Journal of Ethnopharmacology 151 (2014) 984–989
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Evaluation of the antidiarrhoeal activity of the hydroethanolic leaf extract of Pupalia lappacea Linn. Juss. (Amaranthaceae) A.J. Akindele n, O.A. Salako, U.V. Ohonbamu Department of Pharmacology, Therapeutics & Toxicology (PTT), Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, P.M.B. 12003 Lagos, Nigeria
art ic l e i nf o
a b s t r a c t
Article history: Received 7 October 2013 Received in revised form 21 November 2013 Accepted 7 December 2013 Available online 14 December 2013
Ethnopharmacological Relevance: Pupalia lappacea is a medicinal plant found in savannah and woodland localities and forest path sides from Senegal to Southern Nigeria. It has been used in the management of diarrhoea in Nigerian traditional medicine. This study was designed to evaluate the antidiarrhoeal activity of the hydroethanolic leaf extract of Pupalia lappacea (PL). Materials and methods: The antidiarrhoeal activity of PL was evaluated using the normal and castor oilinduced intestinal transit, castor oil-induced diarrhoea, gastric emptying and intestinal fluid accumulation tests in rodents. Results: PL (100–400 mg/kg, p.o.) produced a significant dose-dependent decrease in normal and castor oil-induced intestinal transit compared with the control group (distilled water 10 ml/kg, p.o.). This effect was significantly (P o0.05) 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 and significant 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 the extract 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 the volume of intestinal content in the intestinal fluid accumulation test. Phytochemical analysis of the extract revealed the presence of alkaloids, saponins, and fixed oils and fats. The acute toxicity studies revealed 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 antidiarrhoeal activity possibly mediated by antimuscarinic receptor activity. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pupalia lappacea Antidiarrhoeal activity Castor oil Loperamide Intestinal transit
1. Introduction Diarrhoea is a symptom marked by rapid and frequent passage of semi-solid or liquid faecal material through the gastrointestinal tract. Various types of diarrhoea exist (based on their clinical manifestations), but it is broadly classified into acute and chronic for diagnostic and therapeutic purposes. Acute diarrhoea is the prevalent form of the disease which has a major impact on the morbidity and mortality worldwide in all age groups, particularly in infants and children under the age of three (Hirchhorn, 1980; Muriithi, 1996; Farthings, 2002). In less developed parts of the world (such as Latin America, India and Africa), children may experience between 3 and 10 episodes of diarrhoea yearly (Farthings, 2002). In 2009, diarrhoea was estimated to have caused
n
Corresponding author. Tel.: þ 234 8062359726. E-mail addresses: [email protected], [email protected] (A.J. Akindele). 0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.013
1.1 million deaths in people aged 5 and 1.5 million deaths in children under the age of 5 (World Health Organization, 2009). In terms of etiology, diarrhoea can be classified as infectious and non-infectious (de Hostos et al., 2011). Infectious diarrhoea is caused by a virus, parasite or bacterium while non-infectious diarrhoea can be caused by toxins, chronic diseases or antibiotics (NHDHHS, 2009). Pharmacological models of non-infectious diarrhoea were used in this study. Pupalia lappacea Linn. Juss. (Amaranthaceae) is a plant found in savannah and woodland localities and forest path sides from Senegal to Southern Nigeria, but less common in the western part of the region. Distribution is widespread elsewhere in tropical Africa and in Asia. Pupalia lappacea also commonly known as ram's bur and locally as “Kaimin kadangari” (Hausa, Northern Nigeria), “Agbiriba” (Igbo, South-east Nigeria) or “Emo agbo” (Yoruba, South-west Nigeria) has been used in traditional medicine for the treatment of cough, syphilis, skin diseases and diarrhoea (Odugbemi, 2008). The leaves mixed with palm-oil, are used in Ghana to treat boils (Agyare et al., 2009). The leaves are also used in topical applications to cuts, or used as enema or
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febrifuge (Agyare et al., 2009). In Ivory Coast, a decoction is taken in draught and applied in frictions for oedema of the legs and is used in various remedies for dysentery and diarrhoea. It is used as an antiemetic in the south-western region of Nigeria and the Republic of Benin (Adjanohoun and Akjakidje, 1989). Pupalia lappacea preparations have been reported to have antioxidant activity (Aladedunye and Okorie, 2008), anti-cancer activity (Ravi et al., 2012), antinociceptive and antipyretic activities (Neeharika et al., 2013). However, no scientific report of the antidiarrhoeal activity of Pupalia lappacea is available. The aim of this study is to evaluate the antidiarrhoeal effects of the hydroethanolic leaf extract of Pupalia lappacea (PL) in search for safe, effective and cheap remedies for diarrhoea management.
2.5. Phytochemical analysis
2. Materials and methods
2.5.2. Test for saponins 2 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 distilled water and the mixture was vigorously agitated to obtain a stable froth. The froth was mixed with three drops of olive oil and shaken vigorously. Observation was made for the formation of emulsion. In the haemolysis test, two test tubes were labelled A and B. 0.2 ml solution 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 B contained 0.2 ml of 10% blood in normal saline with 0.2 ml of normal saline. The observation of red supernatant in test tube A, absent in test tube B, confirmed the presence of saponins.
2.1. Reagents and drugs Castor oil (Finest Cold Drawn Commercial Castor oil, UK), loperamide hydrochloride (Imodiums, Janssen Pharmaceutical N.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 the farm of a traditional herbal practitioner in Ogun State, Nigeria, and the plant was identified and authenticated at the herbarium of the Department of Botany, University of Lagos, Lagos, Nigeria, by Mr. T.K. Odewo. A voucher specimen with reference number LUH 5092 was deposited in the herbarium of the department.
2.3. Experimental animals Adult albino Wistar mice (15–30 g) and rats (120–220 g) (8 weeks old) of either sex obtained from the Laboratory Animal Centre of the College of Medicine, University of Lagos, Lagos, Nigeria, were used for the experiments. Animals were maintained under standard environmental conditions (12 h light and 12 h dark cycle; 23–25 1C) and had free access to standard pelleted feed (RAAF Animal Feeds Ltd., Akute, Ogun State, Nigeria) and water adlibitum in accordance with the Guidelines for Care and Use of Laboratory Animals in Biomedical Research (National Academy of Sciences, 2011).
2.4. Preparation of extract The fresh leaves of the plant were collected and air-dried for a period of three weeks. The dried leaves were thereafter ground into powder with an electric blender. The powdered leaves were then macerated in hydroethanol (1:1; 50 g/L) for 48 h. Exhaustive extraction was done. Filtration was carried out after 48 h and the combined filtrate was evaporated to dryness under reduced pressure at 40 1C using a Heidolph rotavapor. The dried hydroethanolic extract had a sticky consistency which was readily soluble in water. It had a dark brown colour and pH of 5.2. The percentage yield of the extract was 3.25%. The dried extract obtained was kept in the refrigerator at 4 1C and reconstituted in distilled water prior to each experimental session.
