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Stem Bark Extract and Fractions Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45 Nigerian Journal of Pharmaceutical Sciences Vol. 17, No1, 2018, ISSN: 0189-823X 2 All Rights Reserved INVESTIGATION OF THE ANTIDIARRHOEAL AND EX-VIVO INTESTINAL ACTIVITY OF HYMENOCARDIA ACIDA (PHYLLANTHACEAE) STEM BARK EXTRACT AND FRACTIONS *1Bafor, E. E., 2Aibangbee, O. F., 1Amaechina F. C., 2Ofeimun J. O., 1Obarisiagbon P. A., 1Okosun I. V., 1Omoruyi O. and 2Ayinde, B. A. 1Department of Pharmacology, Faculty of Pharmacy University of Benin, Edo State Nigeria 2Department of Pharmacognosy, Faculty of Pharmacy University of Benin, Edo State Nigeria *Author for correspondence: [email protected] ABSTRACT Hymenocardia acida is used traditionally in parts of Africa for the management of diarrhoea. This study was therefore aimed at investigating the antidiarrhoeal properties of the stem bark extract and fractions of H. acida in rat and mouse models of diarrhoea and intestinal motility. The stem bark was collected, powdered, macerated in methanol for 72 h, and concentrated under pressure. Liquid-liquid partitioning was performed in succession yielding chloroform and aqueous fractions. The crude methanol extract (MHAS), chloroform fraction (CHAS) and aqueous fraction (AHAS) were then used for the studies. Qualitative phytochemical analyses on MHA were performed using standard procedures. The antidiarrhoeal activity of the extract (100, 200 and 400 mg/kg) was evaluated on intestinal transit, castor oil-induced diarrhoea and castor-oil induced enteropooling with either atropine (5 mg/kg) or loperamide (5 mg/kg) as positive controls. The antispasmodic effect of the extract and fractions were examined on the isolated rat or mouse ileum motility. Several secondary metabolite classes were detected. The crude extract and fractions significantly inhibited (p < 0.01) intestinal transit, castor-oil induced diarrhoea (p < 0.001) and intestinal fluid accumulation (p < 0.001). The extract and fractions also inhibited the amplitude and frequency of spontaneous and ACh- induced intestinal contractions. However CHAS was observed to produce a more potent inhibition of motility. This study has therefore shown that H. acida stem bark exerts anti-diarrhoea and anti-motility activities supporting the traditional use of the plant. Keywords: Acetylcholine; Anti-diarrhoea; Hymenocardia acida; Intestine; Enteropooling INTRODUCTION medications (Baldi et al., 2009). It is however considered a major health problem. Diarrhoea has been described as one of the In several parts of the world, diarrhoea is the most common disorders in the world today major cause of infant mortality. In severe and results from a failure in function of the cases, diarrhoea may lead to dehydration, digestive tract leading to abnormal stools electrolyte imbalance as well as protein loss, (Niemegeers et al., 1981). Impairment of all of which can cause severe health risk gastrointestinal tract functionality has been (Niemegeers et al., 1981). Several attributed to a complex interplay of several pharmaceuticals have been developed for pathophysiological factors and includes diarrhea treatment, but diarrhea continues to factors such as infections, malnutrition, food be a major health challenge to people intolerances, intestinal disorders, and some particularly in tropical and subtropical 33 Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45 countries. Currently, available MATERIALS pharmacological treatments seem insufficient in diarrhea control and Plant materials management due to high cost, adverse The stem bark of H. acida was collected effects as well as therapeutic approaches. from Iwo Town in Osun State, Nigeria and Investigations and search for new treatment identified by Professor B. A. Ayinde of the options from natural products are therefore Department of Pharmacognosy, University encouraged (Mishra et al., 2016). The of Benin. Authentication was carried out by ethnomedicinal approach for the Mr S. A. Odewo; a plant taxonomist at the management of diarrhea is considered Forest Research Institute of Nigeria (FRIN), practical, and cost-effective (Mishra et al., Ibadan, Nigeria where a voucher specimen 2016). Significant research into chemical was deposited and a herbarium number and biological properties of plants is FHI110465 was issued. The stem bark of the therefore encouraged. Plants are used as a plant was cleaned and shade-dried. Further source for antidiarrhoea therapy across drying was achieved by drying in an oven at Africa and one of such plants is 40°C for 30 min prior to milling. As soon as Hymenocardia acida which is used the stem bark material was dry, it was traditionally for the management of ground into fine powder using a milling diarrhoea and