Stem Bark Extract and Fractions

Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45

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

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.

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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 and Mayer’s reagents. extract (MHAS) of H. acida (100, 200 and Presence of saponins was detected based on 400 mg/kg p.o.). Oral administrations were the appearance of froth upon vigorous achieved with the aid of a feeding syringe. shaking. Sterols and terpenes required Charcoal meal suspension (0.5 mL of 17% treatment of the plant material with charcoal in distilled water) was orally petroleum ether and subsequent extraction administered 30 min after initial with chloroform and observed for colour drug/treatment in each group. Thirty minutes changes. Flavonoids were assessed using post charcoal administration, the mice were aluminium chloride reagent and observed humanely killed by cervical dislocation and for yellow colour change. Tannins were the small intestine was immediately isolated assessed using aqueous ferric chloride and a and the total length measured (Adeyemi et

Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45 al., 2009). The distance travelled by the weight of wet stools were observed and charcoal meal from the pylorus to the recorded. caecum was measured and percentage inhibitions of movement calculated (Oben et Intestinal fluid accumulation al., 2006) as a function of the distilled water- Intestinal fluid accumulation was induced in treated negative control. rats by oral administration of castor oil (20 mL/kg) (Adeyemi and Akindele, 2008). Briefly, rats were fasted for 24 h prior to the experiment but with free access to water. They were randomly divided into five Where MDC = Mean distance travelled in control groups consisting of five rats each. Group 1 MDT = Mean distance travelled in test received distilled water (1 mL), group 2 received loperamide (5 mg/kg p.o.) and Castor-oil induced diarrhoea groups 3, 4, and 5 received MHAS (100, Rats were randomly allocated into five 200, and 400 mg/kg p.o. respectively). One groups of 5 animals each and fasted for 18 h hour later, castor oil (20 mL/kg) was then prior to experiments but with free access to administered orally. All oral administrations drinking water ad libitum. The animals were were performed with the aid of a feeding randomly divided into five groups (1 – 5) syringe. The animals were then placed with five rats in each. All administration individually in separate transparent well- were done with the aid of a feeding syringe. ventilated cages and observed. Two hours Group 1 received distilled water (1 mL) and after castor oil administration, the animals served as the negative control, group 2 were humanely sacrificed by cervical received loperamide (5 mg/kg p.o.) and dislocation and the small intestines served as the positive control while groups immediately isolated after ligation of the 3, 4, 5 orally received 100, 200 and 400 pyloric sphincter and the ileo-caecal mg/kg of MHAS respectively and served as junction. Each isolated intestine was the treatment groups. One hour after weighed, the intestinal content was collected administrations of drug/treatment in all in a dry graduated measuring cylinder and groups, castor oil (20 mL/kg p.o.) was then the volume determined. Each intestine was administered to induce diarrhoea and the re-weighed after removal of content. The animals were individually placed in well- weight of the empty intestine was taken and ventilated transparent cages with floor lining the difference between the full and empty of clean paper (Adeyemi et al., 2009; Oben intestine determined. et al., 2006). The animals were observed for defecation every hour up to 3 h, which is the Ex-vivo analyses approximate time taken for solids or liquids to traverse the small bowel (Hansen, 2003) Tissue preparation and the total number of dry or wet faeces as The mice to be used were fasted for 12 h well as the total weight of faecal droplets prior to experiments, and were humanely were recorded from the pre-weighed paper euthanized under anaesthesia by cervical which lined the cages. The paper was dislocation. The intestine from duodenum to replaced every hour. The difference in time colon was gently flushed using saline between castor-oil administration, the solution. A section of the ileum was excretion of the first wet stools, the total carefully isolated, freed from attached number of faecal output, the number of wet mesenteries, and then placed in a petri dish stools excreted by each animal, and the total containing previously warmed and aerated physiological salt solution. The Tyrode’s

Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45 physiological salt solution used for the ex- extract or fraction. A contact time of vivo studies was of the following approximately 5 min was allowed following composition (in mM): NaCl 136.9, each concentration administered. NaHCO311.9, D-glucose 5.6, KCl 2.7, Statistical Analysis MgCl2 1.1, NaH2PO4 0.4 and CaCl2 1.8. The ileum was prepared with approximately 2 Results were expressed as mean ± standard mm length, and placed in a 10 mL organ error of mean (SEM). Statistical analysis of bath containing constantly aerated the data was done using One-way analysis physiological salt solution, and maintained of variance (ANOVA) followed by either at 37°C. Tissues were mounted under an Dunnett's or Tukey’s multiple comparison initial load of 1.0 g, and equilibrated for a test where appropriate. In experiments with minimum of 30 min or till stable regular sufficient datasets the concentration- contractions were obtained. The differential response plots were derived according to the amplitude of contractions generated from formula Y=Bottom + (Top- the longitudinal muscle layers of each tissue Bottom)/(1+10^((X-LogIC50))), where segment were recorded using a 7003E- IC50 is the concentration of antagonist that isometric force transducer (UgoBasile, gives a response half way between minimum Varise, Italy) connected to a 17400 data and maximum response. The number of capsule digital recorder with an inbuilt animals used were represented by n. Results bridge amplifier (UgoBasile, Varese, Italy). were considered significant in all cases Effect of extract and fractions on when P ≤ 0.05. spontaneous ileal contractions RESULTS The effect of the crude extract and fractions (0.01 – 0.3 mg/mL) on spontaneous Phytochemical screening contractions of the isolated ileum were H. acida stem bark extract was observed to investigated. The extract and fractions were yield positive results for the presence of added cumulatively (without washing) and alkaloids, flavonoids, saponins, tannins, responses observed. A contact time of triterpenoids and anthracene derivatives. approximately 3 min was allowed following each concentration administered. At the end Normal intestinal transit of the experiment, the extract or fractions In the absence of any condition or treatment, were washed off and the tissues were the charcoal meal traversed 97.51 ± 0.51 % allowed to recover. The amplitude and of the intestine in control animals, and in the frequency were assessed for each presence of atropine (the positive control), experiment. charcoal meal transit was significantly (p < Effect of extract and fractions on ACh- 0.001) inhibited up to 62.10%. Similarly, the induced ileal contractions crude extract resulted in a dose-dependent To assess the effect on agonist-induced significant inhibition at 100, 200 and 400 stimulation, the extracts and fractions (0.03, mg/kg (p < 0.05, p < 0.01 and p < 0.001 0.3 and 3.0 mg/ml) were examined on respectively). The extract at 400 mg/kg acetylcholine (ACh) – induced ileal produced the most potent inhibition among contractions. Non-cumulative (with washing the doses of extract used in this study (Table after each concentration) additions were 1). performed on ACh (0.55 nM) for each

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Table 1: Effect of H. acida stem bark on normal intestinal transit

Group Dose (mg/kg) Distance travelled by Inhibition (%) charcoal (%) Control (distilled water) 0.2 mL 97.51 ± 0.51 - Atropine 5 37.88 ± 3.02*** 62.10 ± 3.02*** Extract 100 77.43 ± 3.48* 22.56 ± 3.40* Extract 200 74.37 ± 8.15* 25.78 ± 8.13** Extract 400 55.38 ± 7.30*** 44.62 ± 7.22*** Values are indicated as mean ± SEM (n = 5 animals). ***P<0.0001, **P<0.01, *P < 0.05 vs. Control.

(One way ANOVA followed by Dunnett's Multiple Comparison Test).

Castor oil-induced diarrhoea (Table 2). The onset of diarrhoea was also The H. acida stem bark extract at all doses significantly delayed (P < 0.001) by both employed in this study (100, 200 and 400 extract at all doses and loperamide (Table mg/kg) produced a significant dose- 2). Up to 37.57% and 28.17% inhibition was dependent inhibition (P < 0.01) of total produced by the extract on faecal weight and weight and number of faecal droplet in number (Table 3) which was also diarrhoea induced by castor oil (Table 2). comparable to the inhibition produced by The inhibition produced by loperamide, the loperamide (30.12% and 15.66 % positive control drug was observed to be respectively) (Table 3). comparable to that produced by the extract

Table 2: Effect of H. acida stem bark extract on castor-oil induced diarrhoea Group Dose Diarrhoea onset Total weight Number faecal (mg/kg) (min) faecal droplet droplet (3 h) (3 h) Control 0.2 mL 30.82 ± 0.37 7.07 ± 2.04 11.22 ± 3.01 (distilled water) Loperamide 5 60.60 ± 0.54*** 2.12 ± 1.11* 1.64 ± 0.94** Extract 100 47.85 ± 0.81*** 2.72 ± 0.81** 3.27 ± 1.69** Extract 200 53.82 ± 0.62*** 1.11 ± 0.55** 1.23 ± 0.34** Extract 400 56.62 ± 0.53*** 0.82 ± 0.42** 1.02 ± 0.15** Values are indicated as mean ± SEM (n = 5 animals). ***P<0.0001, **P<0.01, *P < 0.05 vs. Control. (One way ANOVA followed by Dunnett's Multiple Comparison Test).