A portion of the dried extract was used for phytochemical screening in order to determine the presence of pharmacologically active constituents using the methods described by Trease and Evans (1989) and Edeoga et al. (2005).
2.5.1. Test for alkaloids 0.5 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 treated with few drops of Mayer's, Dragendorff's, and Wagner's reagent. Observation of turbidity of precipitation was taken as indication for the presence of alkaloids.
2.5.3. Test for phlobatannins Observation of the deposition of a red precipitate upon the boiling of PL with 1% aqueous HCl was taken as indication for the presence of phlobatannins.
2.5.4. Test for reducing sugars 0.5 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 water bath. A brown colouration indicates the presence of reducing sugars.
2.5.5. Test for fixed oils and fats The filter paper and saponification tests were used in this respect. In the filter paper test, a small quantity of PL was pressed between two filter papers. The appearance of oil stain was taken as indication for the presence of fixed oils. In the saponification test, few drops of 0.5 M potassium hydroxide were added to a small quantity of PL along with a drop of phenolphthalein. On a water bath, the mixture was heated for 1–2 h. The presence of fixed oils and 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 divided doses, by oral intubation to a group. The other groups of mice were given the extract intraperitoneally at the dose of 400 and 800 mg/ kg respectively. The general symptoms of toxicity such as restlessness, panting and mortality in each group within 24 h were recorded. The LD50 was estimated using Miller and Tainter's log-probit analysis method (Adeyemi et al., 2010). Animals were further observed for one week for any delayed toxic effects.
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2.7. Normal intestinal transit
2.10. Measurement of gastric emptying
The method used was as described by Hsu (1982). Mice were allotted to five groups containing six animals each. Three groups were separately treated orally with the hydroethanolic extract (100, 200 and 400 mg/kg) while distilled water (10 ml/kg) and loperamide hydrochloride (5 mg/kg) were separately administered orally to the control and standard groups respectively. One hour after treatment, the mice were administered a standard charcoal meal (0.2 ml/mouse, made up of 5% charcoal suspension in 5% methylcellulose) orally. The mice were then sacrificed 30 min after administration of charcoal meal and the small intestine immediately isolated. Peristaltic index for each mouse was expressed as a percentage of the distance travelled by the charcoal meal relative to the total length of the small intestine (Aye-Than et al., 1989).
The method used was that described by Droppleman et al., 1980. Albino rats of either sex (previously fasted for 24 h prior to the experiment), were randomly allotted to two groups of six animals each. The test meal was prepared by mixing 10% charcoal suspension in 5% methylcellulose. Group one received distilled water (10 ml/kg, p.o.) while group two received hydroethanolic extract (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 an analytical balance. The stomach was opened and the stomach contents poured out and rinsed. Excess moisture was removed with cotton wool, and the empty stomach was weighed. The difference between full and empty stomach was subtracted from the weight of 3 ml of the test meal, indicating the quantity emptied from the stomach during the test period.
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 and 400 mg/kg) via the oral route. Another group was given loperamide hydrochloride (5 mg/kg) orally and the control group was given distilled water (10 ml/kg) orally. Thirty minutes later, all groups of mice were administered castor oil (0.2 ml/mouse). In respect of mechanism of action elucidation, four groups comprising 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 administration of hydroethanolic extract of PL at the dose of 400 mg/kg (most effective extract dose in this model). Thirty minutes after the administration of castor oil, the animals were given a standard charcoal meal (0.2 ml/mouse, made up of 5% charcoal suspension in 5% methylcellulose) orally. All the animals in each treatment group were sacrificed 30 min after the administration of the charcoal meal and the small intestine immediately isolated. The peristaltic index, which is the distance travelled by the charcoal meal relative to the total length of small intestine 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 control group was given distilled water (10 ml/kg) orally. Pre-treatment was done one hour before the administration of castor oil (0.2 ml/ mouse). Each mouse was kept for observation under a transparent funnel, the floor of which was lined with paper and observed for four hours (Izzo et al., 1992). The following parameters were observed: time elapsed between the administration of the castor oil and the excretion of the first diarrhoea faeces, the total amount of hard stool, semi-solid stool and watery stool. The weight of watery stool (which comprises of the 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 was assigned: 1 (hard stool), 2 (semi-solid stool), and 3 (watery stool). The in vivo antidiarrhoeal index (ADIin vivo) was expressed according pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi to the formula: ADIin vivo ¼ 3 Dfreq Gmeq P freq (Aye-Than et al., 1989; Adeyemi and Akindele, 2008), where Dfreq is the delay in defaecation time or diarrhoea onset (in % of control), Gmeq is the gut meal travel reduction (in % of control), and Pfreq is the purging frequency, as number of stool reduction (in % of control).
2.11. Intestinal fluid accumulation Albino rats were divided into two groups of six rats each. Intestinal fluid accumulation was induced by oral administration of castor oil (2 ml/rat) (Robert et al., 1976). The rats were fasted for 24 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) was administered intragastrically and 1 h after that, the rats were sacrificed. The rats were then dissected and their small intestines were immediately isolated after ligation at the pyloric sphincter and at the ileo-caecal junction respectively. Each intestine was weighed and the intestinal contents were expelled into measuring cylinder and the volume determined. The intestine was reweighed and the difference between the full and empty intestine was determined. 2.12. Statistical analysis Results were expressed as mean 7SEM. Statistical analysis of the data was done using One-way ANOVA followed by the Tukey's multiple comparison test or Unpaired Student's t-test, where appropriate. Results were considered significant when P o0.05.
3. Results 3.1. Phytochemical analysis Phytochemical analysis carried out revealed the presence of alkaloids, saponins, and fixed oils and fats, and absence of phlobatannins and reducing sugars. 3.2. Acute toxicity study in mice The hydroethanolic extract administered to animals by the oral route did not produce any visible signs of toxicity and no mortality was recorded at a dose of 10,000 mg/kg. The animals were kept under observation for additional seven days and this showed no delayed toxic effect. Administration of the extract via the intraperitoneal route produced writhing and calming of the animals especially at the dose of 800 mg/kg. Mortality was 20% at the dose of 800 mg/kg. This implies that the LD50 for the intraperitoneal route is 4800 mg/kg.
