Antidiarrhoeal and antioxidant properties of ethanol leaf extract of Pseudocedrela kotschyi

1 Department of Pharmacology, College of Medical Sciences, University of Calabar, Nigeria. 2 Department of Pharmacology and Therapeutics, Faculty of Medicine, Ebonyi State University, Abakaliki, Nigeria. 3 Department of Optometry, School of Health, Federal University of Technology, Owerri, Nigeria. 4 Department of Pharmacology and Therapeutics, College of Health Sciences, Abia State University, Uturu, Nigeria.


INTRODUCTION
Diarrhoeal diseases account for over 5-8 million deaths globally, each year in children less than 5 years in developing countries (WHO, 2006;Moszynski, 2007;Khalilur et al., 2015).The incidence of diarrhoeal disease remains high despite the efforts of various governments and international organizations to prevent it.Thus it becomes imperative to identify and investigate available natural products as alternative to currently used antidiarrhoeal agents, which are not free from adverse effects (Akuodor et al., 2014).Medicinal plants have been the most accessible form of therapy for the less privileged in the global population (Espinosa et al., 2012).This has been the justification for the push of the World Health Organization (WHO) for scientific validation of ethnobotanical knowledge of medicinal plants (Ministério da Saúde, 2005).Pseudocedrela kotschyi Schweint.Harms, is a plant that belongs to the family Meliaceae.P. kotschyi grows well on moisture of heavy soils.The decoction of the leaf is used traditionally in the folk medicine in Nigeria for the treatment of a number of diseases and health conditions, including malaria, fever, pains, diabetes and convulsion (Akuodor et al., 2015;Akuodor et al., 2013;Georgewill and Georgewill, 2009;Anuka et al.,1999).The interest in this plant was justified by its potential medicinal value against diseases.Therefore, the aim of this study was to investigate the antidiarrhoeal and antioxidant effects of ethanol leaf extract of P. kotschyi.

Plant collection
The leaves of Pseudocedrela kotschyi were collected from Chaza, Niger State, Nigeria.

Sample extraction
Three hundred and fifty grams (350 g) of the powdered leaves were macerated overnight in 1.5 L of ethanol.The mixture was filtered and dried on a water bath at reduced temperature of 45 o C and the residues obtained were weighed and stored at 4 o C.

Phytochemical screening
Phytocchemical analysis of the ethanol leaf extract was carried out employing standard procedures (Harborne, 1992;Evans, 2005).

Animals
Adult wistar rats (200-250 g) of both sexes obtained from Animal House of Department of Anatomy, Ebonyi State University, Abakaliki, were used for the study.The rats were housed in cages at room temperature and moisture, under naturally illuminated environmental of 12:12 h dark/ light cycle.The animals were fed on standard feed and water ad libatum.A standard protocol was drawn in accordance with the Good laboratory Practice (GLP) regulation [ENV/MC/CHEM (98)] (1997).The National Institute of Health Guide for the care and use of Laboratory Animals was adopted for the animal protocol in this study (NIH, 1978).

Acute toxicity test
The LD 50 of the leaf extract was examined to determine the safety of the agent in rats, in vivo following OECD (2010) method.Dose levels used ranged from 10-5000 mg/kg.The rats were observed for signs of toxicity such as salivation, stretching of the body, weakness, paw licking respiratory distress, coma and death for 24 h and 72 h respectively.

Castor oil induced diarrhoea
Wistar albino rats for this test were first observed for any wet faeces.The droppings were easily distinguished from the normal dry faeces which were regular in shape, hard and did not stain the white transparent paper.Rats that produced wet faeces were not used for the study.The ethanol leaf extract of Pseudocedrela kotschyi (100, 200 and 400 mg/kg), 20 ml/kg normal saline (negative control) or 4 mg/kg loperamide (positive control) were orally administered 60 min before diarrhoea induction.Diarrhoea was induced by oral administration of 1 ml castor oil to 24 h fasted rats.The rats in individual cages were observed for 4 h for the presence or absence of wet faeces.(Akuodor et al., 2010;Akuodor et al., 2012).