Table 3: Percentage inhibition of faecal weight and number by H. acida stem bark extract Group Dose Inhibition of Inhibition faecal (mg/kg) faecal weight number (%) (%) Control 0.2 mL - - (distilled water) Loperamide 5 30.12*** 15.66*** Extract 100 37.57*** 28.17*** Extract 200 15.70*** 10.96** Extract 400 12.67** 9.02* Values are indicated as mean ± SEM (n = 5 animals). ***P<0.0001, **P<0.01, *P < 0.05 vs. Control. (One way ANOVA followed by Dunnett's Multiple Comparison Test).

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Intestinal fluid accumulation inhibition produced by the extract at 400 The extract produced a dose-dependent mg/kg was observed to be more potent than inhibition of intestinal fluid accumulation at the effect produced by loperamide, the all doses used in this study. At 400 mg/mL positive control drug in this study (Table 4). the extract significantly reduced (P < 0.001) This was clearly observed in the percentage the volume of the intestinal content and inhibition of 83.56% and 69.74% produced weight of the intestine (Table 4). by the extract on the content (volume) and Loperamide also produced a significant weight of intestine respectively (Table 5) inhibition (P < 0.01) of intestinal content compared to that of loperamide which was and weight (Table 4). However, the 83.16% and 33.61% respectively (Table 5).

Table 4: Effect of H. acida stem bark extract on intestinal fluid accumulation Group Dose (mg/kg) Volume Weight intestinal content (mL) intestinal content (g) Control (distilled 1.0 mL 2.92 ± 0.25 2.38 ± 0.13 water) Loperamide 5 0.57 ± 0.28** 0.80 ± 0.18** Extract 100 0.99 ± 0.37** 1.07 ± 0.35** Extract 200 0.95 ± 0.39** 1.01 ± 0.39** Extract 400 0.48 ± 0.54*** 0.72 ± 0.12*** Values are indicated as mean ± SEM (n = 5 animals). ***P<0.0001, **P<0.01 vs. Control. (One way ANOVA followed by Dunnett's Multiple Comparison Test).

Table 5: Percentage inhibition of H. acida stem bark extract on intestinal fluid accumulation Group Dose (mg/kg) Inhibition Inhibition intestinal weight of intestinal (%) volume (%) Control (distilled 1.0 mL - - water) Loperamide 5 83.16*** 66.38*** Extract 100 43.03*** 55.04*** Extract 200 69.69*** 57.56*** Extract 400 83.56*** 69.74*** Values are indicated as mean ± SEM (n = 5 animals). ***P<0.0001 vs. Control. (One way ANOVA followed by Dunnett's Multiple Comparison Test).

Effect on spontaneous ileal contractions inhibitory response (IC50) were computed The extract and fractions produced a and CHAS produced the most significant concentration-dependent inhibition in the inhibition (P < 0.05) with an IC50 value of amplitude of spontaneous contractions 0.02 ± 0.01 mg/mL on the amplitude (Table (Figure 1). However, from the 6). The extract and fractions also inhibited concentration-response plot, CHAS was the frequency of spontaneous contractions observed to produce a more potent inhibition (Figure 2) with CHAS producing the most due to the leftward- shift in the significant inhibition (P < 0.05) on the concentration-response plot (Figure 1). The frequency with an IC50 value of 0.008 ± 0.01 concentrations producing 50% of maximum mg/mL (Table 6).

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Table 6: Contractility parameters for inhibitory extract and fractions on spontaneous contractions of the isolated ileum Extract/Fraction IC50 Amplitude IC50 Frequency MHAS 0.06 ± 0.02 0.040 ± 0.002 AHAS 0.08 ± 0.01 0.030 ± 0.007 CHAS 0.02 ± 0.01* 0.008 ± 0.01# Values are indicated as mean ± SEM (n = 5 animals). *P < 0.05 vs. AHAS; #P< 0.05 vs MHAS (One way ANOVA followed by Tukey's Multiple Comparison Test).