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3.3. Normal intestinal transit In control animals, the charcoal meal traversed 72.577 0.63% of the total length of the small intestine. The hydroethanolic extract (100–400 mg/kg) produced a dose-dependent decrease in the intestinal transit with the peak effect of 34.78% inhibition at 400 mg/kg compared to loperamide (5 mg/kg) with inhibition value of 66.93% (Table 1). 3.4. Castor oil-induced intestinal transit The hydroethanolic extract and loperamide (5 mg/kg) caused significant (Po 0.001) reduction in the distance travelled by the charcoal meal in the small intestine, relative to the total length of Table 1 Effect of Pupalia lappacea on normal intestinal transit. Group
Dose (mg/kg)
Peristaltic index (%)
Inhibition (%)
Control PL PL PL Loperamide
– 100 200 400 5
72.577 0.63nnn 66.177 0.53nnn 63.487 0.46nnn 47.337 0.38nnn 24.007 0.48nnn,a
– 8.82 12.53 34.78 66.93
Values are mean7 SEM (n ¼6). nnn
Po 0.001 vs. Control. P o0.001 vs. PL (400 mg/kg) (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL¼ Pupalia lappacea. a
Dose (mg/kg)
Control PL PL PL PL þ Yohimbine PL þ Prazosin PL þ Propranolol PL þ Pilocarpine Loperamide
100 200 400 400 10 400 1 400 1 400 1 5
the small intestine, compared to the control mice. The highest percentage inhibition produced by the extract (44.60%) was seen at the dose of 400 mg/kg and this was significantly (P o0.01) less than the effect (63.84%) produced by loperamide (5 mg/kg). Also, there was no significant difference when the peristaltic index of the extract at 400 mg/kg was compared with that of yohimbineþ extract (400 mg/kg), prazosinþextract (400 mg/kg) and propranolol þextract (400 mg/kg). However, there was a significant (P o0.05) difference when the peristaltic index of the extract (400 mg/kg) was compared with that of pilocarpine þextract 400 mg/kg (Table 2). 3.5. Castor oil-induced diarrhoea As shown in Table 3, 4 h after the administration of castor oil all groups produced watery stool. However, there was a dosedependent significant (P o0.001) delay in the onset of diarrhoea produced by the hydroethanolic extract with the peak effect produced at the dose of 400 mg/kg (173.67 min) compared with control (59.33 min.). Similarly, there was reduction in the number of wet stools, total number of stools, total weight of wet stools and total weight of all stools and diarrhoea score for all doses of the hydroethanolic extract when compared with control. Also, the hydroethanolic extract produced its highest percentage protection in terms of reduction in diarrhoea score and in vivo antidiarrhoeal index at the dose of 400 mg/kg (62.68% and 56.95% respectively), but this was less than that produced by loperamide (84.69% and 77.75% respectively). 3.6. Gastric emptying
Table 2 Effect of Pupalia lappacea on castor oil-induced intestinal transit. Group
987
Peristaltic index (%)
Inhibition (%)
82.06 56.05nnn 53.36nnn 45.46nnn,a 44.05
– 31.70 34.97 44.60 46.32
34.00
58.57
44.99
45.18
60.28
26.54
29.67nnn,b
63.84
3.7. Intestinal fluid accumulation In the fluid accumulation test, the extract at 400 mg/kg significantly (P o0.01) reduced both the weight and volume of Table 4 Effect of Pupalia lappacea on gastric emptying.
Values are mean7 SEM (n ¼6). a
As shown in Table 4, the extract (400 mg/kg) significantly (P o0.01) increased the quantity of test meal emptied (2.08 70.10 g) compared to that of control (1.56 7 0.10 g).
P o0.05 vs. PLþ Pilocarpine. b Po 0.01 vs. PL (400 mg/kg). nnn Po 0.001 vs. Control (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL¼Pupalia lappacea.
Group
Dose (mg/kg)
Weight of intestinal content (g)
Quantity of intestinal content emptied (g)
Control PL
– 400
1.49 70.10 0.97 70.10nn
1.56 7 0.10 2.08 7 0.10nn
Values are mean 7 SEM (n¼ 6). nn
Po 0.01 vs. Control (Unpaired Student's t-test.) PL¼Pupalia lappacea.
Table 3 Effect of Pupalia lappacea in castor oil-induced diarrhoea in mice. Group
Dose (mg/kg)
Control PL 100 PL 200 PL 400 Loperamide 5
Onset of diarrhoea (min)
Number of wet stools
Total number of stools
Total weight of wet stools (g)
59.337 2.68 129.50 7 4.99nnn 137.83 7 9.16nnn 173.677 6.17nnn 218.337 5.24nnn,a
9.177 0.83 7.50 7 0.76 6.50 7 0.67 3.177 0.48nnn 1.50 7 0.43nnn
13.83 7 0.31 0.34 7 0.03 10.007 0.86nn 0.25 7 0.03 nnn 9.177 0.79 0.22 7 0.02nn 5.50 7 0.76nnn 0.157 0.01nnn 2.177 0.40nnn 0.137 0.01nnn
Total weight of all stools (g)
Diarrhoea Score
Percentage protection
In vivo antidiarrhoeal index (%)
0.38 7 0.03 0.28 7 0.02n 0.25 7 0.02nn 0.177 0.01nnn 0.147 0.01nnn
34.83 7 1.40 26.337 2.39n 23.677 2.26nn 13.007 2.01nnn 5.337 1.28nnn
– 24.40 32.06 62.68 84.69
– 28.18 35.36 56.95 77.75
Values are mean7 SEM (n ¼6). n
Po 0.05. Po 0.01. nnn Po 0.001 vs. Control. a P o0.001 vs. PL 400 mg/kg (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL ¼Pupalia lappacea. nn
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Table 5 Effect of Pupalia lappacea on intestinal fluid accumulation. Group
Dose (mg/kg)
Weight of intestinal content (g)
Volume of intestinal content (ml)
Control PL
– 400
2.05 7 0.14 1.53 7 0.26
1.65 7 0.08 1.187 0.09nn
Values are mean7 SEM (n ¼6). nn
Po 0.01 vs. Control (Unpaired Student's t-test). PL¼ Pupalia lappacea.
intestinal content (1.53 7 0.26 g and 1.18 70.09 ml respectively) compared with that of control (2.05 7 0.14 g and 1.65 70.08 ml respectively) as shown in Table 5.
