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Journal of Ethnopharmacology
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Antidiarrheal activity of Pyrenacantha staudtii Engl. (Iccacinaceae) aqueous leaf extract in rodents
Emmanuel O. Awe , Simeon O. Kolawole, Kamoru O. Wakeel, Oyindamola O. Abiodun
Department of Pharmacology & Therapeutics, College of Health Sciences, Ladoke Akintola University of Technology, P.M.B. 4400 Ogbomoso, Nigeria

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relervance: Pyrenacantha staudtii Engl. (Icacinaceae) is a plant which is traditionally used for the treatment of blemnorrhea, hernia, insomnia, intestinal pain and diarrhea in Nigeria. Therefore the core aim of the present study is to evaluate antidiarrheal activity of Pyrenacantha staudtii aqueous extract (PSE). Materials and methods: The antidiarrheal activity was evaluated using castor oil-induced diarrhea method. The effects of Pyrenacantha staudtii aqueous extract on gastrointestinal motility, intestinal transit and enteropooling were also examined in rodents. The acute toxicity effect of the aqueous extract of Pyrenacantha staudtii was also investigated. Results: Pyrenacantha staudtii aqueous extract (PSE, 100400 mg/kg, p.o.) produced dose-dependent and signicant (P < 0.050.01) protection of rats and mice against castor oil-induced diarrhea, inhibited intestinal transit, and delayed gastric emptying. PSE, produced dose-dependent and signicant (P < 0.050.01) antimotility effect, caused dose-related inhibition of castor-oil-induced enteropooling in animals, comparable to atropine (1 mg/kg, p.o.). Like loperamide (10 mg/kg, p.o.), PSE, dose-dependently and signicantly (P < 0.050.01) delayed the onset of castor-oil induced diarrhea, decreased the frequency of defecation, and reduced the severity of diarrhea in the rodents. Compared with control animals, PSE, dose-dependently and signicantly (P < 0.050.01) decreased the volume of castor oil-induced intestinal uid secretion, and reduced the number, weight and wetness of fecal droppings. Conclusion: The ndings of this study indicate that PSE possesses antidiarrheal property in rats and mice. These ndings conrm the ethnomedicinal use of Pyrenacantha staudtii leaf as a valuable natural remedy for the treatment, management and/or control of diarrhea. 2011 Elsevier Ireland Ltd. All rights reserved.

Article history: Received 11 January 2011 Received in revised form 13 April 2011 Accepted 29 April 2011 Available online xxx Keywords: Pyrenacantha staudtii Engl. Icacinaceae Aqueous extract Antidiarrheal activity

1. Introduction Since ancient times, diarrhea has been recognized as one of the most important health problems aficting mankind, particularly those populations in socio-economically backward, and developing, third-world countries. Globally, diarrhea has been estimated to kill about 2.2 million people annually, the majority of whom are infants and children below the age of 5 years (Venkatesan et al., 2005; Gutierrez et al., 2008). Diarrhea is a disorder characterized by discharge of semisolid or watery fecal matter from the bowel three or more times in a day (Hirchhorn, 1980; Snyder and Merson, 1982; Mbagwu and Adeyemi, 2008; Suleiman et al., 2008). It involves an increase in the uidity, volume and frequency of bowel movements, increased frequency of bowel sound, wet stools and abdominal pain, accompanied by increased secretion and decreased absorp-

Corresponding author. Tel.: +234 07061368413. E-mail address: awemman@yahoo.co.uk (E.O. Awe). 0378-8741/$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.04.068

tion of uid, and thus loss of water and electrolytes (Fontaine, 1988; Field et al., 1989; Avery and Synder, 1990; Longe and Dipiro, 1999). Generally, the treatment of diarrhea is non-specic, and is usually aimed at reducing the discomfort and inconvenience of frequent bowel movements (Brunton, 2008; Suleiman et al., 2008). In order to overcome the menace of diarrhea in developing countries, especially the discomfort and inconvenience of frequent bowel movements, the World Health Organization (WHO) has introduced a program for diarrheal control which involves the use of traditional herbal medicines. Several African medicinal plants have been reported to be useful in the treatment, management and/or control of diarrhea (Abdullahi et al., 2001; Aniagu et al., 2005; Agunu et al., 2005; Suleiman et al., 2008; Adeyemi and Akindele, 2008; Mbagwu and Adeyemi, 2008). Pyrenacantha staudtii (Engl.) Engl. (Icacinaceae) is called Ahara in Yoruba, nhia in Igbo and Ohogbo among Edos of Nigeria. The plant occurs as an arborescent, 6 m long and 510 cm in diameter. It is widely distributed in the tropical Africa and found in secondary jungles of southern Nigeria, Western Cameroon and across central

Please cite this article in press as: Awe, E.O., et al., Antidiarrheal activity of Pyrenacantha staudtii Engl. (Iccacinaceae) aqueous leaf extract in rodents. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.04.068

