Indian Journal of Experimental Biology Vol. 42, August 2004, pp. 787-791

Effect of aqueous leaf extract of on gastrointestinal tract in rodents

F Abdulrahman", I S In yang", J Abbahb*, L Bindab, S Amosb & K Gamanielb "Department of Chemistry, University of Maiduguri, Borno State, Nigeria bDepartment of Pharmacology and Toxicology, National Institute for Pharmaceutical Research and Development, Garki, Abuja, Nigeria Received 23 May 2003; revised 29 March 2004

Effect of th e aqueous leaf extract of l.gabonensis on the gastrointestinal tract was investigated on isolated rabbit jejunum, guinea pig ileum, gastrointestinal motility, castor oil-induced diarrhoea in mice and castor oil-induced fluid accumulation in rats. The results showed that the extract exhibited a concentration-dependent relaxation of spontaneous pendular movement of isolated rabbit jejunum and guinea pig ileum, and attenuated both acetylcholine-induced contraction of rabbit jejunum and hi stamine-induced contraction of guinea pig ileum. The extract ( 100, 200 and 400 mg/kg) also caused a significant dose-dependent decrease of gastrointestinal motility in mice (40. 12, 39.45 and 37.45%), intestinal fluid accumulation in rats (71.43, 81.63 and 83.27%), and remarkably protected mice against castor oil-induced diarrhoea [58.33, 75 and 91.67% (Di Carlo score)] respectively. Preliminary phytochemical screening of the aqueous leaf extract of /. gabonensis revealed th e presence of saponins, tannins, phenols and phlobatanins. Keywords: Diarrhoea, Gastrointestinal tract, lrvingia gabonensis

7 IPC Code: In t. Cl. , A 6 1 P

Diarrhoea is a condition characterized by an abnormal popularly known as wild or African . increase in liquidity, frequency of defecation and The plant occurs freely in many parts of Africa. Local stool weight exceeding 200 g daily and containing 60- names include goron biri (Rausa), Ogbono (Ibo). The 95 % water. It is a common disease in tropics and in plant is commonly used as food supplement. In 1 some parts of the world • It is a prime cause of illness Nigeria, oily seeds of the plant are used in soup as a 1 5 and mortality especially in infants and children • seasoning for various native dishes . Diarrhoea occurs at all ages of human, but its In addition, a decoction of the leaf is employed in incidence is higher in children between 6 month and 3 southern Nigeria to treat diarrhoea (personal 2 year . On a worldwide basis, 750 million cases are communication/oral testimony). Recently, Raji et a/. 6 reported in children below 5 years in Asia, Latin have reported anti-ulcer properties of /. gabonensis. 3 America and Africa resulting in 4-5 million deaths • The present study was designed to evaluate the anti­ However, there are seasonal vartat10ns in the diarrhoeal effect of/. gabonensis. 2 incidence of diarrhoea . In an effort to tackle the problems of diarrhoea, the World Health Organization Materials and Methods (WHO) has established a diarrhoea disease control Animals-New Zealand rabbits (1.5-3.0 kg), Swiss programme (DDC) which includes studies of albino mice (18-25g), Adult Wistar rats (180-220g) traditional medicinal practices together with the and adult guinea pigs (300-400g) of either sex were evaluation of health education and prevention maintained at the Animal Facility Centre, National 3 approaches .4 . In most parts of the developing Institute for Pharmaceutical Research and countries, particularly Africa, the use of herbal Development (NIPRD), Ahuja. These animals were remedies in management of diarrhoea is a common fed with standard diet (Ladokun Feeds Ltd, Ibadan) practice. Irvingia gabonensis () is a and given water ad libitum. Experimental procedures were conducted following *Correspondent author Phone: 234-9-5239089; Fax: 234-9-5231043 the Principles of laboratory animal care (NIH E-mail: [email protected] Publication 85-23, revised 1985), as adopted by the 788 INDIAN J EXP BIOL, AUGUST 2004