Intestinal transit test
The wistar albino rats used for this test were deprived of food for 24 h but had access to water.The rats were grouped into 5 of 6 per cage.Rats in group 1 were administered with normal saline (negative control).Group 2, 3 and 4 were administered with 100, 200 and 400 mg/kg of the leaf extract, while group 5 received 5 mg/kg of atropine.After 30 min, the animals were orally dosed with 1 ml of freshly prepared charcoal meal (10 % in tragacanth).The rats were sacrificed 30 min later and gastrointestinal tract removed.The distance travelled by the marker (charcoal meal) from the pylorus to caecum was measured and expressed as the percentage of the total length of the small intestine (Abere et al., 2010;Akuodor et al., 2014).

Intestinal fluid accumulation
The rats used for this test were starved for 24 h prior to the study but allowed access to water.The rats were grouped into 5 of 6 per cage.Rats in group 1 were administered with normal saline (negative control).Group 2, 3 and 4 were administered with 100, 200 and 400 mg/kg of the leaf extract, while group 5 received 4 mg/kg of loperamide respectively.After 30 min, each rat was orally challenged with 1 ml of castor oil.They were sacrificed (30 min after) and the whole stomach contents were milked into a measuring cylinder and the volume measured (Mujumdar et al., 2005).

DPPH radical scavenging activity
DPPH radical scavenging activity of the ethanol leaf extract of Pseudocedrela kotschyi was determined according to the method described by Kaneria et al. (2012) with slight modification.The stock solution of the extract was diluted to final concentrations of 250, 200, 150, 100 and 50 μg/mL in methanol.1 mL of a 0.3 mM DPPH methanol solution was added to 2.5 mL solution of the extract and was allowed to react at room temperature for 30 min under complete dark.The absorbance of the resulting mixture after the reaction was taken at 517 nm using UV visible spectrophotometer.

Statistical Analysis.
The results are presented as mean ±standard error of mean (SEM).The one-way ANOVA test was used to analyze and compare the data, while p< 0.05 was considered as statistically significant.

Phytochemical screening of P. kotschyi
Phytochemical screening of the crude extract of P. kotschyi revealed the presence of alkaloids, flavonoids, terpenoids, tannins, saponins, steroids, glycosides and reducing sugar.

Acute toxicity test
The ethanol leaf extract of P. kotschyi did not produce any mortality up to the oral dose of 5000 mg/kg in rats, thus the LD 50 was indeterminable.There were no significance changes in behaviour, mood and motor activity.

Effect of castor Oil-Induced Diarrhoea
In castor oil induced diarrhoeal test, the ethanol leaf extract of P. kotschyi showed a marked antidiarrhoeal effect in the rats (Table 1).In both doses used (100, 200 400 mg/kg), the extract produced significant (p<0.05)decrease defecation in rats.The result is comparable to the effect produced by the standard antidiarrhoeal drug, loperamide (4 mg/kg).

Effect on intestinal transit Test
The ethanol extract of P. kotschyi decreased the gastrointestinal distance travelled by the charcoal meal in the rats noticeably compared with the control group.Atropine (5mg/kg) produced a marked decrease in the propulsion of charcoal meal through gastrointestinal tract (Table 2).

Effect on castor oil induced intestinal fluid accumulation
In this test, ethanol leaf extract of P. Kotschyi at both doses used (100, 200, 400 mg/kg produced significant (p<0.05) and dose dependent reduction in intestinal fluid volume (Table 3).The standard drug loperamide (4mg/kg) also significantly inhibited (p<0.05) the intestinal fluid accumulation.

Effect on DPPH radical scavenging test
The leaf extract of P. kotschyi at concentrations used (50-250 µg/ml), showed strong DPPH free radical scavenging activity (Table 4).The result of DPPH scavenging activity in this study indicates that the plant was potentially active.