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Africa to Uganda and Angola (Daziel, 1948). Traditionally, the plant is used for the treatment of blemnorrhea, intestinal pain and hernia (Daziel, 1948; Oliver-Bever, 1986; Iwu, 1993), and diarrhea (Samuel Olasore, Personal communication). The leaf extract of the plant has been reported for its anti-inammatory and antiulcerogenic activities (Akubue et al., 1983), smooth muscle relaxant activity (Aguwa and Okunji, 1986), anticonvulsant and analgesic activities (Awe et al., 2005). Phytochemically the plant extract was reported to contain saponins and alkaloids (Okunji and Iwu, 1988). The core aim of the present study was, therefore, to examine the antidiarrheal activity of Pyrenacantha staudtii in rodents. 2. Materials and methods 2.1. Ethical considerations Experimental procedures and protocols used in this study were approved by the Ethics committee of the Ladoke Akintola University of Technology, Nigeria and conform to the Guide to the care and use of animals in research and teaching (NIH publications number 85-93 revised in 1985) 2.2. Plant materials Fresh leaves of Pyrenacantha staudtii Engl. (Icacinaceae) were collected at Ilesha in the southern part of Nigeria in June 2008. The leaves were identied by Mr. TK Odewo, a taxonomist at the herbarium of the Federal Institute of Forest Research Ibadan, where voucher specimen was deposited with voucher number 106886. 2.3. Preparation of plant extract Pyrenacantha staudtii fresh leaves (1 kg) were air-dried at room temperature. The air-dried leaves of the plant were milled into ne powder in a Waring commercial blender. The powdered leaf was macerated in distilled water and extracted twice, on each occasion with 2.5 l of distilled water at room temperature (27 1 C) for 48 h (with occasional shaking). The combined aqueous extract solubles were ltered and concentrated to dryness under reduced pressure at 60 1 C in a rotary evaporator. Freezedrying and solvent elimination of the resulting aqueous extract nally gave 35.23 g (i.e., 3.52% yields) of a light brown, powdery crude Pyrenacantha staudtii leaf aqueous extract (PSE). Without any further purication, aliquot portions of the plant crude extract residue were weighed and dissolved in distilled water (at room temperature) for use on each day of our experiments. 2.4. Animals Healthy, male and female Balb C mice (Mus domesticus) weighing 2025 g, and healthy young adult, Wistar rats (Rattus norvegicus) of both sexes weighing 250300 g, were used. The animals were kept and maintained under laboratory conditions of temperature and humidity with a 12 h light/dark cycles. They were allowed free access to food (standard pellet diet) and drinking tap water ad libitum. The animals were divided into plant extract, reference drug-treated test and distilled water-treated control groups of 8 animals per group. All the animals were fasted for 16 h, but still allowed free access to tap water, before the commencement of our experiments. 2.5. Acute toxicity testing The median lethal dose (LD50) of PSE was determined in the mice, using a modied method of Lorke (1983). Mice fasted for 16 h were randomly divided into groups of 8 mice per group. Graded

doses of PSE (100, 200, 400, 800, 1600 and 3200 mg/kg) were separately administered orally (p.o.) to the mice in each of the test groups. Each of the mice in the control group was treated with distilled water (10 ml/kg p.o.) only. The mice in both the test and control groups were then allowed free access to food and water, and observed over a period of 48 h for signs of acute toxicity. Deaths (caused by the extract) within this period was noted and doseresponse plots were constructed from which the median lethal dose (LD50) of the leaf aqueous extract was determined, following the method of Bliss (1935). 2.6. Evaluation of antidiarrheal activity of PSE 2.6.1. Castor oil-induced diarrhea in rats Diarrhea was induced in rats by oral administration of castor oil (3 ml/kg, p.o.) as previously described by Awouters et al. (1978), Mukherjee et al. (1998), Aniagu et al. (2005), Mbagwu and Adeyemi (2008) and Suleiman et al. (2008) with some modications. Before the commencement of our experiments, the rats used were fasted for 16 h (during which time the animals were given free access to drinking tap water ad libitum) and randomly allocated into six groups of 6 rats per group. Each rat was subsequently separately placed in a plastic cage lined at the bottom with a transparent sheet. Group 1 rats were individually treated with 10 ml/kg (p.o.) of distilled water (the vehicle in which PSE and loperamide were dissolved) only. Group 25 rats received 10 ml/kg (p.o.) of Pyrenacantha staudtii leaf aqueous extract equivalent to PSE doses of 100, 200 and 400 mg/kg, respectively. Group 6 rats were individually treated with 10 mg/kg (p.o.) of loperamide, a standard antidiarrheal agent. Thirty minutes after oral pretreatment of the animals with distilled water, doses of PSE and loperamide, each rat in each of the six groups was given 3 ml/kg of castor oil orally. Thereafter, the animals were observed for 6 h for the presence of characteristic diarrheal droppings. The following parameters were observed: the time elapsed between the administration of castor oil and the excretion of the rst diarrheic feces, the total number of fecal output and the number of diarrheic stools excreted by each animal in 6 h, as well as the total weight of the diarrheic feces in that period of time. Severity of the castor-oil induced diarrhea was noted and recorded as scores. A numerical score based on stool consistency, using the method of Dicarlo et al. (1994), was assigned as follows: normal stool (or lack of diarrhea) = 1, semi-solid stool = 2, and watery stool/feces = 3, respectively. The corresponding percentages and purging indices were calculated, the latter by comparison with the control group of rats. The in vivo antidiarrheal index (ADI in vivo) was thereafter calculated according to the formula described in detail earlier by Aye-Than et al. (1989), and Mbagwu and Adeyemi (2008). The onset of diarrhea is measured as the time interval (in minutes) between the administration of castor oil and the appearance of the rst diarrheic stool. The fecal matter was collected and weighed over the entire observation period of 6 h. The changes in body weight between the pre- and post-experiment periods were also determined. Percentage inhibition of defecation score and fecal weight were calculated by using [control mean treated(test)mean] 100] control mean 2.6.2. Castor oil-induced enteropooling, intestinal transit and intestinal uid in rats As described above, the rats used were fasted for 16 h (during which time the animals were given free access to drinking tap water ad libitum) and randomly allocated into six groups of 6 rats per group. Each rat in each group was subsequently separately placed in a plastic cage lined at the bottom with a transparent sheet. Group