Ethical review committee of National Institute for investigated. Responses were recorded isometrically Pharmaceutical Research and Development on an Ugo Basile Unirecorder (7050), through 8 Plant material-Leaves of lrvillgia gabonensis isometric transducer (7004) . were collected from Etim Osam, Akpabuyo, Cross Studies on guinea pig ileum-Adult guinea pigs River State, Nigeria in September 2002. The plant were killed and bled out. Their abdomens were was identified and authenticated by Mallam Ibrahim opened and the ceacum exposed. For this experiment, Muazzam and Grace Ugbabe of the National Institute the terminal portions (2-3 em in length) were used for Pharmaceutical Research and Development after discarding the 10 em portion nearest to the (NIPRD), Abuja. A voucher specimen of the plant ileoceacal junction. The tissue ·was suspended in 5395 was deposited at NIPRD herbarium. organ bath (20 ml) containing Tyrode solution at Preparation of extract-The freshly collected 37°±1 °C aerated with 0 2 (95%) and C02 (5 %). This leaves of I. gabonensis were washed with water and solution was comprised (mM) - NaCI, 136.8; KCI, dried under sun. The dried leaves were ground using a 2.7; CaCI2. 1.3; NaHC03, 12.0; MgCb. 0.5; Na2P04, mechanical grinder. The powder (84 g) was then cold 0.14; and glucose, 5.5. The initial tension was 0.5g. macerated with 1.5 I of water for 24 hr with An equilibration period (60min) was allowed during continuous shaking using GMB shaker. The extract which the physiological solution was changed for was filtered through Whatman paper (No. I) and the every 15min. At the end of the equilibration period, filtrate was dried completely over water bath to give a effect of graded concentrations of histamine, extract solid residue (14.29% w/w yield). and mepyramine were investigated. Contact time for Drug-Acetylcholine chloride, histamine, atropine, each concentration of drug was 60 sec, which was and castor oil were obtained from Sigma Chemical followed by washing three times. The tissue was Company, USA; mepyramine from May & Baker, allowed a resting period of 15min in between drug Pharmaceuticals, Nigeria. addition. Inhibitory effect of the extract and Phytochemical tests-The freshly prepared extract mepyramine on histamine-induced contraction was was subjected to a standard phytochemical screening also established. Responses were recorded on Ugo 7 9 test for various constituents . The extract was Basile Unirecorder (7050) . screened for the presence of alkaloids, glycosides, Studies on gastrointestinal motility test in saponins, tannins, flavonoids and phlobatanins etc mice--Effect of the extract on small intestinal transit using conventional protocols. in unanaesthetised mice were tested using the 3 17 charcoal method · . In brief, overnight fasted mice Studies on rabbit jejunum-The rabbits were killed were randomly divided into four groups of 6 animals by a blow to the head, exsanguinated and their abdomens were opened. Segments of the jejunum each. Mice in group I were given normal saline (20 about 2-3 em long were removed and dissected free of ml/kg) intraperitoneally (ip), while those in groups II, adhering mesentery. The intestinal content was III and IV received the extract (100, 200 and 400 removed by flushing with Tyrode's solution mg/kg; ip) respectively. Five minutes after drug administration, 0.5ml of charcoal suspension (5 %) in comprising (mM)- NaCI, 136.8; KCI, 2.7; CaCb. 1.3; suspension of tragacanth powder (1 0%) was NaHC03. 12.0; MgCb. 0.5; Na2P04. 