DISCUSSION
This study was conducted to establish the potential pharmacological properties of Pseudocedrela kotschyi based on its use in traditional medicine.Diarrhoea can be described as the abnormally frequent defecation due to a disturbance in the transport of water and electrolytes in the intestines.Castor oil produces diarrhoea due to its active component, ricinolic acid which increases peristaltic activity in small intestine leading to changes in the electrolyte permeability of the intestinal mucosal membrane (Akuodor et al., 2010).Several mechanisms have been proposed to explain the diarrhoeal effect of castor oil which includes inhibition of intestinal Na+ K+ ATPase activity, thus decreasing normal fluid absorption, activation of adenylate cyclase or mucosal cAMP-mediated active secretion (Capasso et al., 1994;Imam et al., 2012).Castor oil is usually metabolized into ricinolic acid in the gut, and this causes irritation and inflammation in the intestinal mucosa leading to the release of prostaglandins (Khalilur et al., 2015).The released prostaglandins initiate vasodilatation, smooth muscle contraction, and mucus secretion in the small intestines.Inhibitors of prostaglandins biosynthesis delay castor oil induced diarrhoea (Akuodor et al., 2011).The leaf extract of Pseudocedrela kotschyi significantly reduced the amount of faeces in castor oil induced diarrhoea in rats at the doses used (100, 200 and 400 mg/kg).The leaf extract exhibited comparable characteristics as the reference drug, Loperamide which at present is one the most potent and widely employed antidiarrhoeal drugs.Apart from regulating the gastrointestinal tract, Loperamide has been reported to slow down transit in the intestine, decrease colon flow rates and any effect on colonic motility (Camilleri, 2004;Akuodor et al., 2011).The antimuscarinic agent (Atropine) and different doses of the leaf extract reduced the propulsive movement of the charcoal plug dose dependently.This is possible due to its anticholinergic activity.Moreso, our results significantly demonstrated an inhibition of castor oil induced enteropooling with reduction of the volume of intraluminal contents.These results suggest that the ethanol leaf extract of P. kotschyi contain antidiarrheal properties.Previous report on the phytochemical screening of P. kotschyi leaves has shown the presence of alkaloids, tannin, cardiac glycosides, steroids, flavoniods and saponins (Akuodor et al., 2014).Plants containing these secondary metabolites have been reported to possess antidiarrhoeal activities.However, previous reports have also shown that flavonoids have ability to inhibit intestinal motility and water and electrolytes secretion (Di Carlo et al., 1993).It could therefore be suggested that the secondary metabolites present in P. kotschyi leaves are responsible for the observed biological activities.Results show that P. kotschyi leaf possesses potential antioxidant activity.
Compounds such as alkaloids, flavonoids, terpenoids and vitamins which have the ability to scavenge free radicals are produced by the natural machinery of the plants (Cait et al., 2003).Ingestion of natural antioxidants through food and medicine prepared from plants can reduce the complications connected with the presence of free radicals (Veerapur et al., 2009).The results further suggest that P. kotschyi leaf could be used in food industry as natural antioxidant.In conclusion, the findings of the present study provide convincing evidence that ethanol leaf extract of P. kotschyi possesses remarkable antidiarrheal and antioxidant activities.The Antidiarrheal, antimotility antienteropooling effects are rapid, long lasting and statistically significant at both doses.However, further chemical studies are required to isolate the bioactive compounds and elucidate the precise mechanisms responsible for the observed pharmacological activities of this medicinal plant.
The plant was identified by a taxonomist in the Department of Medicinal plant Research and Traditional Medicine, National Institute for Pharmaceutical Research & Development (NIPRD), Abuja, Nigeria where voucher specimens were deposited with the number, NIPRD/H/6542.The international plant name index is Meliaceae Pseudocedrela kotschyi-Bot.Jahrb.Syst.22 (1): 154.1895 (19 Nov. 1895) (IK).

Table 1 :
Effect of ethanol leaf extract of Pseudocedrela kotschyi on castor oilinduced diarrhoea in rats.

Table 2 :
Effect of ethanol leaf extract of Pseudocedrela kotschyi on intestinal transit time in rats.

Table 3 :
Effect of ethanol leaf extract of Pseudocedrela kotschyi on castor oilinduced intestinal fluid accumulation in rats.