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1 rats were individually treated with 10 ml/kg (p.o.) of distilled water (the vehicle in which PSE was dissolved) only. Groups 25 rats received 10 ml/kg (p.o.) of Pyrenacantha staudtii leaf aqueous extract equivalent to PSE doses of 100, 200 and 400 mg/kg, respectively, followed immediately by oral administration of 3 ml/kg of castor oil. Group 6 rats were individually treated with 10 mg/kg (p.o.) of loperamide, followed by oral administration of 3 ml/kg of castor oil. Thirty minutes following oral treatments of the animals with distilled water, graded doses of PSE, loperamide and castor oil, each animal in the six groups was given 3 ml/kg (p.o.) of 10% activated charcoal in physiological saline (0.9%, w/v sodium chloride solution). The intestinal transit and volume of intestinal uid in the rats were then measured as described in detail by Dicarlo et al. (1994), with some modications. Forty minutes after charcoal meal, the rats were anaesthetized by ether inhalation and euthanized by cervical dislocation. The abdomen of each rat was cut open, and the whole length of the intestine from the pylorus to the caecum was ligated, dissected and carefully removed. The distance of the charcoal plug from pylorus to caecum, i.e., peristaltic index (PI) which is the distance traveled by charcoal meal relative to the entire length of the small intestine was determined and expressed as a percentage of the total length of the small intestine. The weight of the full intestine was determined. The intestinal content was expelled into a graduated measuring cylinder and its volume was determined. The weight of the empty intestine was taken, and the difference between the full and empty intestine was thus calculated. Percentage inhibitions of intestinal transit of the charcoal meal by loperamide (10 mg/kg, p.o.) and PSE (100, 200 and 400 mg/kg, p.o.) were calculated as a function of the distilled water-treated negative control by using the formula: percentage transit inhibition = To Tt 100 To

in normal saline) orally. The animals were subsequently anaesthetized by ether inhalation and euthanized by cervical dislocation 30 min later, and the abdomen opened. The small intestine of each mouse was dissected out from the pylorus to the caecum, and the total distance traversed by the charcoal plug along the small intestine was determined in both the control and treated test mice groups (Aniagu et al., 2005). For each animal group, the results were expressed as percentage of the distance traveled from the pylorus to the caecum (Gamaniel and Akah, 1996). 2.7. Data and statistical analysis Data obtained are presented as means (S.E.M.) for the number of animals in each group (n = 6). Data obtained from distilled water-treated control mice and rats were used as baseline values. In all cases, the results obtained from extract- and reference drugtreated test animal groups were compared with those obtained from distilled water (vehicle)-treated control animal groups. The differences between the data obtained from test animal groups and the data obtained from vehicle-treated control animal groups, were subjected to one-way analysis of variance (ANOVA); 95% condence interval and followed by Dunnetts post hoc test (Dunnett and Goldsmith, 1993). In all cases, statistical signicance was established at values of P 0.05. 3. Results 3.1. Acute toxicity study Oral administrations of graded doses of Pyrenacantha staudtii leaf aqueous extract in mice gave an LD50 value of 2 155 80 mg/kg. 3.2. Castor oil-induced diarrhea in rats Three-and-a-half hours after oral administration of castor oil, all the rats in the control group of animals produced copious diarrhea. Pretreatment of the rats (in the treated test groups) with Pyrenacantha staudtii leaf aqueous extract (PSE, 100, 200 and 400 mg/kg, p.o.) dose dependently and signicantly (P < 0.050.01) delayed the onset of diarrhea, reduced the frequency of diarrhea, reduced the frequency of defecation and the wetness of the fecal droppings (reduction in the number of wet stools and total stools), and decreased the weight of wet stools and the general diarrhea score, including the hard, mild and copious stools (Table 1). The standard antidiarrheal drug, loperamide (10 mg/kg, p.o.), produced a more marked, signicantly greater (P < 0.001) inhibitory effects on all the diarrheal parameters examined than the highest dose of PSE (400 mg/kg, p.o.) used (Table 1). 3.3. Normal intestinal transit in mice In the control group of mice, charcoal meal traversed the farthest distance of the total length of small intestine. In the Pyrenacantha staudtii leaf aqueous extract-treated test groups of mice, PSE (100, 200 and 400 mg/kg, p.o.) dose-dependently and signicantly (P < 0.050.01) decreased the normal intestinal propulsive movement and transit of charcoal meal through the small intestine (Table 2). Loperamide (10 mg/kg, p.o.) produced greater antimotility effect than the highest dose of PSE (400 mg/kg, p.o.) used (Table 2). 3.4. Castor oil-induced intestinal transit in rats In the control group of rats used, charcoal meal traveled faster than in the control group of mice used for the above normal intestinal transit study. Thirty minutes after intra-gastric administration in the control group of rats, the charcoal meal traversed 94.13%

