Evaluation of the anti-diarrheal activity of methanol extract and its fractions of Urena sinuata L . ( Borss ) leaves

1 Department of Pharmacy, University of Chittagong, Chittagong-4331, Bangladesh. 2 Drug Management and Policy, Division of Pharmaceutical Sciences, Kakuma-machi, Kanazawa 920-1192, Japan. 3 Laboratory of Vaccinology and Applied Immunology, Kanazawa University School of Pharmacy, Kakuma-machi, Kanazawa 920-1192, Japan. 4 Department of Pharmacy, BGC Trust University Bangladesh, Chittagong-4000, Bangladesh. 5 Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331, Bangladesh.


INTRODUCTION
Diarrhoeal disease is often a leading source of mortality and morbidity, especially among children in developing countries causing a major healthcare problem (Chitme et al., 2004).Microorganisms including Shigella flexneri, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Aeromonas hydrophila, Plesiomonas shigelloides are only to be major causative agents of diarrhoea in humans (Brenden et al., 1988;Janda and Abbott, 2010;Umer et al., 2013).Candida albicans has also been recognised to cause diarrhoea in humans (Krause et al., 2001).It was reported that, in fact, due to diarrhoea, that 50% of deaths attributed to diarrhoea combined with respiratory disease were due to diarrhoea, and that one-third of deaths due to combined measles with diarrhoea or respiratory disease were due to diarrhoea.With these assumptions, using the most recently available national verbal autopsy data (1999)(2000)(2001)(2002)(2003), diarrhoea accounted for 5.4 deaths per 1,000 children in Bangladesh (Baqui et al., 2001;Luby et al., 2008).The sickness is characterised by a discharge of semi-solid or watery fecal matter on the bowels three or even more times every day (Hirschhorn, 1980;Snyder and Merson, 1982).It requires a rise in the fluidity, volume and frequency of going number two or three, abdominal pain associated with increased secretion and decreased absorption of fluid thereby loosing water and electrolytes (Fontaine; Field et al., 1989).
However, regardless of the use of vast spectrum of approaches for diarrhoeal management, the greater parts of an individual in developing countries make use of herbal drugs to the management of diarrhoea.WHO has encouraged studies for treatment and protection against diarrhoeal diseases depending on traditional medical practices (Atta and Mouneir, 2004).Therefore, medicinal plants represent a promising source for the discovery of new anti-diarrhoeal agents (Maikere-Faniyo et al., 1989).Urena sinuata L. (Family: Malvaceae) is often a wild shrubby plant by folk medicinal use in its native areas.It is additionally known while using synonyms U. lobata, U. morifolia, U. moricata, U. paradoxaand U. swartzii.Various varieties of this plant grow in different tropical and subtropical areas throughout the world.The roots are sweet, slightly cooling, anti-rheumatic and antipyretic.A decoction on the stem and roots is employed in Brazil like a remedy of severe windy colic.A poultice prepared from the roots and leaves are needed as an emollient and are particularly given for snakebites, sprains and bruises.The flowers are administered as expectorant in dry and inveterate coughs.An infusion from the flowers is needed as a gargle raw throat bronchitis (Kirtikar, 1965).
In India the fundamental is utilized just as one external application for lumbago.It's for reproductive purposes in the Pacific, Trinidad and Tobago, China and India (Browner, 1985) for specific human problems of both the genders (Lans, 2007).It is just a popular diuretic in Assam, and in addition used as an abortifacient.In Philippines, the fundamental is considered as emollient, refrigerant and maturant; the leaves are prescribed in inflammation on the intestines and also the bladder.A decoction of dried root is utilized in enteritis, dysentery, rheumatic pains and tonsillitis.
Anyway, some view the rose like a medicinal plant, some deem a weed, but others utilize its fiber (Aramina fiber) for assorted purposes in Madagascar, Nigeria and Western Sudan, Chad, Central African Republic, Zaire and Gabon, which is told proof against damage by termites and water (Anon, 1976;Ahmed, 2009).However, house elevators its biological activity on diarrhoea remains to be scanty.For this reason the flower was considered for your investigation of its anti-diarrhoeal activity.

Drugs and chemicals
Methanol was bought from SIGMA® (Sigma-Aldrich®, St Louis, USA), while loperamide manufactured by Square Pharmaceuticals Ltd., Bangladesh, was bought from local pharmacy.Castor oil was purchased from WELL's Heath Care, Spain.All the chemicals and reagents were analytical grade.

Ethical statement
All experimental protocols were in compliance with Dhaka University Ethics Committee (approval number AE-DUEC 2012/118) on research in animals as well as internationally accepted principles for laboratory animal use and care (Zimmermann).