Effect on ACh-induced ileal contractions MHAS inhibited the amplitude of contractions at the concentrations used and significant inhibition (P < 0.05) was observed at 0.3 mg/ml (Figure 3). AHAS inhibited the amplitude of contractions but was not statistically significant (Figure 4) while CHAS significantly inhibited contractions at 0.03 mg/mL (P < 0.05) and 0.3 mg/mL (P < 0.01) (Figure 5). The effect on frequency of ACh-induced contractions was also observed. MHAS significantly Figure 1. Concentration-response curves showing effect inhibited the frequency of contractions at of the extracts and fractions on the amplitude of both concentrations of 0.03 and 0.3 mg/mL spontaneous intestinal contractions. An inhibition of the amplitude was observed. MHAS = methanol crude stem (Figure 6). AHAS significantly inhibited the bark extract of H. acida; AHAS = aqueous stem bark frequency at 0.03 mg/mL (P < 0.05) and 0.3 fraction of H. acida; CHAS = chloroform stem bark mg/mL (P < 0.01) (Figure 7) and CHAS fraction of H. acida. n= 3 animals. similarly inhibited the frequency of contractions at 0.03 mg/mL (P < 0.01) and at 0.3 mg/mL (P < 0.001) (Figure 8).

Figure 2. Concentration-response curves showing effect Figure 3. Bar plots showing the effect of MHAS (0.03 of the extracts and fractions on the frequency of spontaneous intestinal contractions. An inhibition of the and 0.3 mg/mL) on the amplitude of ACh-induced frequency was observed. MHAS = methanol crude stem intestinal contractions. A decrease in the amplitude of bark extract of H. acida; AHAS = aqueous stem bark contractions was observed. MHAS = methanol crude stem fraction of H. acida; CHAS = chloroform stem bark bark extract of H. acida. n = 3 animals. *P < 0.05; ns = fraction of H. acida. n= 3 animals. not significant.

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DISCUSSION Phytochemical analysis revealed the presence of some secondary metabolite classes which have been reported to exert anti-diarrhoea activity (Bais et al., 2014; Kavitha et al., 2004; Taiwo and Igbeneghu, 2014; Teke et al., 2010). The crude stem bark extract of H. acida was found in this study to inhibit normal intestinal transit which was similar to what was observed with atropine, a muscarinic receptor blocker. Normal intestinal transit can also be referred to as gastrointestinal tract (GIT) motility in the absence of any interference which may Figure 4. Bar plots showing the effect of AHAS (0.03 be disease, obstruction or chemicals and 0.3 mg/mL) on the amplitude of ACh-induced (Hansen, 2003). intestinal contractions. A slight decrease in the amplitude of contractions was observed. AHAS = aqueous stem bark fraction of H. acida. n = 3 animals.

Figure 6. Bar plots showing the effect of MHAS (0.03 and 0.3 mg/mL) on the frequency of ACh- induced intestinal contractions. A decrease in the frequency of contractions was observed. MHAS =

Figure 5. Bar plots showing the effect of CHAS methanol crude stem bark extract of H. acida. n = 3 (0.03 and 0.3 mg/mL) on the amplitude of ACh- animals. ***P < 0.001 induced intestinal contractions. A decrease in the amplitude of contractions was observed. CHAS = chloroform stem bark fraction of H. acida. n = 3 animals. *P < 0.05; *** P < 0.001.

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absence of a disease condition. Atropine, an Atropa belladonna alkaloid is known to be an antagonist for muscarinic receptors and blocks the parasympathetic postganglionic muscarinic receptors resulting in decreased secretions and decreased GIT motility among other effects (Bouziri et al., 2011; Lochner and Thompson, 2016; Mirakhur and Dundee, 1980). This supports the inhibition of GIT motility by atropine observed. Diarrhoea is characterised by an increase in Figure 7. Bar plots showing the effect of AHAS stool frequency and a change in stool (0.03 and 0.3 mg/mL) on the frequency of ACh- induced intestinal contractions. An inhibition of the consistency. However, both symptoms can frequncy of contractions was observed. AHAS = occur in functional disorders such as aqueous stem bark fraction of H. acida. n = 3 irritable bowel syndrome and it is animals. *P < 0.05; ** P < 0.01. considered more precise therefore to define diarrhoea as an increase in stool weight (Farthing, 2004), for this reason, these parameters mentioned were observed in this study. Diarrhoea occurs as a result of increased intestinal secretion or decreased intestinal absorption, but in some diseases diarrhoea may result from a combination of these mechanisms (Farthing, 2004). In this study, castor oil was used to induce diarrhoea in animal models. Castor oil also known as Oleum Palmae Christi, is obtained from the seeds of Ricinus communis (Scarpa and Guerci, 1982). Castor oil is a triglyceride characterized by a high content of the hydroxylated unsaturated fatty acid ricinoleic acid [(9Z,12R)-12- Figure 8. Bar plots showing the effect of CHAS hydroxyoctadec-9-enoic acid] (Tunaru et al., (0.03 and 0.3 mg/mL) on the frequency of ACh- 2012). After oral ingestion of castor oil, induced intestinal contractions. A decrease in the ricinoleic acid is released by lipases in the frequency of contractions was observed. CHAS = chloroform stem bark fraction of H. acida. n = 3 intestinal lumen, and considerable amounts of ricinoleic acid are absorbed in the animals. **P < 0.01; *** P < 0.001. intestine (Watson and Gordon, 1962). The GIT motility functions to facilitate digestion released ricinoleic acid induces a strong and proper absorption of intestinal content laxative effect (Burdock et al., 2006; Garry and fluid (Camilleri, 2004). Altered transit et al., 2000) and possibly associated pro might result from abnormal motility of the stimulation of water and electrolyte bowel (Camilleri, 2004). Inhibition by the secretion due to release of nitric oxide (NO) extract therefore suggests ability of the under conditions of mucosa damage in extract to alter GIT function even in the diarrhoea (Capasso et al., 1994). Ricinoleic