where To is the mean length traversed by charcoal meal in distilled water-treated control rats; and Tt is the mean length traversed by charcoal meal in PSE- and loperamide-treated test rats. 2.6.3. Castor oil-induced enteropooling, intestinal transit and intestinal uid in mice The experimental procedures and protocols described in detail above were repeated using adult Balb C mice of both sexes (2530 g). Percentage inhibitions of intestinal transit of the charcoal meal by loperamide (10 mg/kg, p.o.) and PSE (100. 200 and 400 mg/kg, p.o.) were calculated as a function of the distilled watertreated negative control by using the formula: percentage transit inhibition = To Tt To 100

where To is the mean length traversed by charcoal meal in distilled water-treated control mice; and Tt is the mean length traversed by charcoal meal in PSE- and loperamide-treated test mice. 2.6.4. Gastrointestinal motility of Pyrenacantha staudtii against atropine in mice. Balb C adult mice of both sexes (weighing 2530 g) were randomly divided into six groups of 6 mice each. Prior to the experiments, the mice were fasted for 16 h (during which time the animals were given free access to drinking tap water ad libitum). Group 1 mice individually received distilled water (10 ml/kg, p.o.) only, while Groups 25 mice individually received 10 ml/kg (p.o.) of Pyrenacantha staudtii leaf aqueous extract equivalent to PSE doses of 100, 200 and 400 mg/kg, respectively. Group 6 mice were treated with atropine sulphate (1 mg/kg, p.o.) Thirty minutes after distilled water, PSE, or atropine sulphate administration, each mouse was given 3 ml/kg of activated charcoal meal (10% activated charcoal

Please cite this article in press as: Awe, E.O., et al., Antidiarrheal activity of Pyrenacantha staudtii Engl. (Iccacinaceae) aqueous leaf extract in rodents. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.04.068

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E.O. Awe et al. / Journal of Ethnopharmacology xxx (2011) xxxxxx Dose (ml/kg) or (mg/kg) 10 100 200 400 10 Total no. of stools 13.5 4.3 7.0 2.5 4.8 1.1 2.8 0.7 0.0 Rats with wet stools (%) 90.20 50.00 34.14 9.80 0.0 No. of wet stools 10.9 3.6 6.4 1.9 3.1 0.9 1.9 0.6 0.0 Percentage protected 0.0 50.00* 64.60** 90.20*** 100.00***

Table 1 Effects of Pyrenacantha staudtii leaf aqueous extract (PSE, 50400 mg/kg p.o.) on castor oil-induced diarrhea in rats. Treatment group Control (distilled water) Extract

Loperamide

One-way ANOVA + Dunnetts post hoc test n = 6. * P < 0.05. ** P < 0.01. *** P < 0.001 vs control. Table 2 Effects of Pyrenacantha staudtii leaf aqueous extract (PSE, 50400 mg/kg p.o.) on normal intestinal transit in mice. Treatment Control (distilled water) Extract Dose (ml/kg) 10 100 200 400 10 Peristaltic index (%) 86.88 62.80 43.73 20.92 4.79 Inhibition of transit (%) 0.0 27.89* 50.29* 76.66** 95.30***

Loperamide One-way ANOVA + Dunnett s post hoc test n = 6. * P < 0.05. ** P < 0.01. *** P < 0.001 vs control.