Plant material
The plant was collected from University of Chittagong campus in 2011.The plant was taxonomically identified by Dr. Shaikh Bokhtear Uddin, Professor, Department of Botany, University of Chittagong.The sample specimens of the identified plants have been preserved in the national herbarium with the mentioned accession numbers.The plant leaves were thoroughly washed with water and were dried in a hot air oven at room temperature (25 ± 1) °C for 7 days in two days interval and at 40°C for the next 2 days.

Extraction of the plant material and sample preparation
Weighed (600 g of the dried and powdered) sample was soaked in 1500 mL of 99% Methanol (Merck KGaA, Germany) in clean, sterilized and flat-bottomed glass container.Afterwards, it was sealed and maintained for 15 days accompanying occasional stirring and agitation.The complete mixture was then subjected to coarse filtration on a piece of clean, white sterilized cotton material and Whatman® filter paper.The extract was obtained by evaporation using rotary evaporator (Bibby RE-200, Sterilin Ltd., UK) at 4 rpm and 65ºC temperature.It rendered a gummy concentrate of greenish black color.The gummy concentrate was designated as crude extract, or Methanol extract.Then the crude methanolic extract was dried by freeze drier and preserved at +4°C (yield 13.56% w/w).
After that, solvent-solvent partitioning was done by using the protocol designed by Kupchan and Tsou (1973) (Kupchan et al., 1973) and modified version of Wagenen et al., (1993).The crude extract was (5 g) was triturated by dissolved in 10% aqueous methanol (methanol: water; 9:1 v/v) to make the mother solution which was partitioned off successively by four solvents such as nhexane, chloroform, ethyl acetate and hydro-methanolin order of increasing polarity by using separating funnel (Figure 1).The amount of extract found after partitioning and their physical appearances are shown in Table 1.All the four fractions of each plant extract were dried by evaporating respective solvent using rotary evaporator.All extracts were stored at 4ºC in airtight containers till further analysis (Bibi et al., 2011).

Experimental Animal
Albino Wistar rats (Rattus norvigicus) of either sex weighing 120-150 g were also used for the present study.They were purchased from the animal house of Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.They were maintained in the animal house of North South University, Bashundhara, Dhaka-1229, Bangladesh for experimental purpose.The animals were maintained under controlled conditions of temperature (23 ± 2ºC), humidity (50 ± 5%) and 12 h light-dark cycles.All the animals were acclimatized for seven days before the study.The animals were randomized into experimental and control groups and housed individually in sanitized polypropylene cages containing sterile paddy husk as bedding.They had free access to standard pellets as basal diet and water ad libitum.Animals were habituated to laboratory conditions for 48 h prior to experimental protocol to minimize if any of non-specific stress.

Antidiarrheal activity Castor oil induced diarrhoea
Castor oil-induced diarrhoea was done according to the method of Soba et al. (Shoba and Thomas, 2001) and Uddin et al (Uddin et al., 2005).Rats of either sex were divided into twelve groups of four rats each.The animals were fasted for 18 h prior to the test.Group I was treated with normal saline (2 mL/kg), which served as control; while Group II received loperamide (5 mg/kg).Groups III, IV, V, VI, and VII received orally 200 or 400 mg/kg of methanol, n-hexane, chloroform, ethyl acetate and hydro-methanol respectively received methanol and petroleum ether extracts (200 mg/kg).All doses were administered orally.After 1 h, all groups received 1 mL of castor oil orally.
Then animals were placed in cages lined with adsorbent papers and observed for 4 h for the presence of diarrhoea defined as watery (wet), unformed stool.The control group result was considered as 100%.The activity of each group was expressed as percent inhibition (%) of diarrhoea.The percent inhibition of defecation was calculated as follows:

% Inhibition of defecation= [(A-B)/A] ×100
Where A indicated mean number of defecation caused by castor oil; B indicated mean number of defecation caused by drug or extract.

Castor oil induced enteropooling
The castor oil-induced enteropooling was carried out according to the method of Robert et al.Rats of either sex were divided into twelve groups of four rats each.The animals were fasted for 18 h prior to the test.Group I was treated with normal saline (2 mL/kg), which served as control; while Group II received loperamide (5 mg/kg).
Groups III, IV, V, VI, and VII received orally 200 or 400 mg/kg of methanol, n-hexane, chloroform, ethyl acetate and hydro-methanol respectively received methanol and petroleum ether extracts (200 mg/kg).One hour later, all the rats were challenged with 1 mL of castor oil orally.After 1 h of castor oil received, the rats were sacrificed and the small intestine from the pylorus to the caecum was isolated.Then the intestinal contents were weighed and volume measured by graduated tube (Robert et al., 1976).