4. Discussion Diarrhoea is the frequent passage of liquid faeces and it involves both an increase in the motility of the gastrointestinal tract, along with increased secretion and decreased absorption of fluid, and loss of electrolytes (particularly sodium) and water (Rang et al., 2003). Therefore, to restore personal comfort and convenience, many patients require antidiarrhoeal therapy and treatment is carried out to achieve, among other objectives, increased resistance to flow (segmental contraction, decreased propulsion and peristalsis) and increased mucosal absorption or decreased secretion (Burks, 1991; Akindele and Adeyemi, 2006). Approaches that are used in the treatment of severe acute diarrhoea include maintenance of fluid and electrolyte balance, use of anti-infective agents, and use of spasmolytic or other antidiarrhoeal agents (Rang et al., 2007). In this context, the investigation of the antidiarrhoeal effect of Pupalia lappacea in this study comprised the evaluation of its effect on intestinal transit and fluid accumulation. Its effect on gastric emptying was also investigated based on the fact that the action of drugs like atropine and morphine in reducing intestinal propulsion is aided by their ability to delay gastric emptying (Izzo et al., 1999; Akindele and Adeyemi, 2006). Major classes of drugs used in the pharmacologic therapy of diarrhoea include antimotility and spasmolytic agents (e.g. opiates like diphenoxylate and loperamide, and muscarinic receptor antagonists like atropine, hyoscine and propantheline); antisecretory agents (e.g. bismuth compounds and octreotide); adsorbents and bulk agents including preparations containing kaolin, chalk, charcoal, methylcellulose and activated attapulgite; bile acid sequestrants (e.g. cholestyramine, colestipol and colesevalam); probiotics; anti-infectives (e.g. fluoroquinolones [ciprofloxacin or levofloxacin], azithromycin and erythromycin); and miscellaneous agents like α2--adrenergic receptor antagonists (e.g. clonidine), calcium channel blockers (e.g. verapamil and nifedipine), berberine (plant alkaloid with antimicrobial, antimotility and antisecretory effects) and calmodulin inhibitors (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 their findings reported that herbal treatments remain important as home remedy for diarrhoea and that despite the availability of simple and cheap treatments for diarrhoea (ORT), healers and patients in many communities still rely on locally available phytomedicines. The phytochemical analysis of the hydroethanolic leaf extract of Pupalia lappacea revealed the presence of bioactive compounds such as alkaloids, saponins, and fixed oils and fats. Some of these phytoconstituents have been reported to have inhibitory activity on intestinal motility in a dose related manner (Carlo et al., 1994).
The acute toxicity study also revealed that the extract is safe when administered orally, having produced neither obvious morbidity nor mortality at a dose of 10,000 mg/kg (Clarke and Clarke, 1977). However, administration through the intraperitoneal route caused mortality with a median lethal dose greater than 800 mg/kg since only 20% mortality was recorded at this dose. Also, the animals exhibited certain characteristics like calming, suggesting that the extract might have sedative properties. The extract produced a dose-dependent decrease in the propulsive movement of the standard charcoal meal in the small intestine both in the normal and castor oil-induced intestinal transit models. In both models, the effect was observed to peak at the dose of 400 mg/kg (34.78% and 44.60% inhibition respectively). This inhibitory action of the extract on intestinal transit will delay the passage of gastrointestinal contents allowing faeces to become desiccated, thus further retarding movement through the colon. The extract was more effective in the castor-oil induced intestinal transit test than in the normal intestinal transit test, suggesting that the extract might be more effective in a diseased state than in a normal state. Yohimbine (an α2-adrenergic receptor antagonist), prazosin (an α1-adrenergic receptor antagonist), propranolol (a non-selective beta receptor antagonist) and pilocarpine (a nonselective muscarinic receptor agonist) were employed in this study to elucidate the mechanism of action of Pupalia lappacea using the castor oil-induced intestinal transit model. Yohimbine, prazosin and propranolol had no significant influence on the action of the extract (400 mg/kg) on intestinal propulsion. This suggests that the extract had little or no activity on alpha and beta adrenergic receptors. However, pilocarpine significantly altered the inhibitory action of the extract (400 mg/kg) on intestinal propulsion. This suggests that the extract probably has muscarinic receptor activity. Going by the results obtained in the castor oil-induced diarrhoea test, the hydroethanolic leaf extract of Pupalia lappacea caused a dose-dependent significant delay in the onset of diarrhoea, decrease in frequency of purging (reduction of number of wet stools), decrease in weight of wet stools and decrease in severity of diarrhoea (diarrhoea score). Also, the extract increased the amount of test meal emptied in the gastric emptying model, and this suggests that unlike atropine, the effect of the extract on intestinal propulsion may not be due to delay in gastric emptying time. 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 is summed up by the calculation of the ADIin vivo (in vivo antidiarrhoeal index). The higher the ADIin vivo value, the greater the effectiveness in the treatment of diarrhoea. The extract produced a dose-dependent increase in ADIin vivo with a maximum of 56.95% produced at the dose of 400 mg/kg, a value lower than that elicited by loperamide 5 mg/kg (77.75%). 5. Conclusion The results obtained in this study suggest that the hydroethanolic leaf extract of Pupalia lappacea possesses antidiarrhoeal property through antimotility and antisecretory effects possibly mediated by its activity on muscarinic receptors. This validates the claims made by traditional medicine practitioners about its possession of antidiarrhoeal activity. Further studies on the plant extract would entail the evaluation of antimicrobial effects.
Acknowledgements The authors are grateful to Mr. M. Chijioke, of the Department of Pharmacology, Therapeutics and Toxicology, for his technical assistance during the study.