Table 3 Effects of Pyrenacantha staudtii leaf aqueous extract (PSE, 50400 mg/kg p.o.) on normal intestinal transit in rats. Treatment Control (distilled water) Extract Dose (ml/kg) 10 100 200 400 10 Peristaltic index (%) 93.23 62.54 28.63 12.85 3.85 Inhibition of transit (%) 0.0 43.51* 69.40** 86.49** 94.24***

Loperamide One-way ANOVA + Dunnett s post hoc test n = 6. * P < 0.05. ** P < 0.01. *** P < 0.001 vs control.

of the total length of the small intestine (Table 3). In the PSEtreated test groups of rats, PSE (100, 200 and 400 mg/kg, p.o.) dose-dependently and signicantly (P < 0.050.01) decreased the propulsive movement and transit of charcoal meal through the small intestine. Loperamide (10 mg/kg, p.o.) produced greater antimotility effect than the highest dose of PSE (400 mg/kg, p.o.) used (Table 3). 3.5. Intestinal motility and transit in mice (charcoal study) Compared with the control group of animals, in the treated test groups of mice, PSE (100, 200 and 400 mg/kg, p.o.) dose-dependently and signicantly (P < 0.050.01) decreased the propulsive movement and transit of charcoal meal through the gastrointestinal tract (GIT) Atropine sulphate (1 mg/kg, p.o.) produced greater antimotility effect than the highest dose of PSE (400 mg/kg, p.o.) used (Table 4).

3.6. Castor oil-induced enteropooling in rats Oral administration of castor oil (3 ml/kg, p.o.) produced a marked and signicant (P < 0.01) increase in the intestinal uid volume of castor oil-treated groups of rats compared to control group of animals treated with distilled water (10 ml/kg, p.o.) only. Compared with the control group of rats, pretreatment of the test groups of rats with PSE (100, 200 and 400 mg/kg, p.o.) dose-dependently and signicantly (P < 0.050.01) inhibited castor oil-induced uid accumulation in rats (Table 5). The standard antidiarrheal drug, loperamide (10 mg/kg, p.o.), produced a more marked and significantly greater (P < 0.001) inhibitory effects on castor oil-induced uid accumulation than the highest dose of PSE (400 mg/kg, p.o.) used (Table 5). The intestinal uids of the animals pretreated with the aqueous extract (PSE) and loperamide were found to be more viscous than those of the distilled water-treated control rats.

Table 4 Effects of Pyrenacantha staudtii leaf aqueous extract (PSE, 100400 mg/kg p.o.) on castor oil-induced intestinal propulsion in rats (Charcoal meal study). Treatment Control (distilled water) Extract Dose (mg/kg) 10 100 200 400 1 Intestinal length (cm) 41.4 41.6 41.9 40.7 41.2 1.4 1.80 1.92 1.75 1.56 Distance traveled by CP 37.2 27.6 21.3 15.1 12.6 4.10 2.78 1.66 1.25 0.56 P.I. (%) 40.71 62.40 49.82 36.63 28.42 Inhibition of motility (%) 0.00 31.80* 45.07* 59.60** 68.73**

Atropine

ANOVA + Dunnett s post hoc test n = 6. C.P. = Charcoal plug; P.I. = Peristaltic index. * P < 0.05. ** P < 0.01 vs control.

Please cite this article in press as: Awe, E.O., et al., Antidiarrheal activity of Pyrenacantha staudtii Engl. (Iccacinaceae) aqueous leaf extract in rodents. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.04.068

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E.O. Awe et al. / Journal of Ethnopharmacology xxx (2011) xxxxxx 5 Dose (ml/kg) or (mg/kg) 3 100 200 1.2 0.2 10 Intestinal uid (ml) 5.3 1.6 3.3 0.6 2.3 0.4 77.5** 0.5 0.1 Inhibition of Intestinal uid volume (ml) (%) 0.0 37.9* 56.9* 90.6***

Table 5 Effects of Pyrenacantha staudtii leaf aqueous extract (PSE, 50400 mg/kg p.o.) on castor oil-induced intestinal enteropooling and uid accumulation in mice. Treatment Control (Castor oil) Extract v400 Loperamide

One-way ANOVA + Dunnett s post hoc test n = 6. * P < 0.05. ** P < 0.01. *** P < 0.001 vs control.