Gastrointestinal motility test
This test was performed according to the method previously described using charcoal as a diet marker (Meite, 2009).Rats of either sex were divided into twelve groups of four rats each.The animals were fasted for 18 h prior to the test.Group I was treated with normal saline (2 mL/kg), which served as control; while Group II received loperamide (5 mg/kg).Groups III, IV, V, VI, and VII received orally 200 or 400 mg/kg of methanol, n-hexane, chloroform, ethyl acetate and hydro-methanol respectively received methanol and petroleum ether extracts (200 mg/kg).After 1 h of drug administration, all animals were received 1 mL of charcoal meal (10% charcoal suspension in 5% gum acacia) orally.
One hour later, all animals were sacrificed, and the distance covered by the charcoal meal in the intestine from the pylorus to the caecum was measured and expressed as percentage of distance moved (Marona and Lucchesi, 2004).

Statistical analysis
All analyses were carried out in triplicates.Data were presented as mean ± SEM.The significance of difference between the control and treated groups was determined using twoway analysis of variance (ANOVA), followed by Student's t-test.P value of 0.05 or 0.01 was considered as significant.

Castor oil induced diarrhoea
In castor oil induced diarrhoea test, ethyl acetate showed considerable anti-diarrheal effect in rats.Methanol extract significantly inhibited the frequency of defecation (41.75%) when compared with untreated control rats (P < 0.01).The fraction decreased the entire number of wet feces produced upon administration of castor oil when compared to the castor oil treated rats.
The crude methanol extract, chloroform fraction and hydro methanol fraction are also showed significant effect (P < 0.05) with an inhibition of defection 38.28%, 27.18%, 37.73% respectively.The results are shown in Table 1.

Castor oil induced enteropooling
All of the extracts showed noticeable effect in castor oil induced entropooling test in the rats.The intestinal volume was decreased by 56.64% for hydro methanol extract and 60.69 % for loperamide (5 mg/kg).
The values were statistically more significant (P < 0.01).Other fractions and crude, chloroform, ethyl acetate, n-hexane and methanol are also significantly inhibited intestinal fluid accumulation (P < 0.05) and the effect of the extract was less potent in comparison to the standard drug.All the data are shown in Table 2.

Gastrointestinal motility test
The gastrointestinal distance travelled by the charcoal meal in the rats significantly (P < 0.01) lessened by methanol, hydro methanol and ethyl acetate fractions compared with the control group (Table 3).Loperamide (5 mg/kg) produced a marked decrease (42.30%) in the propulsion of charcoal meal through gastrointestinal tract.

DISCUSSION
Diarrhoea is frequently considered a consequence of altered motility and fluid accumulation within the intestinal tract.The act of using castor oil as being a diarrheal inductor has become largely studied, and it is active component is ricinoleic acid, which produces an irritating and inflammatory action on the intestinal mucosa, leading to the discharge of prostaglandins.This disorder enhances the permeability in the mucosal cells and provokes alterations in electrolyte transport thus causing diarrhea (Watson and Gordon, 1962;Mascolo et al., 1993;Kase et al., 1999;Tunaru et al., 2012).Prostaglandins on the E series are viewed as for being good diarrheogenic agents in experimental animals and world.The inhibitors of prostaglandins biosynthesis are therefore considered to delay using castor oil-induced diarrhoea (Tunaru et al., 2012).The present study sought to assess the antidiarrheal activity of U. sinuata.Our study showed that methanol extract of U. sinuata and its different fraction significantly inhibited castor oil-induced diarrhoea in rats, as shown by the significant reduction of the number of diarrhoeal and total faeces.Plant extracts containing tannin, flavonoids, alkaloids, saponins and steroids have been reported to possess antidiarrheal activity (Umer et al., 2013).The leaves of U. sinuata comprise numerous alkaloids which may be responsible for its consequence.The assumed effects were noticeable considering with the standard drug, loperamide at 5 mg/kg.In our study, we have shown the experimental data for one dose (200 mg/kg) only, for both extract and fractions.Loperamide, apart from governing the alimentary tract, is usually reported to reduce transit from the intestine, reduce colon flow rates and consequently produce any profit on colonic motility (Camilleri, 2004).Both extract and fractions moderately reduced intestinal transit from the decline in the length traveled by charcoal meal.The antimuscarinic drug and atropine decreased the propulsive movement in the charcoal meal study because anticholinergic effect (Rahman et al., 2013).From the result, we established that the extracts suppressed the propulsion of charcoal meal by increasing the absorption of water and electrolytes.

CONCLUSION
Our results exhibit that the leaves extract of U. sinuata and its fractions contains bioactive natural substances with antidiarrheal properties.These attributes may give a justification for the use of U. sinuata in diarrhoea management by traditional healers.But further studies are also required to identify the phytoconstituents responsible for these bioactivities and to establish the mechanism of action of antidiarrheal activity.