PL 400 mg/kg (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL¼Pupalia lappacea.

Table 4Effect of Pupalia lappacea on gastric emptying.

Group Dose(mg/kg)

Weight of intestinalcontent (g)

Quantity of intestinalcontent emptied (g)

Control – 1.4970.10 1.5670.10PL 400 0.9770.10nn 2.0870.10nn

Values are mean7SEM (n¼6).nn Po0.01 vs. Control (Unpaired Student's t-test.) PL¼Pupalia lappacea.

A.J. Akindele et al. / Journal of Ethnopharmacology 151 (2014) 984–989 987

intestinal content (1.5370.26 g and 1.1870.09 ml respectively)compared with that of control (2.0570.14 g and 1.6570.08 mlrespectively) as shown in Table 5.

4. Discussion

Diarrhoea is the frequent passage of liquid faeces and itinvolves both an increase in the motility of the gastrointestinaltract, along with increased secretion and decreased absorption offluid, and loss of electrolytes (particularly sodium) and water(Rang et al., 2003). Therefore, to restore personal comfort andconvenience, many patients require antidiarrhoeal therapy andtreatment is carried out to achieve, among other objectives,increased resistance to flow (segmental contraction, decreasedpropulsion and peristalsis) and increased mucosal absorption ordecreased secretion (Burks, 1991; Akindele and Adeyemi, 2006).Approaches that are used in the treatment of severe acutediarrhoea include maintenance of fluid and electrolyte balance,use of anti-infective agents, and use of spasmolytic or otherantidiarrhoeal agents (Rang et al., 2007). In this context, theinvestigation of the antidiarrhoeal effect of Pupalia lappacea inthis study comprised the evaluation of its effect on intestinaltransit and fluid accumulation. Its effect on gastric emptying wasalso investigated based on the fact that the action of drugs likeatropine and morphine in reducing intestinal propulsion is aidedby their ability to delay gastric emptying (Izzo et al., 1999;Akindele and Adeyemi, 2006). Major classes of drugs used in thepharmacologic therapy of diarrhoea include antimotility andspasmolytic agents (e.g. opiates like diphenoxylate and lopera-mide, and muscarinic receptor antagonists like atropine, hyoscineand propantheline); antisecretory agents (e.g. bismuth compoundsand octreotide); adsorbents and bulk agents including prepara-tions containing kaolin, chalk, charcoal, methylcellulose and acti-vated attapulgite; bile acid sequestrants (e.g. cholestyramine,colestipol and colesevalam); probiotics; anti-infectives (e.g. fluor-oquinolones [ciprofloxacin or levofloxacin], azithromycin anderythromycin); and miscellaneous agents like α2--adrenergicreceptor antagonists (e.g. clonidine), calcium channel blockers(e.g. verapamil and nifedipine), berberine (plant alkaloid with anti-microbial, antimotility and antisecretory effects) and calmodulininhibitors (e.g. zaldaride maleate) (Pasricha, 2006; Rang et al.,2007; Mims and Curry, 2008).

Martinez et al. (1998) and Heinrich et al. (2005) based on theirfindings reported that herbal treatments remain importantas home remedy for diarrhoea and that despite the availability ofsimple and cheap treatments for diarrhoea (ORT), healersand patients in many communities still rely on locally availablephytomedicines.

The phytochemical analysis of the hydroethanolic leaf extractof Pupalia lappacea revealed the presence of bioactive compoundssuch as alkaloids, saponins, and fixed oils and fats. Some of thesephytoconstituents have been reported to have inhibitory activityon intestinal motility in a dose related manner (Carlo et al., 1994).

The acute toxicity study also revealed that the extract is safewhen administered orally, having produced neither obvious mor-bidity nor mortality at a dose of 10,000 mg/kg (Clarke and Clarke,1977). However, administration through the intraperitoneal routecaused mortality with a median lethal dose greater than 800 mg/kgsince only 20% mortality was recorded at this dose. Also, the animalsexhibited certain characteristics like calming, suggesting that theextract might have sedative properties.

The extract produced a dose-dependent decrease in the pro-pulsive movement of the standard charcoal meal in the smallintestine both in the normal and castor oil-induced intestinaltransit models. In both models, the effect was observed to peak atthe dose of 400 mg/kg (34.78% and 44.60% inhibition respectively).This inhibitory action of the extract on intestinal transit will delaythe passage of gastrointestinal contents allowing faeces to becomedesiccated, thus further retarding movement through the colon.The extract was more effective in the castor-oil induced intestinaltransit test than in the normal intestinal transit test, suggestingthat the extract might be more effective in a diseased state than ina normal state. Yohimbine (an α2-adrenergic receptor antagonist),prazosin (an α1-adrenergic receptor antagonist), propranolol (anon-selective beta receptor antagonist) and pilocarpine (a non-selective muscarinic receptor agonist) were employed in this studyto elucidate the mechanism of action of Pupalia lappacea using thecastor oil-induced intestinal transit model. Yohimbine, prazosinand propranolol had no significant influence on the action of theextract (400 mg/kg) on intestinal propulsion. This suggests thatthe extract had little or no activity on alpha and beta adrenergicreceptors. However, pilocarpine significantly altered the inhibitoryaction of the extract (400 mg/kg) on intestinal propulsion. Thissuggests that the extract probably has muscarinic receptor activity.

Going by the results obtained in the castor oil-induced diar-rhoea test, the hydroethanolic leaf extract of Pupalia lappaceacaused a dose-dependent significant delay in the onset of diar-rhoea, decrease in frequency of purging (reduction of number ofwet stools), decrease in weight of wet stools and decrease inseverity of diarrhoea (diarrhoea score). Also, the extract increasedthe amount of test meal emptied in the gastric emptying model,and this suggests that unlike atropine, the effect of the extract onintestinal propulsion may not be due to delay in gastric emptyingtime. In the intestinal fluid accumulation test, the extract (400 mg/kg) significantly reduced the volume of intestinal content.

The effect of Pupalia lappacea on all the diarrhoea indicators issummed up by the calculation of the ADIin vivo (in vivo antidiar-rhoeal index). The higher the ADIin vivo value, the greater theeffectiveness in the treatment of diarrhoea. The extract produced adose-dependent increase in ADIin vivo with a maximum of 56.95%produced at the dose of 400 mg/kg, a value lower than that elicitedby loperamide 5 mg/kg (77.75%).

5. Conclusion

The results obtained in this study suggest that the hydroetha-nolic leaf extract of Pupalia lappacea possesses antidiarrhoealproperty through antimotility and antisecretory effects possiblymediated by its activity on muscarinic receptors. This validates theclaims made by traditional medicine practitioners about its pos-session of antidiarrhoeal activity. Further studies on the plantextract would entail the evaluation of antimicrobial effects.

Acknowledgements

The authors are grateful to Mr. M. Chijioke, of the Departmentof Pharmacology, Therapeutics and Toxicology, for his technicalassistance during the study.

Table 5Effect of Pupalia lappacea on intestinal fluid accumulation.

Group Dose(mg/kg)

Weight of intestinalcontent (g)

Volume of intestinalcontent (ml)

Control – 2.0570.14 1.6570.08PL 400 1.5370.26 1.1870.09nn

Values are mean7SEM (n¼6).nn Po0.01 vs. Control (Unpaired Student's t-test). PL¼Pupalia lappacea.

A.J. Akindele et al. / Journal of Ethnopharmacology 151 (2014) 984–989988

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