4. Discussion The LD50 value of 2 155 80 mg/kg, p.o., obtained for PSE in the mice used for acute toxicity study probably suggests that the plant extract is relatively safe in, or non-toxic to, mice. However, the results of the present laboratory animal study indicate that Pyrenacantha staudtii leaf aqueous extract (PSE) possesses antidiarrheal activity in rodents. Previous studies have reported anti-inammatory and antiulcerogenic activities (Akubue et al., 1983), antischitosomosis (Okunji and Iwu, 1986), antispasmodic activity (Aguwa and Okunji, 1986), anticonvulsant and analgesic activities (Awe et al., 2005) of Pyrenacantha staudtii extract in various experimental animal paradigms. PSE (100400 mg/kg, p.o.) dose dependently and signicantly inhibited all the diarrheal parameters (onset, frequency and severity of diarrhea, total number of stools, number of wet stools, weight of wet stools, etc.) measured. Furthermore, the extract dose-dependently and signicantly decreased normal intestinal propulsive movement and transit in rodents comparable to those of the standard antidiarrheal drugs, loperamide (10 mg/kg, p.o.) and atropine (1 mg/kg, p.o.). Loperamide and atropine inhibited gastrointestinal motility (propulsion), reduced intestinal uid secretion and accumulation, and delayed gastric emptying, thus ameliorating diarrhea. Castor oil is known to cause water and electrolyte permeability changes in the intestinal mucosal membranes, resulting in uid and watery luminal content that ow rapidly through the small and large intestines (Gaginella et al., 1975; Mbagwu and Adeyemi, 2008). The induction of diarrhea by castor oil is attributed to its active ingredient ricinoleic acid (Gaginella et al., 1975), which stimulates the production of several mediator substances that include prostaglandins, nitric oxide, and platelet activating factor, cAMP and tachykinins (Izzo et al., 1999). The induction of diarrhea by castor oil can also be attributed to the liberation of prostaglandins by colonic cells (Awouters et al., 1978). In castor oil-induced diarrheic animals, PSE dose-dependently and signicantly delayed the onset of copious diarrhea, decreased the frequency of purging, reduced the number and weight of wet stools, and inhibited the severity of diarrhea (diarrhea score) generally. Since the extract was capable of inhibiting the castor oil induced diarrhea, we can suppose that the prostaglandin, nitric oxide, and platelet activating factor, cAMP and tachykinins synthesis inhibition can be involved in the mechanism of action. In the enteropooling study, the plant extract dose-dependently and signicantly reduced both the weight and volume of the animal intestinal contents. The intraluminal uid accumulation induced by castor oil was blocked by the extract in a dose-related manner. Clinically, diarrhea may result from disturbed bowel function, in which case, there is impaired intestinal absorption, excessive intestinal secretion of water and electrolytes, and a rapid bowel transit (Gurgel et al., 2001; Mbagwu and Adeyemi, 2008). According to Mbagwu and Adeyemi (2008), antidiarrheal index (ADI) is a measure of the combined effects of the different components of

diarrhea, including purging frequency, onset of diarrheal stools, and frequency of intestinal movement. Activation of the sympathetic innervations of the intestine results in inhibition of peristaltic activity and a reduction in tone. Earlier study has shown that PSE has smooth muscle relaxant activity; the effect of which is thought to be due to inhibition of calcium mobilization (Aguwa and Okunji, 1986). It has been proposed by Reynolds et al. (1984) that calcium antagonism is responsible, at least in part, for the antidiarrheal effect of loperamide and other compounds with calcium-antagonist properties like verapamil and diltiazem which exhibit spasmolytic properties on guinea pig ileum (Staneva-Stoytcheva and Venkova, 1992). Since Pyrenacantha staudtii, dose-dependently and signicantly decreased normal intestinal propulsive movement and transit in rodents comparable to the standard antidiarrheal drug, loperamide, it is not unreasonable, therefore, to suggest that the calcium antagonist property of Pyrenacantha staudtii leaf aqueous extract may be due at least in part to its antidiarrheal effect observed in this study. Furthermore, it has been observed that PSE relaxed guinea-pig isolated ileum, and antagonized acetylcholine (ACh) and histamine (HT)-induced contractions of the ileum in a concentrationdependent manner (Aguwa and Okunji, 1986). These observations tend to suggest that the anti-motility effect of PSE could be partly mediated via blockade of muscarinic cholinoceptors and/or histaminic receptors on mammalian intestine. The possibility also exists that muscarinic cholinoceptor antagonists (such as atropine), can synergistically potentiate the antidiarrheal effect of PSE by enhancing the extracts effect on both intestinal secretion and motility. The remarkable dose-related reductions in castor oil-induced diarrhea produced by PSE in rats is a clear evidence of the antidiarrheal efcacy of the extract, when compared with loperamide (10 mg/kg, p.o.), a standard anti-diarrheal drug. Atropine (1 mg/kg, p.o.), an antimuscarinic drug, and different doses of PSE (100, 200 and 400 mg/kg, p.o.) decreased propulsive movements in the charcoal meal study, the effect of which is less than that of atropine. The signicant inhibition of the castor oil-induced enteropooling by PSE in rats suggests that the extract probably produces relief in diarrhea through its spasmolytic and antienteropooling effects. These ndings are in consonance with the observations reported for aqueous root extract of Terminalia avicennoides Guill & Perr (Combretaceae) (Abdullahi et al., 2001), aqueous root extract of Guiera senegalensis JF Gmel. (Combretaceae) (Gamaniel, 2005), aqueous leaf extract of Byrsocarpus coccineus Schum. & Thonn. (Connaraceae) (Akindele and Adeyemi, 2006), aqueous plant extract of Mezoneuron benthamianum Baill. (Fabaceae) (Mbagwu and Adeyemi, 2008). Although the exact mechanism of the antidiarrheal action of PSE could not be established in this study, a number of investigators have shown polyphenolic compounds (e.g., coumarins), avonoids, triterpenoids, saponins, and a host of other plant secondary metabolites possess antidiarrheal properties in various experimental animal models (Dicarlo et al., 1994; Abdullahi et al.,