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Izzo, A.A., Mascolo, N., Capasso, R., Gormano, M.P., De-Pasquale, R., Capasso, F., 1999. Inhibitory effect of cannabinoid agonists on gastric emptying in rat. Arch. Pharmacol. 360, 221–223. Martinez, H., Ryan, G.W., Guiscafre, H., Gutierrez, G., 1998. An intercultural comparison of home case management of acute diarrhea in Mexico: implications for program planners. Arch. Med. Res. 29, 351–360. Mims, B.C., Curry Jr., C.E., 2008. Constipation, diarrhoea, and irritable bowel syndrome. In: Brown, M., Malley, J., Pancotti, R., Brown, R.Y. (Eds.), Pharmacotherapy Principles and Practice, (Eds.) McGraw-Hill USA, pp. 307–321. Muriithi, A.W., 1996. Control of diarrhoea disease programmes: the Kenyan situation. Afr. Med. J. 281, 189–195. National Academy of Sciences, 2011. Guide for the care and use of laboratory animals, 8th Ed. National Academies Press, Washington, D.C.,USA. Neeharika, Vajrala, Humaira, Fatima, Bommineni, Madhava Reddy, 2013. Evaluation of antinociceptive and antipyretic effect of Pupalia lappacea Juss. Int. Curr. Pharma. J. 2 (2), 23–28. New Hampshire Department of Health and Human Services (NHDHHS), 2009. Diarrhea (Infectious Diarrhea). Disease handbook for childcare providers. Division of Public Health Services, Communicable Disease Control Section. Revised April, 2009. 〈www.dhhs.nh.gov/dphs/cdcs/documents/diarrhea.pdf〉 (accessed 31.10.13). Odugbemi, T., 2008. Textbook of Medicinal plants from Nigeria. University of Lagos Press p. 106 118, and 232. Pasricha, P.J., 2006. Treatment of disorders of bowel motility and water flux; antiemetics; agents used in biliary and pancreatic disease. In: Brunton, L.L., Lazo, J.S., Parker, K.L. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics (eleventh edition). McGraw-Hill, USA, pp. 983–1019. Rang, H.P., Dale, M.M., Ritter, J.M., Moore, P.K., 2003. The gastrointestinal tract, Pharmacology, 5th ed. Churchill Livingstone, Edinburgh, pp. 367–379. Ravi, A., Alvala, M., Sama, V., Kalle, A.M., Irlapati, V.K., Reddy, B.M., 2012. Anticancer activity of Pupalia lappacea on chronic myeloid leukemia k562 cells. Daru J. Pharma. Sci. 20 (1), 86, http://dx.doi.org/10.1186/2008-2231-20-86. Rang, H.M., Dale, M.M., Ritter, J.M., Flower, R.J., 2007. The gastrointestinal tract. In Rang and Dale's Pharmacology, 6th Ed. Churchill Livingstone Elsevier Limited, pp. 385–396. Robert, A., Nozamis, J.E., Lancaster, C., Harcha, A.J., Klepper, M.S., 1976. Enteropooling assay: a test for diarrhoea produced by prostaglandins. Prostaglandins 11, 809–828. Trease, G.E., Evans, W.C., 1989. Pharmacognosy, 13th ed. Brailliar Tiridel Can. Macmillian Publishers, London. World Health Organization, 2009. 〈http://www.whqlibdoc.who.int〉. Diarrhoea: Why children are still dying and what can be done.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Evaluation of the antidiarrhoeal activity of the hydroethanolic leaf extract of Pupalia lappacea Linn. Juss. (Amaranthaceae) A.J. Akindele n, O.A. Salako, U.V. Ohonbamu Department of Pharmacology, Therapeutics & Toxicology (PTT), Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, P.M.B. 12003 Lagos, Nigeria
art ic l e i nf o
a b s t r a c t
Article history: Received 7 October 2013 Received in revised form 21 November 2013 Accepted 7 December 2013 Available online 14 December 2013
Ethnopharmacological Relevance: Pupalia lappacea is a medicinal plant found in savannah and woodland localities and forest path sides from Senegal to Southern Nigeria. It has been used in the management of diarrhoea in Nigerian traditional medicine. This study was designed to evaluate the antidiarrhoeal activity of the hydroethanolic leaf extract of Pupalia lappacea (PL). Materials and methods: The antidiarrhoeal activity of PL was evaluated using the normal and castor oilinduced intestinal transit, castor oil-induced diarrhoea, gastric emptying and intestinal fluid accumulation tests in rodents. Results: PL (100–400 mg/kg, p.o.) produced a significant dose-dependent decrease in normal and castor oil-induced intestinal transit compared with the control group (distilled water 10 ml/kg, p.o.). This effect was significantly (P o0.05) 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 and significant 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 the extract 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 the volume of intestinal content in the intestinal fluid accumulation test. Phytochemical analysis of the extract revealed the presence of alkaloids, saponins, and fixed oils and fats. The acute toxicity studies revealed 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 antidiarrhoeal activity possibly mediated by antimuscarinic receptor activity. & 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pupalia lappacea Antidiarrhoeal activity Castor oil Loperamide Intestinal transit
1. Introduction Diarrhoea is a symptom marked by rapid and frequent passage of semi-solid or liquid faecal material through the gastrointestinal tract. Various types of diarrhoea exist (based on their clinical manifestations), but it is broadly classified into acute and chronic for diagnostic and therapeutic purposes. Acute diarrhoea is the prevalent form of the disease which has a major impact on the morbidity and mortality worldwide in all age groups, particularly in infants and children under the age of three (Hirchhorn, 1980; Muriithi, 1996; Farthings, 2002). In less developed parts of the world (such as Latin America, India and Africa), children may experience between 3 and 10 episodes of diarrhoea yearly (Farthings, 2002). In 2009, diarrhoea was estimated to have caused
n
Corresponding author. Tel.: þ 234 8062359726. E-mail addresses: [email protected], [email protected] (A.J. Akindele). 0378-8741/$ - see front matter & 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.013
1.1 million deaths in people aged 5 and 1.5 million deaths in children under the age of 5 (World Health Organization, 2009). In terms of etiology, diarrhoea can be classified as infectious and non-infectious (de Hostos et al., 2011). Infectious diarrhoea is caused by a virus, parasite or bacterium while non-infectious diarrhoea can be caused by toxins, chronic diseases or antibiotics (NHDHHS, 2009). Pharmacological models of non-infectious diarrhoea were used in this study. Pupalia lappacea Linn. Juss. (Amaranthaceae) is a plant found in savannah and woodland localities and forest path sides from Senegal to Southern Nigeria, but less common in the western part of the region. Distribution is widespread elsewhere in tropical Africa and in Asia. Pupalia lappacea also commonly known as ram's bur and locally as “Kaimin kadangari” (Hausa, Northern Nigeria), “Agbiriba” (Igbo, South-east Nigeria) or “Emo agbo” (Yoruba, South-west Nigeria) has been used in traditional medicine for the treatment of cough, syphilis, skin diseases and diarrhoea (Odugbemi, 2008). The leaves mixed with palm-oil, are used in Ghana to treat boils (Agyare et al., 2009). The leaves are also used in topical applications to cuts, or used as enema or
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febrifuge (Agyare et al., 2009). In Ivory Coast, a decoction is taken in draught and applied in frictions for oedema of the legs and is used in various remedies for dysentery and diarrhoea. It is used as an antiemetic in the south-western region of Nigeria and the Republic of Benin (Adjanohoun and Akjakidje, 1989). Pupalia lappacea preparations have been reported to have antioxidant activity (Aladedunye and Okorie, 2008), anti-cancer activity (Ravi et al., 2012), antinociceptive and antipyretic activities (Neeharika et al., 2013). However, no scientific report of the antidiarrhoeal activity of Pupalia lappacea is available. The aim of this study is to evaluate the antidiarrhoeal effects of the hydroethanolic leaf extract of Pupalia lappacea (PL) in search for safe, effective and cheap remedies for diarrhoea management.
2.5. Phytochemical analysis
2. Materials and methods
2.5.2. Test for saponins 2 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 distilled water and the mixture was vigorously agitated to obtain a stable froth. The froth was mixed with three drops of olive oil and shaken vigorously. Observation was made for the formation of emulsion. In the haemolysis test, two test tubes were labelled A and B. 0.2 ml solution 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 B contained 0.2 ml of 10% blood in normal saline with 0.2 ml of normal saline. The observation of red supernatant in test tube A, absent in test tube B, confirmed the presence of saponins.