Bafor et al., Nig. Journ. Pharm. Sci., March, 2018, Vol. 17 No.1, P33-45 acid has been reported to release prostanoids an outer longitudinal and an inner circular from intestinal tissue under ex vivo layer, oriented at 90 degree angles to each conditions (Capasso et al., 1987). Ricinoleic other (Hansen, 2003). The circular layer acid has also been shown to be a selective mediates the basic contractile pattern, agonist of prostaglandin E2 subtype 3 segmentation (mixing and propulsion). The receptor (EP3) and prostaglandin E2 subtype longitudinal muscle probably does not have 4 receptor (EP4) receptors (Tunaru et al., potent propulsive capabilities, but shortens 2012). PGE2 and EP receptors have indeed the gut length and accelerates transit been implicated in the regulation of (Hansen, 2003). The ileum which was intestinal functions (Dey et al., 2006). The utilized in this study is the proximal section stem bark extract of H. acida was observed of the small intestine characterized by slow to inhibit diarrhoea induced by castor oil in wave propagation velocity which delays this study, suggesting blockade of either EP3 propulsion and permits absorption of more receptors or interaction with other slowly digested and absorbed substances mechanisms that inhibit motility and/or fluid such as fats, bile, and fat soluble vitamins retention. (Hansen, 2003). The propulsive movement of the ileum was inhibited by both the crude Altered GIT motility has been suggested to extract and its fractions with a more intense influence absorption of fluid by increasing effect observed with the chloroform fraction. or decreasing the exposure of luminal Acetylcholine (ACh), is a major regulator of content to the absorptive surface (Camilleri, GIT motility (Nelson et al., 1996) and 2004). To investigate possible activity of the stimulates intestinal phasic motor activity by effect on fluid absorption, the extract enhancing spike potential activity, resulting activity on intestinal fluid accumulation was in a contractile wave passing down the gut observed. The extract was observed to (Makhlouf, 1995) mainly through inhibit fluid accumulation and to reduce muscarinic M1 and M3 receptor contractile intestinal weight. This activity was similar mechanisms (Olsson and Holmgren, 2001). to that observed with loperamide, the Again, the extract and fractions inhibited positive control drug of this model. both amplitude and frequency of ACh- Loperamide is an orally-active drug which induced intestinal contractions, lending reduces gastrointestinal motility in animals credence to an inhibition of GIT motility by and man through interaction with opiate the extract and fractions. It was also receptors (Heel et al., 1978). The observed that the chloroform fraction observation with the extract further supports produced a more potent inhibition of ACh. interaction of the extract with GIT motility. That the chloroform fraction appeared more To further examine the extract effect on potent in this study suggests that the more intestinal motility, the extract together with active constituent inhibiting GIT motility is its aqueous and chloroform fractions were contained in the chloroform fraction and examined on the isolated ileum. The extract should therefore be prioritised for future and fractions attenuated the amplitude and characterization studies. frequency of intrinsic (spontaneous) intestinal contractions. The small intestinal wall has two organized regions of muscles: Conclusion the muscularis externa and the muscularis mucosa. The muscularis externa is the major The stem bark of H. acida has been shown effector of contractile activity. It consists of in this study to attenuate acute diarrhoea induced by castor oil and inhibit intestinal

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