Please cite this article in press as: Awe, E.O., et al., Antidiarrheal activity of Pyrenacantha staudtii Engl. (Iccacinaceae) aqueous leaf extract in rodents. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.04.068

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E.O. Awe et al. / Journal of Ethnopharmacology xxx (2011) xxxxxx Brunton, L.L., 2008. Agents for Control of Gastric Acidity and Treatment of Peptic Ulcers Goodman Gilmans The Pharmacological Basis of Therapeutics, 11th ed. McGaw-Hill, New York, pp. 623652. Daziel, J.M., 1948. The useful plants of West Tropical Africa. London Crown Agents for Overseas Government and Administration. Mill Bank, London, p. 45. Dicarlo, G.D., Mascolo, N., Izzo, A.A., Capasso, F., Autore, G., 1994. Effects of quercetin on gastrointestinal tract in rats and mice. Phytotherapy Research 8, 4245. Dunnett, C., Goldsmith, C., 1993. In: Buncher, C.R., Tsay, J.Y. (Eds.), Statistics in the Pharmaceutical Industry. , 2nd ed. Marcel Dekker, New York, pp. 397433. Field, M., Rao, M.C., Chang, E.B., 1989. Intestinal electrolyte transport and diarrhoea disease. New England Journal of Medicine 321, 800806. Fontaine, O., 1988. Bacterial diarrhea and treatment. Lancet 331, 12341235. Gaginella, T.S., Stewart, J.J., Olsen, W.A., Bass, P., 1975. Action of ricinoleic acid and structurally related fatty acid on gastrointestinal tract. II. Effect on water and electrolyte absorption in vitro. Journal of Pharmacology and Experimental Therapeutic 195, 355361. Gamaniel, K.S., 2005. Anti-diarrhoeal and ulcer-protective effects of the aqueous root extract of Guiera senegalensis in rodents. Journal of Ethnopharmacology 97, 549554. Gamaniel, K.S., Akah, P.A., 1996. Analysis of the gastrointestinal relaxing effect of the stem-bark extract of Gongronema latifolium. Phytomedicine 2, 293296. Gurgel, L.A., Silva, R.M., Santos, F.A., Martins, D.T.O., Mattos, P.O., Rao, V.S.N., 2001. Studies on the antidiarrhoeal effect of dragons blood from Croton urucarana. Phytotherapy Research 15, 319322. Gutierrez, R.M.P., Mitchell, S., Solis, R.V., 2008. Psidium guajava: A review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology 117, 127. Hirchhorn, N, 1980. The treatment of acute diarrhoea in children. An historical physiological perspective. American Journal of Clinical Nutrition 33, 637663. Iwu, M.M., 1993. Handbook of African Medicinal Plants. CRC Press Inc, New York, p. 27. Izzo, A.A., Mascolo, N., Capassco, R., Germano, M.P., De pasuele, R., Caspassco, F., 1999. Inhibitory effect of cannabinoid agonists on gastric emptying in rat. Archives of Pharmacology 360, 221223. Longe, R.L., Dipiro, J.T., 1999. Diarrhoea and constipation. In: Dipiro, J.T., Talbert, R.L., Hayes, P.E. (Eds.), Pharmacotherapy: A Pathophysiologic Approach. , 4th ed. Appleton & Lange, Stamford, CT, pp. 606612. Lorke, D., 1983. A new approach to practical acute toxicity testing. Archives of Toxicology 54, 275287. Mbagwu, H.O.C., Adeyemi, O.O., 2008. Anti-diarrhoeal activity of the aqueous extract of Mezoneuron benthamianum Baill (Caesalpinaceae). Journal of Ethnopharmacology 116, 1620. Mukherjee, P.K., Saha, K., Murugesan, T., Mandal, S.C., Pal, M., Saha, B.P., 1998. Screening of antidiarrheal prole of some plant extracts of specic regions of West Bengal, India. Journal of Ethnopharmacology 60, 8589. Okunji, C.O., Iwu, M.M., 1986. Control of schistosomiasis using Nigerian medicinal plants. Annual Review Nutrition 6, 433458. Okunji, C.O., Iwu, M.M., 1988. Phytochemical screening of Pyrenacantha staudtii. International Journal of Crude Drug Research 4, 246252. Oliver-Bever, B., 1986. Medicinal Plants in Tropical West Africa. Cambridge University Press, London, p. 61. Reynolds, I.J., Gould, R.J., Snyder, S.H., 1984. Loperamide: blockade of calcium channels as a mechanism for antidiarrheal effects. Journal of Pharmacology and Experimental Therapeutics 231, 628632. Staneva-Stoytcheva, D., Venkova, K., 1992. Effects of the calcium antagonists diltiazem, verapamil and nitrendipine on the contractile responses of guinea-pig isolated ileum to electrical stimulation or carbachol. Journal of Pharmacology and Pharmacy 44, 321325. Snyder, J.D, Merson, M.H., 1982. The magnitude of the global problem of acute diarrhoeal disease: a review of acute surveillance data. Bulletin of World Health Organization 60, 604613. Suleiman, M.M., Dzenda, T., Sani., C.A., 2008. Antidiarrhoeal activity of the methanol stem- bark extract of Annona senegalensis Pers. (Annonaceae). Journal of Ethnopharmacology 116, 125130. Venkatesan, N., Thiyagarajan, V., Narayanan, S., Arul, A., Raja, S., Kumar, S.G.V., Rajarajan, T., Perianayagam, J.B., 2005. Antidiarrhoeal potential of Asparagus racemosus wild root extracts in laboratory animals. Journal of Pharmacy and Pharmacological Science 8, 3945.