2.1. Reagents and drugs Castor oil (Finest Cold Drawn Commercial Castor oil, UK), loperamide hydrochloride (Imodiums, Janssen Pharmaceutical N.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 the farm of a traditional herbal practitioner in Ogun State, Nigeria, and the plant was identified and authenticated at the herbarium of the Department of Botany, University of Lagos, Lagos, Nigeria, by Mr. T.K. Odewo. A voucher specimen with reference number LUH 5092 was deposited in the herbarium of the department.
2.3. Experimental animals Adult albino Wistar mice (15–30 g) and rats (120–220 g) (8 weeks old) of either sex obtained from the Laboratory Animal Centre of the College of Medicine, University of Lagos, Lagos, Nigeria, were used for the experiments. Animals were maintained under standard environmental conditions (12 h light and 12 h dark cycle; 23–25 1C) and had free access to standard pelleted feed (RAAF Animal Feeds Ltd., Akute, Ogun State, Nigeria) and water adlibitum in accordance with the Guidelines for Care and Use of Laboratory Animals in Biomedical Research (National Academy of Sciences, 2011).
2.4. Preparation of extract The fresh leaves of the plant were collected and air-dried for a period of three weeks. The dried leaves were thereafter ground into powder with an electric blender. The powdered leaves were then macerated in hydroethanol (1:1; 50 g/L) for 48 h. Exhaustive extraction was done. Filtration was carried out after 48 h and the combined filtrate was evaporated to dryness under reduced pressure at 40 1C using a Heidolph rotavapor. The dried hydroethanolic extract had a sticky consistency which was readily soluble in water. It had a dark brown colour and pH of 5.2. The percentage yield of the extract was 3.25%. The dried extract obtained was kept in the refrigerator at 4 1C and reconstituted in distilled water prior to each experimental session.
A portion of the dried extract was used for phytochemical screening in order to determine the presence of pharmacologically active constituents using the methods described by Trease and Evans (1989) and Edeoga et al. (2005).
2.5.1. Test for alkaloids 0.5 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 treated with few drops of Mayer's, Dragendorff's, and Wagner's reagent. Observation of turbidity of precipitation was taken as indication for the presence of alkaloids.
2.5.3. Test for phlobatannins Observation of the deposition of a red precipitate upon the boiling of PL with 1% aqueous HCl was taken as indication for the presence of phlobatannins.
2.5.4. Test for reducing sugars 0.5 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 water bath. A brown colouration indicates the presence of reducing sugars.
2.5.5. Test for fixed oils and fats The filter paper and saponification tests were used in this respect. In the filter paper test, a small quantity of PL was pressed between two filter papers. The appearance of oil stain was taken as indication for the presence of fixed oils. In the saponification test, few drops of 0.5 M potassium hydroxide were added to a small quantity of PL along with a drop of phenolphthalein. On a water bath, the mixture was heated for 1–2 h. The presence of fixed oils and 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 divided doses, by oral intubation to a group. The other groups of mice were given the extract intraperitoneally at the dose of 400 and 800 mg/ kg respectively. The general symptoms of toxicity such as restlessness, panting and mortality in each group within 24 h were recorded. The LD50 was estimated using Miller and Tainter's log-probit analysis method (Adeyemi et al., 2010). Animals were further observed for one week for any delayed toxic effects.
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2.7. Normal intestinal transit
2.10. Measurement of gastric emptying
The method used was as described by Hsu (1982). Mice were allotted to five groups containing six animals each. Three groups were separately treated orally with the hydroethanolic extract (100, 200 and 400 mg/kg) while distilled water (10 ml/kg) and loperamide hydrochloride (5 mg/kg) were separately administered orally to the control and standard groups respectively. One hour after treatment, the mice were administered a standard charcoal meal (0.2 ml/mouse, made up of 5% charcoal suspension in 5% methylcellulose) orally. The mice were then sacrificed 30 min after administration of charcoal meal and the small intestine immediately isolated. Peristaltic index for each mouse was expressed as a percentage of the distance travelled by the charcoal meal relative to the total length of the small intestine (Aye-Than et al., 1989).
The method used was that described by Droppleman et al., 1980. Albino rats of either sex (previously fasted for 24 h prior to the experiment), were randomly allotted to two groups of six animals each. The test meal was prepared by mixing 10% charcoal suspension in 5% methylcellulose. Group one received distilled water (10 ml/kg, p.o.) while group two received hydroethanolic extract (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 an analytical balance. The stomach was opened and the stomach contents poured out and rinsed. Excess moisture was removed with cotton wool, and the empty stomach was weighed. The difference between full and empty stomach was subtracted from the weight of 3 ml of the test meal, indicating the quantity emptied from the stomach during the test period.
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 and 400 mg/kg) via the oral route. Another group was given loperamide hydrochloride (5 mg/kg) orally and the control group was given distilled water (10 ml/kg) orally. Thirty minutes later, all groups of mice were administered castor oil (0.2 ml/mouse). In respect of mechanism of action elucidation, four groups comprising 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 administration of hydroethanolic extract of PL at the dose of 400 mg/kg (most effective extract dose in this model). Thirty minutes after the administration of castor oil, the animals were given a standard charcoal meal (0.2 ml/mouse, made up of 5% charcoal suspension in 5% methylcellulose) orally. All the animals in each treatment group were sacrificed 30 min after the administration of the charcoal meal and the small intestine immediately isolated. The peristaltic index, which is the distance travelled by the charcoal meal relative to the total length of small intestine 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 control group was given distilled water (10 ml/kg) orally. Pre-treatment was done one hour before the administration of castor oil (0.2 ml/ mouse). Each mouse was kept for observation under a transparent funnel, the floor of which was lined with paper and observed for four hours (Izzo et al., 1992). The following parameters were observed: time elapsed between the administration of the castor oil and the excretion of the first diarrhoea faeces, the total amount of hard stool, semi-solid stool and watery stool. The weight of watery stool (which comprises of the 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 was assigned: 1 (hard stool), 2 (semi-solid stool), and 3 (watery stool). The in vivo antidiarrhoeal index (ADIin vivo) was expressed according pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi to the formula: ADIin vivo ¼ 3 Dfreq Gmeq P freq (Aye-Than et al., 1989; Adeyemi and Akindele, 2008), where Dfreq is the delay in defaecation time or diarrhoea onset (in % of control), Gmeq is the gut meal travel reduction (in % of control), and Pfreq is the purging frequency, as number of stool reduction (in % of control).