0.14; and glucose, 5.5. The tissue was mounted in organ bath administered per oral (po) to each mouse. All the (20 ml) containing Tyrode's solution at 37°±1 oc and mice were killed 30 min after treatment. The abdomen opened and the distance travelled by the charcoal plug aerated with air. A load of 0.5 g was applied. An from pylorus to ceacum was determined and equilibration period (60 min) was allowed during expressed as a percentage of the total length of the which the physiological solution was changed for 10 small intestine • every 15 min. After equilibration period, the effects of graded concentrations of acetylcholine, atropine and Studies on castor oil-illduced diarrhoea-This the extract were evaluated. The contact time for each experiment was carried out using mice. The animals concentration of drug was 60sec, which was followed were fasted for 16 hr prior to experiment and were by washing three times. The tissue was allowed a randomly divided into five groups having 6 animals in resting period of 15 min in between drug addition. each group. Animals in group I were pretreated with Inhibitory effect of the extract and atropine on normal saline (20 mllkg; ip), groups II, Ill and IV acetylcholine-induced contraction was also animals received the extract (100, 200 and 400 mg/kg; ABDULRAHMAN et a!.: EFFECT OF/. GABONENSIS EXTRACT ON GASTROINTESTINAL TRACT 789 ip) respecti vely, while group V animals were atropine (5 .0 x 10"9M) on acetylcholine-induced administered loperamide (5 mg/kg; ip). After 30 min contraction (Fig. 2). of drug administration, diarrhoea was induced by oral Effect on guinea pig ileum- The extract caused a 11 administration of castor oil to mi ce (0.5 mllanimal) • concentration-dependent inhibition of guinea pi g The animals were pl aced in individual cages and over ileum. The extract also attenu ated the histamine­ 7 6 clean filter paper. Four hour after oil challenge, the mediated (4.5 X 10· - 7.2 X 10· M) contraction of mice were inspected (by an observer unaware of the guinea pi g il eum in a concentrati on related manner 9 particul ar treatment) for the characteri stic droppings; similar to th at of mepyramine (2.49 X 10· M) (Fig. 3). their absence was recorded as a protecti on from Effect on gastrointestinal transit studies-The diarrhoea 12 and the percentage protecti on 3 12 extract signi ficantly decreased the gastrointestin al calcul ated • • di stance travell ed by th e charcoal plug in mi ce, In addition, onset time and severity of diarrhoea compared with the control. The effect appeared to be were noted. Severity were recorded as scores using Di 13 dose related (Table 1). Carl o et a!. scoring method ( ++ fo r copi ous, + mild Effect on castor oil test-The extract significantl y and 0 for lack of di arrhoea). protected mice again st castor oil-induced diarrhoea Studies 0 11 castor oil-induced fluid accumulation­ The method of Di Carl o et al. 13 was used fo r thi s 12 0~------, study. Briefly , overnight fasted rats were randoml y r::: 100 di vided into fo ur groups having 5 animals in each .!2 1U )( 80 group. Animals in group I were administered with CG normal saline (20mllkg; ip), wh ile those in groups II , a; 0:: 60 iV III , and IV received th e ex tract (I 00, 200 and 400 E ";( mg/kg; ip) respectively. After 30 min of CG 40 ad ministrati on of normal saline and extract, castor oil :::E ~ (2 mllrat; po) was given. The animals were killed by 20 inhalati on of chloroform 30 min later. The small in testine was li gated at both pylori c sphincter and 0.05 0.1 0.2 0.4 0.8 1.6 3.2 il eoceacal juncti ons. The entire small intestine was Extract Concentration (mg/ml) di ssected out. The contents of the small intestine were expell ed into a graduated measuring cylinder and th e Fig. !-Concentrati on-dependent relaxant effect of the aqueous volume of contents was recorded. extract of /. gabonensis on the spontaneous contracti on of th e rabbit jej unum [*P < 0.05 Wilcoxan' s rank sum test] . Statistical analysis-The results were expressed as median and range. Chi-square test was used for di arrhoea study, while Wilcoxan's rank sum test was 120 used fo r analysis of gastrointestinal transit test, c: -Acetylcholine 0 100 studies on intraluminal fluid accumulati on, isolated -.