2001; Agunu et al., 2005; Aniagu et al., 2005; Akindele and Adeyemi, 2006; Mbagwu and Adeyemi, 2008; Suleiman et al., 2008). Since Pyenacantha staudtii is known to contain saponins and alkaloids (Okunji and Iwu, 1988), it is not unreasonable to speculate that some of these chemical compounds could have contributed to the observed antidiarrheal effect of the plant leaf aqueous extract. The results obtained from the present experimental laboratory animal study show that, PSE probably produces antidiarrheal activity through a combination of its ability to inhibit gastrointestinal acetylcholine and histamine release, and also its calcium-antagonist effect. In conclusion, the experimental evidence obtained in the present laboratory animal study indicates that PSE possesses antidiarrheal activity in rats and mice. These ndings could therefore lend pharmacological credence to or conrm the basis for the use of Pyrenacantha staudtii leaf in traditional medicine for the treatment and/or management of diarrhea in some rural communities of Nigeria. Acknowledgements The authors are grateful to Mr. Aibare, of the Department of Chemistry, University of Ibadan, Nigeria for his assistance in the extraction of Pyrenacantha staudtii leaf, and to Mr.O.O. Olaniran for his technical assistance. References
Abdullahi, A.L., Agho, M.O., Amos, S., Gamaniel, K.S., Wambebe, C., 2001. Antidiarrhoeal activity of the aqueous extract of Terminalia avicennoides roots. Phytotherapy Research 15, 431434. Adeyemi, O.O., Akindele, A.J., 2008. Antidiarrhoeal activity of the ethyl acetate extract of Baphia nitida (Papilionaceae). Journal of Ethnopharmacology 116, 407412. Aguwa, C.N., Okunji, C.O., 1986. Gastrointestinal studies of Pyrenacanthia staundti leaf extract. Journal of Ethnopharmacology 5, 4555. Akubue, P.I., Mittal, G.C., Aguwa, C.N., 1983. Preliminary study of some Nigerian Medicinal Plants. Journal of Ethnopharmacology 8, 63163. Agunu, A., Yusuf, S., Andrew, G.O., Zezi, A.U., Abdurahman, E.M., 2005. Evaluation of ve medicinal plants used in diarrhoea treatment in Nigeria. Journal of Ethnopharmacology 101, 2730. Akindele, A.J., Adeyemi, O.O., 2006. Evaluation of the antidiarrhoeal activity of Byrsocarpus coccineus. Journal of Ethnopharmacology 108, 2025. Aniagu, S.O., Binda, L.G., Nwinyi, F.C., Orisadipe, A., Amos, S., Wambebe, C., 2005. Antidiarrheal and ulcer-protective effects of the aqueous root extract of Guiera senagalensis in rodents. Journal of Ethnopharmacology 97, 549554. Awe, E.O., Makinde, J.M., Olajide, O.A., Wakeel, O.K., Adeloye, A.O., 2005. Evaluation of analgesic, anticonvulsant and hypnotic activities of Pyrenacantha staudtii. African Journal of Traditional Complementry and Alternative Medicine 2, 122128. Avery, M.E., Synder, J.D., 1990. Oral therapy for acute diarrhoea. Antenn. Journal of Medicine 323, 891894. Awouters, F., Niemegeers, C.J.E., Lenaerts, F.M., Janssen, P.A.J., 1978. Delay of castor oil diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin biosynthesis. Journal of Pharmacy and Pharmacology 30, 4145. Aye-Than, J.H., Kulkarni, W., Tha, S.J., 1989. Antidiarrhoeal efcacy of some Burmese indigenous drug formulations in experimental diarrhoea models. International Journal of Crude Drug Research 27, 195200. Bliss, T., 1935. The calculation of the dose-mortality curve. Annual Applied Biology 22, 134167.

Please cite this article in press as: Awe, E.O., et al., Antidiarrheal activity of Pyrenacantha staudtii Engl. (Iccacinaceae) aqueous leaf extract in rodents. J. Ethnopharmacol. (2011), doi:10.1016/j.jep.2011.04.068