2.11. Intestinal fluid accumulation Albino rats were divided into two groups of six rats each. Intestinal fluid accumulation was induced by oral administration of castor oil (2 ml/rat) (Robert et al., 1976). The rats were fasted for 24 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) was administered intragastrically and 1 h after that, the rats were sacrificed. The rats were then dissected and their small intestines were immediately isolated after ligation at the pyloric sphincter and at the ileo-caecal junction respectively. Each intestine was weighed and the intestinal contents were expelled into measuring cylinder and the volume determined. The intestine was reweighed and the difference between the full and empty intestine was determined. 2.12. Statistical analysis Results were expressed as mean 7SEM. Statistical analysis of the data was done using One-way ANOVA followed by the Tukey's multiple comparison test or Unpaired Student's t-test, where appropriate. Results were considered significant when P o0.05.
3. Results 3.1. Phytochemical analysis Phytochemical analysis carried out revealed the presence of alkaloids, saponins, and fixed oils and fats, and absence of phlobatannins and reducing sugars. 3.2. Acute toxicity study in mice The hydroethanolic extract administered to animals by the oral route did not produce any visible signs of toxicity and no mortality was recorded at a dose of 10,000 mg/kg. The animals were kept under observation for additional seven days and this showed no delayed toxic effect. Administration of the extract via the intraperitoneal route produced writhing and calming of the animals especially at the dose of 800 mg/kg. Mortality was 20% at the dose of 800 mg/kg. This implies that the LD50 for the intraperitoneal route is 4800 mg/kg.
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3.3. Normal intestinal transit In control animals, the charcoal meal traversed 72.577 0.63% of the total length of the small intestine. The hydroethanolic extract (100–400 mg/kg) produced a dose-dependent decrease in the intestinal transit with the peak effect of 34.78% inhibition at 400 mg/kg compared to loperamide (5 mg/kg) with inhibition value of 66.93% (Table 1). 3.4. Castor oil-induced intestinal transit The hydroethanolic extract and loperamide (5 mg/kg) caused significant (Po 0.001) reduction in the distance travelled by the charcoal meal in the small intestine, relative to the total length of Table 1 Effect of Pupalia lappacea on normal intestinal transit. Group
Dose (mg/kg)
Peristaltic index (%)
Inhibition (%)
Control PL PL PL Loperamide
– 100 200 400 5
72.577 0.63nnn 66.177 0.53nnn 63.487 0.46nnn 47.337 0.38nnn 24.007 0.48nnn,a
– 8.82 12.53 34.78 66.93
Values are mean7 SEM (n ¼6). nnn
Po 0.001 vs. Control. P o0.001 vs. PL (400 mg/kg) (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL¼ Pupalia lappacea. a
Dose (mg/kg)
Control PL PL PL PL þ Yohimbine PL þ Prazosin PL þ Propranolol PL þ Pilocarpine Loperamide
100 200 400 400 10 400 1 400 1 400 1 5
the small intestine, compared to the control mice. The highest percentage inhibition produced by the extract (44.60%) was seen at the dose of 400 mg/kg and this was significantly (P o0.01) less than the effect (63.84%) produced by loperamide (5 mg/kg). Also, there was no significant difference when the peristaltic index of the extract at 400 mg/kg was compared with that of yohimbineþ extract (400 mg/kg), prazosinþextract (400 mg/kg) and propranolol þextract (400 mg/kg). However, there was a significant (P o0.05) difference when the peristaltic index of the extract (400 mg/kg) was compared with that of pilocarpine þextract 400 mg/kg (Table 2). 3.5. Castor oil-induced diarrhoea As shown in Table 3, 4 h after the administration of castor oil all groups produced watery stool. However, there was a dosedependent significant (P o0.001) delay in the onset of diarrhoea produced by the hydroethanolic extract with the peak effect produced at the dose of 400 mg/kg (173.67 min) compared with control (59.33 min.). Similarly, there was reduction in the number of wet stools, total number of stools, total weight of wet stools and total weight of all stools and diarrhoea score for all doses of the hydroethanolic extract when compared with control. Also, the hydroethanolic extract produced its highest percentage protection in terms of reduction in diarrhoea score and in vivo antidiarrhoeal index at the dose of 400 mg/kg (62.68% and 56.95% respectively), but this was less than that produced by loperamide (84.69% and 77.75% respectively). 3.6. Gastric emptying
Table 2 Effect of Pupalia lappacea on castor oil-induced intestinal transit. Group
987
Peristaltic index (%)
Inhibition (%)
82.06 56.05nnn 53.36nnn 45.46nnn,a 44.05
– 31.70 34.97 44.60 46.32
34.00
58.57
44.99
45.18
60.28
26.54
29.67nnn,b
63.84
3.7. Intestinal fluid accumulation In the fluid accumulation test, the extract at 400 mg/kg significantly (P o0.01) reduced both the weight and volume of Table 4 Effect of Pupalia lappacea on gastric emptying.
Values are mean7 SEM (n ¼6). a
As shown in Table 4, the extract (400 mg/kg) significantly (P o0.01) increased the quantity of test meal emptied (2.08 70.10 g) compared to that of control (1.56 7 0.10 g).
P o0.05 vs. PLþ Pilocarpine. b Po 0.01 vs. PL (400 mg/kg). nnn Po 0.001 vs. Control (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL¼Pupalia lappacea.
Group
Dose (mg/kg)
Weight of intestinal content (g)
Quantity of intestinal content emptied (g)
Control PL
– 400
1.49 70.10 0.97 70.10nn
1.56 7 0.10 2.08 7 0.10nn
Values are mean 7 SEM (n¼ 6). nn
Po 0.01 vs. Control (Unpaired Student's t-test.) PL¼Pupalia lappacea.
Table 3 Effect of Pupalia lappacea in castor oil-induced diarrhoea in mice. Group
Dose (mg/kg)
Control PL 100 PL 200 PL 400 Loperamide 5
Onset of diarrhoea (min)
Number of wet stools
Total number of stools
Total weight of wet stools (g)
59.337 2.68 129.50 7 4.99nnn 137.83 7 9.16nnn 173.677 6.17nnn 218.337 5.24nnn,a
9.177 0.83 7.50 7 0.76 6.50 7 0.67 3.177 0.48nnn 1.50 7 0.43nnn
13.83 7 0.31 0.34 7 0.03 10.007 0.86nn 0.25 7 0.03 nnn 9.177 0.79 0.22 7 0.02nn 5.50 7 0.76nnn 0.157 0.01nnn 2.177 0.40nnn 0.137 0.01nnn
Total weight of all stools (g)
Diarrhoea Score
Percentage protection
In vivo antidiarrhoeal index (%)
0.38 7 0.03 0.28 7 0.02n 0.25 7 0.02nn 0.177 0.01nnn 0.147 0.01nnn
34.83 7 1.40 26.337 2.39n 23.677 2.26nn 13.007 2.01nnn 5.337 1.28nnn
– 24.40 32.06 62.68 84.69
– 28.18 35.36 56.95 77.75
Values are mean7 SEM (n ¼6). n
Po 0.05. Po 0.01. nnn Po 0.001 vs. Control. a P o0.001 vs. PL 400 mg/kg (One way ANOVA followed by the Tukey's Multiple Comparison Test). PL ¼Pupalia lappacea. nn
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Table 5 Effect of Pupalia lappacea on intestinal fluid accumulation. Group
Dose (mg/kg)
Weight of intestinal content (g)
Volume of intestinal content (ml)
Control PL
– 400
2.05 7 0.14 1.53 7 0.26
1.65 7 0.08 1.187 0.09nn
Values are mean7 SEM (n ¼6). nn
Po 0.01 vs. Control (Unpaired Student's t-test). PL¼ Pupalia lappacea.