-Ext 0.8 mg/ml nftl il eum of guinea pi gs and jejunum of rabbits, and ... 80 -+-Ext 1.6 mg/ml "E -+-Atropine resul ts were regarded as ~ i g ni f i ca nt at P < 0.05. 0 (.) 60 iii E Results ")( 40 ftl Phytochemical test-The extract gave positive tests :::E :::!! 20 - fo r saponins, tanni ns, phenols, alk aloids and 0 phl obatani ns. 0 Effect on rabbit jejunum-Acetylcholine (2.5x l o·9 -8.6 -8.3 -8 -7.6 -7 .3 -7 - I.Ox i0-7 M) caused a concentrati on-dependent Log concentration (M) of Acetylcholine contraction of rabbit jejunum, while the extract produced a concentrati on-dependent inhibition of Fig. 2-Effect of the aqueous extract of I. gabonensis on the spontaneous contraction of jejunum (Fi g. I). The contractil e response induced by acetylcholine on rabbit jejunum. Acetylcholine (ACH) alone (• ), ACH + I. gabonensis 0.8 mg/ml extract also attenu ated the acetylcholine-medi ated ( • ), ACH + I. gabonensis 1.6 mg/ml ( + ) and ACH + atropine contraction of rabbit jejunum similar to the effect of (• ) [*P < 0.05 Wi lcoxan's rank sum test] . 790 INDIAN 1 EXP BIOL, AUGUST 2004 when compared with the control as evident in the actlvtty of the extract is provided by the results of prolongation of onset, decrease in severity and gastrointestinal transi t studies in which the extract reduction in the animal population with diarrhoea. significantly inhibits the gastrointestinal motility in Loperamide (Smg/kg; ip) also significantly protected mice, dose-dependently. Agents that reduce intestinal 13 mice against castor oil- induced diarrhoea. The extract moti lity are known to possess anti-diarrhoea effect . effect was dose-dependent (Tables 2, 3). Our results also showed that intra-luminal fluid Effect on castor oil-induced fluid accumulation accumulation induced by castor oi l was significantly test-There was dose-dependent decrease in intestinal inhibited in a dose-related fashion. Enteropooling fluid accumulation. The extract significantly reduced assay has been proposed as a model for investigating (62 to 84%) the intestinal fluid accumulation diarrhoea produced by prostaglandins, and drugs compared to control (Table 4). 1 20 ~------. c: 0 100 -Histamine Discussion :;::; C.) __.._ Ext 0.8 mg/ml The results of the present study suggested that the ...nl 80 c: -+-Ext 1.6 mg/ml aqueous extract of Irvingia gabonensis had significant -0 (.) 60 . anti-diarrhoeal effect. The extract, in a dose­ iii E dependent manner, significantly protected mice ")( 40 against diarrhoea induced experimentally by castor oil nl == 20 in terms of severity and onset, and the population of 0~ 16 animals with diarrhoea. Castor oil is a ricinoleate 0 and its diarrhoea inducing property is known to be -6.3 -6 -5.7 -5.4 -5.1 -4 .9 due to its active metabolite ricinoleic acid which log concentration (M ) of Histamine increases peristaltic activity and alters permeability of the intestinal mucosa membrane to electrolytes, Fig. 3--Effect of the aqueous extract of /. gabonensis on the particul arly Na+ and Cr (ref. 17) and water, effects contractile response induced by hi stami ne on guinea pig ileum. associated with endogenous stimul ation of Histamine (hist) alone (• ), hi st + I. gabonensis 0.8 mg/ml ( .._ ), 14 hist + I. gabonensis 1.6 mg/ml ( + ) and hi st + mepyramine (• ) prostaglandin release • Release of prostaglandins is [*P < 0.05 Wilcoxan's rank sum test]. also a major cause of arachidonic acid-induced 18 diarrhoea . Further evidence of the anti-diarrhoeal Table 2-Effect of aqueous leaf extract of I. gabonensis on Table !-Effect of aqueous leaf extract of /. gabonensis on castor oil- (0.5ml/ mouse) induced diarrhoea in mice: gastrointestinal transit in mice. population of animals with diarrhoea