dysentery in different parts of machine (Christy Turner, Suffolk, UK) and Africa (Irvine, 1961). kept in air-tight containers till needed. Hymenocardia acida Tul of the Animals Phyllanthaceae family, is a small tree of Albino Sprague Dawley rats (106 – 211 g) about 6 m high, widespread in tropical and albino mice (18 – 20 g) of either sex Africa and widely distributed in the were purchased from the Animal Centre savannah region of Nigeria (Burkill, 1985). Ogbomosho, Oyo State, Nigeria and The plant is known as “Enache” by the maintained at the Animal House Department Idoma people of North Central Nigeria and of Pharmacology and Toxicology, Faculty of as “Janyaro” among the Hausas in Nigeria Pharmacy, University of Benin, Edo state, (Abu and Uchendu, 2011). There have been Nigeria. They were housed in plastic cages some scientific reports on the effects of the at an environmentally controlled room stem bark on several conditions. The ethanol temperature of approximately 27 ± 5°C and stem bark extract has been reported to have environmentally controlled lighting anti-fertility effects (Hyacinth and Nwocha, conditions of approximately 11 h/13 h light 2011), the root and stem bark decoction is and dark cycles. Relative humidity ranged used in the treatment of diabetes (Igoli et al., from 85-88%. The animals were 2004), the stem bark has also been reported acclimatized to these conditions. Handling to have antispermatogenic effect (Abu and was done as much as possible according to Uchendu, 2010), as well as vasorelaxant and standards of the Public Health Service antihypertensive effect (Manga et al., 2013). policy on humane care and use of However there has been no report on the Laboratory Animals (National research effect of the stem bark on diarrhoea. This council 2010). The animals were maintained study is therefore aimed at investigating the on standard diet of animal pellets and clean activity of the H. acida stem bark on tap water. Ethical approval was obtained diarrhoea and intestinal motility in animal from the Ethical Committee, Faculty of (mice and rats) models. Pharmacy, University of Benin, Nigeria. 34 Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45 Drugs and reagents colour change to green or black was Chloroform, methanol, and phytochemical watched for. Assessment for anthracene was analysis reagents were obtained from BDH performed using ammonium chloride and chemicals, UK, acetylcholine (Sigma shaken vigorously. Aldrich, UK), castor oil (Bell, Sons & Co. (Druggists) Ltd., Merseyside, UK), activated Liquid-liquid partitioning of the extract charcoal (General Carbon Co., NJ USA), The crude extract (34 g) was dissolved in a loperamide HCl (Shine Pharmaceuticals, combination of methanol and water (1:4) Gujarat, India). Salts for the physiological and then partitioned with chloroform (100 x solution were obtained from BDH 3 mL) in a separating funnel with occasional chemicals, England, UK. stirring. The chloroform (CHAS) and aqueous phases (AHAS) were collected and METHODS concentrated to dryness over a water bath set Preparation of plant extract at 40⁰C for the chloroform fraction, and The powdered stem bark material (1500 g) 60⁰C for the aqueous fraction. The dried was macerated in methanol (3 L) for 72 h fractions were weighed and stored in the and then filtered. The filtrate obtained was refrigerator at 4°C till needed. The concentrated to dryness with the aid of a chloroform fraction yielded 15 g (44.12 % water bath set at 70°C and the resulting w/w) while the aqueous fraction yielded 12 extract (MHAS) was weighed to give 192.90 g (35.29 % w/w). g with a percentage yield of 12.86 % w/w. Pharmacological Studies Phytochemical qualitative analysis The stem bark extract was analysed for Normal intestinal transit phytochemical compounds using standard A modified protocol earlier described was procedures earlier described (Harborne, used (Hsu, 1982; Akindele et al., 2014). 1998; Odebiyi and Sofowora, 1978; Trease Briefly, mice were randomly allotted to five and Evans, 1989). Briefly, phytochemical groups containing five animals each. The screening was performed for the presence of animals were fasted 18 h prior to glycosides, saponins, flavonoids, alkaloids, experiments but allowed unlimited access to tannins, anthracene derivatives and water ad libitum. Group 1 was the positive triterpenoids. Presence of glycosides was control and received atropine (5 mg/kg p.o.). assessed using α – naphthol and Group 2 was the negative control and concentrated tetraoxosulphate VI acid. The received 0.2 mL distilled water. Groups 3, 4 Fehling’s test for reducing sugars was also and 5 were the treatment groups and performed. Alkaloids were detected by using received the methanol crude stem bark Dragendorff’s
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