intestinal content (1.53 7 0.26 g and 1.18 70.09 ml respectively) compared with that of control (2.05 7 0.14 g and 1.65 70.08 ml respectively) as shown in Table 5.
4. Discussion Diarrhoea is the frequent passage of liquid faeces and it involves both an increase in the motility of the gastrointestinal tract, along with increased secretion and decreased absorption of fluid, and loss of electrolytes (particularly sodium) and water (Rang et al., 2003). Therefore, to restore personal comfort and convenience, many patients require antidiarrhoeal therapy and treatment is carried out to achieve, among other objectives, increased resistance to flow (segmental contraction, decreased propulsion and peristalsis) and increased mucosal absorption or decreased secretion (Burks, 1991; Akindele and Adeyemi, 2006). Approaches that are used in the treatment of severe acute diarrhoea include maintenance of fluid and electrolyte balance, use of anti-infective agents, and use of spasmolytic or other antidiarrhoeal agents (Rang et al., 2007). In this context, the investigation of the antidiarrhoeal effect of Pupalia lappacea in this study comprised the evaluation of its effect on intestinal transit and fluid accumulation. Its effect on gastric emptying was also investigated based on the fact that the action of drugs like atropine and morphine in reducing intestinal propulsion is aided by their ability to delay gastric emptying (Izzo et al., 1999; Akindele and Adeyemi, 2006). Major classes of drugs used in the pharmacologic therapy of diarrhoea include antimotility and spasmolytic agents (e.g. opiates like diphenoxylate and loperamide, and muscarinic receptor antagonists like atropine, hyoscine and propantheline); antisecretory agents (e.g. bismuth compounds and octreotide); adsorbents and bulk agents including preparations containing kaolin, chalk, charcoal, methylcellulose and activated attapulgite; bile acid sequestrants (e.g. cholestyramine, colestipol and colesevalam); probiotics; anti-infectives (e.g. fluoroquinolones [ciprofloxacin or levofloxacin], azithromycin and erythromycin); and miscellaneous agents like α2--adrenergic receptor antagonists (e.g. clonidine), calcium channel blockers (e.g. verapamil and nifedipine), berberine (plant alkaloid with antimicrobial, antimotility and antisecretory effects) and calmodulin inhibitors (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 their findings reported that herbal treatments remain important as home remedy for diarrhoea and that despite the availability of simple and cheap treatments for diarrhoea (ORT), healers and patients in many communities still rely on locally available phytomedicines. The phytochemical analysis of the hydroethanolic leaf extract of Pupalia lappacea revealed the presence of bioactive compounds such as alkaloids, saponins, and fixed oils and fats. Some of these phytoconstituents have been reported to have inhibitory activity on intestinal motility in a dose related manner (Carlo et al., 1994).
The acute toxicity study also revealed that the extract is safe when administered orally, having produced neither obvious morbidity nor mortality at a dose of 10,000 mg/kg (Clarke and Clarke, 1977). However, administration through the intraperitoneal route caused mortality with a median lethal dose greater than 800 mg/kg since only 20% mortality was recorded at this dose. Also, the animals exhibited certain characteristics like calming, suggesting that the extract might have sedative properties. The extract produced a dose-dependent decrease in the propulsive movement of the standard charcoal meal in the small intestine both in the normal and castor oil-induced intestinal transit models. In both models, the effect was observed to peak at the dose of 400 mg/kg (34.78% and 44.60% inhibition respectively). This inhibitory action of the extract on intestinal transit will delay the passage of gastrointestinal contents allowing faeces to become desiccated, thus further retarding movement through the colon. The extract was more effective in the castor-oil induced intestinal transit test than in the normal intestinal transit test, suggesting that the extract might be more effective in a diseased state than in a normal state. Yohimbine (an α2-adrenergic receptor antagonist), prazosin (an α1-adrenergic receptor antagonist), propranolol (a non-selective beta receptor antagonist) and pilocarpine (a nonselective muscarinic receptor agonist) were employed in this study to elucidate the mechanism of action of Pupalia lappacea using the castor oil-induced intestinal transit model. Yohimbine, prazosin and propranolol had no significant influence on the action of the extract (400 mg/kg) on intestinal propulsion. This suggests that the extract had little or no activity on alpha and beta adrenergic receptors. However, pilocarpine significantly altered the inhibitory action of the extract (400 mg/kg) on intestinal propulsion. This suggests that the extract probably has muscarinic receptor activity. Going by the results obtained in the castor oil-induced diarrhoea test, the hydroethanolic leaf extract of Pupalia lappacea caused a dose-dependent significant delay in the onset of diarrhoea, decrease in frequency of purging (reduction of number of wet stools), decrease in weight of wet stools and decrease in severity of diarrhoea (diarrhoea score). Also, the extract increased the amount of test meal emptied in the gastric emptying model, and this suggests that unlike atropine, the effect of the extract on intestinal propulsion may not be due to delay in gastric emptying time. 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 is summed up by the calculation of the ADIin vivo (in vivo antidiarrhoeal index). The higher the ADIin vivo value, the greater the effectiveness in the treatment of diarrhoea. The extract produced a dose-dependent increase in ADIin vivo with a maximum of 56.95% produced at the dose of 400 mg/kg, a value lower than that elicited by loperamide 5 mg/kg (77.75%). 5. Conclusion The results obtained in this study suggest that the hydroethanolic leaf extract of Pupalia lappacea possesses antidiarrhoeal property through antimotility and antisecretory effects possibly mediated by its activity on muscarinic receptors. This validates the claims made by traditional medicine practitioners about its possession of antidiarrhoeal activity. Further studies on the plant extract would entail the evaluation of antimicrobial effects.
Acknowledgements The authors are grateful to Mr. M. Chijioke, of the Department of Pharmacology, Therapeutics and Toxicology, for his technical assistance during the study.
A.J. Akindele et al. / Journal of Ethnopharmacology 151 (2014) 984–989
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