Distance travell ed(%) Drug/Cone. (ml/kg; ip) Mice with Protection(%) Drug/Cone. (ml/kg; i.p.) Median Range diarrhoea Normal saline 20 6/6 0 Normal saline 20 68.86 18.25 Extract 100 3/6 50* Extract 100 40.12 14.75* 200 2/6 66.67* 200 39.45 14.23* 400 1/6 83.33* 400 37.45 12.55* Loperamide 5 0/6 100*

*Statistical difference between treated groups and control. *Statistical difference between treated gro ups and control.

Table 3--Effect of aqueous leaf extract of I. gabonensis on castor oil-induced diarrhoea

Drug/Cone. (ml/kg; ip) Diarrhoea score Total Score % inhibition ++ + 0

Normal saline 20 6 0 0 12 Extract 100 2 0 5 58.33 * Extract 200 I 0 3 75.00 * Extract 400 0 I 0 I 91.67 * Loperamide 5 0 0 0 0 100 *

*Statistical difference between treated groups and control. ABDULRAHMAN eta/.: EFFECT OF/. CABONENSIS EXTRACT ON GASTROINTESTINAL TRACT 791

Table 4--Effect of aqueous leaf extract of/. gabonensis on Hunter, 1 C Swartzwelder and D F Clyde (W B Saunders castor oil-induced intestinal fluid accumulation Co., Philadelphia) 1976. 2 WHO, The management and prevellfion of acute diarrhoea: Drug/Cone. (ml/kg; ip) Volume of Range % Practice guidelines, 3rd edition (Geneva, WHO) 1993. intestinal inhibition 3 Abdullahi A L, Agho, M 0 , Amos S, Gamaniel K S & fluid (ml) Wambebe C, Antidiarrhoeal activity of the aqueous extract of Terminalia avicennoides roots. Phytother Res, 15 (200 I) Normal saline 20 2.45 2.7 431. Extract 100 0.70 0.8 71.43* 4 Syder 1 H & Merson M H, The magnitude of the global 200 0.45 0.31 81.63* problem of acute diarrhoeal disease: A review of active 400 0.41 0.3 83.27* surveillance data. Bull WHO 60 ( 1982) 605. 5 Dalziel 1 M, Th e useful of west tropical Africa, The *Statistical difference between treated groups and control. crown agents for the colonies (Royal botanical garden, Kew) 1937,312. affecting intestinal secretion also possess anti- 6 Raji Y, Ogunwande I A, Adesola 1 M. & Bolarinwa A F, d1arr. h oea I act1v1ty. . 13· 17 . F urt h ermore, t h e extract Anti-diarrhogenic and anti-ulcer properties of lrvingia inhibited spontaneous and agonist (acetylcholine) gabonensis in rats. Pharmaceut Bioi, 39 (5) (200 I) 340. mediated contraction of rabbit jejunum. It is well 7 Trease G E & Evans M C, Textbook of pharmacognosy, 12th edition (Bailliere: London) 1983,343. known that acetylcholine stimulates smooth muscles 8 Amos S, Okwuasaba F K, Gamaniel K, Akah P & of the gastrointestinal tract through actions on the Wambebe C, Inhibitory effects of the aqueous extract of muscarinic receptors. Effect of the extract this way is Pavetta crasipes leaves on gastrointestinal and uterine indicative of possible cholinergic involvement. smooth muscle preparations isolated from rabbits, guinea Similarly, the extract blocked the contractions pigs and rats, 1 Etlmopharmacol, 61 (1998) 209. 9 Tarfa F D, Amos S, Temple V 1, Binda L, Emeje M, induced by histamine on guinea pig ileum, suggesting Obodozie 0, Wambebe C & Gamaniel K, Effect of the that histaminergic receptors might be involved in the aqueous extract of African Mistletoe, Tapinanthus observed effect as well. The above finding is, sessifolius (P. Beauv) Van Teigh leaf on gastrointestinal therefore, suggestive of non-specific mechanism of muscle activity, Indian 1 Exp Bioi, 40 (2002) 571. action of anti-diarrhoeal activity of the extract. I 0 Akah P A & Offiah V N, Gastrointestinal effects of Allamanda cathartica leaf extracts, lnt 1 Pharmacogn, 30 Though, the compound responsible for the observed (1992) 213. actions is unknown, tannins present in the extract II Diurno M V, Izzo A A, Mazzini 0 , Balognese A & Capasso might have made the intestinal mucosa more resistant F, Anti-diarrhoeal activity of new thiazolidinones related to 15 and reduced the secretion . The overall data loperamide, 1 Ph arm Phannaco/, 48 ( 1986) 760. presented suggested that the aqueous extract of I. 12 Izzo A A, Mascolo N, DiCarlo G & Capasso F, NG- Nitro- 1-arginine methyl ester modulates intestinal secretion and gabonensis possesses anti-diarrhoea activity. Further motility produced by carbachol, Eur 1 Phannaco /, 271 studies to isolate the active components are in (1994) 31. progress in our laboratory. 13 DiCarlo G D, Mascolo N, Izzo A A & Capasso F, Effects of quercertin on the gastrointestinal tract in rats and mice, Acknowledgement Phytother Res, 8 ( 1994) 42. This work was supported by a grant from the 14 Luderer 1 R, Demers L M, Normides C T & Hayes A H, University of Maiduguri in collaboration with the Mechanism of action of castor oil: A biochemical link to the National Institute for Pharmaceutical Research and prostaglandins in Advances in Prostaglandin and Thromboxane Research, edited by Samuelsson P W (Raven Development. The authors are grateful to Augustine Press, NY) 1980. Iyagba, Tags Zachariah, Hauwa Abdullahi and 15 Tripathi K D, Essentials of medicinal pharmacology (Jaype Charles Balogun for their technical/secretarial Brothers, Medical Publishers (P), New Delhi), 225. assistance. 16 Mckeon T A, Lin 1 1 & Stafford A E, Biosynthesis of ricinoleate in castor oil, Adv Exp Med Bioi ( 1999) 46437. References 17 Adzu B, Amos S, Amizan M B & Gamaniel K, Evaluation 1 Weber D M, The diarrhoeal disease and food-borne of the anti-diarrhoeal effects of Zizyphus spina-christi stem 1 illnesses in Tropical medicine, 5 h edition, edited by G W bark in rats, Acta Trop 87 (2003) 245.