Bangladesh Journal of Pharmacology Research Article EffectEffectEffect ofofof ethanolicethanolicethanolic seedseedseed extractextractextract ofofof CrotonCrotonCroton penduliflorouspenduliflorouspenduliflorous Hutch.Hutch.Hutch. on on on loperamideloperamideloperamide---induced constipated induced constipated induced constipated malemalemale ratsratsrats BJP Introduction Constipation is a highly prevalent, chronic functional gastrointestinal disorder affecting 3-15% of the general population (Higgins and Johanson, 2004; Muller- Lissner, 2009; Wintola et al., 2010; Meite et al., 2010). Etiological factors of constipation include metabolic problems, fibre deficiency, anorectal problems and drugs. Constipation which can be managed with diver- se drugs such as senna, correctol, exlax, senokot and gaviscon are also associated with shortcomings like high cost, slow and low efficacy over time. Further- more, almost half of the patients with constipation are not satisfied with the efficacy of orthodox laxatives in improving quality of life (Bengtsson and Ohlsson, 2005; Johanson and Kralstein, 2007). Therefore, the continu- ous search for a more natural, effective, affordable and readily available laxative in botanicals is imperative. One plant of interest is Croton penduliflorus. C. penduliflorus (family:Euphorbiaceae) is an important medicinal plant in southern Nigeria where it is known as aworoso by the Yorubas. It is extensively used as a remedy for several stomach complaints including constipation (Adesogan, 1981). The seeds are roasted, ground and incorporated in a ball of ‘’fufu’’ (Nigerian food made from cassava) to induce purgation (Azuzu et al., 1989). It is also claimed to be used in the manage- ment of cancer, constipation, d i a b e t e s , d y s e n t e ry, w o u n d s , f e v e r , hypercholesterolemia, hyperten- sion, inflammation, intestinal worms, malaria, pain, fibroid, ulcers and weight loss (Salatino et al., 2007; A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2012; 7: 42-46 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index; ISSN: 1991-0088 Abstract The laxative effect of ethanolic seed extract of Croton penduliflorus was evaluated in loperamide-induced constipated male rats on daily basis for 7 days. The loperamide significantly (p<0.05) reduced the feed and water intake and the fecal parameters (number, water content and weight). The feed and water intake, number, water content and weight of fecal pellets were significantly reduced in both the constipated and 200 mg/kg body weight treated animals whereas these parameters increased in the 50 mg/kg body weight. The extract at 100 mg/kg body weight produced values that compared with the unconstipated and senokot-treated animals. The body weight was not significantly altered in the 50, 100 and senokot-treated group whereas it was increased significantly in the constipated animals. The GIT ratio decreased in both the loperamide and 200 mg/kg body weight of the extract whereas the ratio at the 50 and 100 mg/kg body weight of the extract compared well with control animals. Overall, the ethanolic seed extract of C. penduliflorus produced a profound laxative activity against loperamide- induced constipated rats. Article Info Received: 15 March 2012 Accepted: 19 April 2012 Available Online: 8 May 2012 DOI: 10.3329/bjp.v7i1.10120 Cite this article: Ashafa AOT, Yakubu MT, Ogbe AAA, Abass AA. Effect of ethanolic seed extract of Croton penduliflorus Hutch. on loperamide-induced consti- pated male rats. Bangladesh J Phar- macol. 2012; 7: 42-46. Effect of ethanolic seed extract of Croton penduliflorus Hutch. on loperamide-induced constipated male rats Anofi Omotayo Tom Ashafa1, Musa Toyin Yakubu2, Abdul-Azeez Adeola Ogbe3 and Aishat Abidemi Abass3 1Department of Plant Sciences, University of the Free State, QwaQwa campus, Phuthaditjhaba 9866, South Africa; 2Phytomedicine, Toxicology and Reproductive Biochemistry Research Laboratory, Department of Biochemistry, University of Ilorin, Ilorin, Nigeria; 3Department of Botany, Lagos State University, Ojo Campus, Lagos, Nigeria. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Odugbemi, 2008; Babalola, 2009; Ojokuku et al., 2011). In addition, the plant has also been claimed to have abortifacient and contraceptive activities (Adjanohoun et al., 1991; Odesanmi et al., 2006). Previous reports have shown that C. penduliflorus seed oil contain purgative principles, antimicrobial, anti- venom, antiparalytical, rubefacient and antitumor acti- vities (Azuzu et al., 1988; 1989; Ojokuku et al., 2011). Acute toxicity study of the seed oil at the doses of 250, 600 and 800 mg/kg in mice adversely affected certain organs of the animals (Azuzu and Chineme, 1988; Ojokuku et al., 2010). Despite all these studies which focussed mainly on the seed oil, there is none in the open scientific literature that has addressed the laxative effect of the ethanolic extract of C. penduliflorus seeds in rats. Therefore, the present study investigated the effect of the solvent extract of C. penduliflorus seeds at the ethnobotanically informed doses of 50, 100 and 200 mg/kg body weight on loperamide-induced consti- pated rats. Materials and Methods Plant materials C. penduliflorus seeds were purchased at Iyana-Iba herbal market in Ojo Lagos State, Nigeria. The seeds were authenticated by Prof. O. A. Oke of the Depar- tment of Botany, Lagos State University, Nigeria. A voucher specimen (AshMed. 2010/LHB02) was deposi- ted in the Departmental Herbarium. Drugs and chemicals Loperamide hydrochloride was a product of Jiangxi Xier Kangtai Pharmaceutical Co., Pingxiang, Jiangxi, China, while carmine and carboxymethylcellulose were from Sigma Chemical Co., USA. Senekot was a product of Reckitt Benckiser Pharmaceutical (Pty) Ltd, South Africa. All other chemicals and reagents used were of analytical grade. Animals Male, albino rats (Rattus norvegicus) of Wistar strain with a mean weight of 160.4 ± 3.9 g were bred in the animal house of the Department of Biochemistry, Lagos State University, Nigeria. The animals were housed individually in clean aluminum cages placed in a well- ventilated house with optimum condition (temperature 22 ± 3°C, photoperiod; 12 hours natural light and 12 hours dark; humidity; 45-50%). The acclimatization was for 7 days during which the animals were allowed free access to commercial rat chow (Lagos State Agro- Products, Agric Farm, Ojo, Lagos) and water ad libitum. The cleaning of the cages was done daily. Animal handling were in accordance with the guidelines on the use and care of animals described by European Treaty Series (ETS, 2005). Preparation of ethanolic extract The dried seeds were pulverised and 40 g of the material was extracted in 500 mL of ethanol with constant shaking for 24 hours. This was filtered using Whatman No 1 (70 mm) filter paper and thereafter concentrated using a rotary evaporator at 45°C to give a yield of 3.4 g. This was reconstituted in normal saline to give the required doses of 50, 100 and 200 mg/kg body weight for the experiment. Induction of constipation in the rats Constipation was induced in the animals by oral administration of 1 mL of loperamide (3 mg/kg body weight in 0.9% sodium chloride for 3 days) (Bustos et al., 1991). The passage of reduced, hard and dry fecal pellets by the rats was used as a measure of constipation. Experimental design A total of 36 Wistar rats were grouped into six (1-6) comprising of six animals each. Animals in Group 1 (non-constipated control) were administered with 1 mL of normal saline while those in groups 2, 3, 4, 5 and 6 were all induced with constipation and administered with 1 mL each of normal saline, 5 mg/kg body weight of senokot, 50, 100 and 200 mg/kg body weight/day of ethanolic seed extract of C. penduliflorus respectively. The administration was done once daily for 7 days using metal oropharyngeal cannula. Indices of constipation monitored The water intake, feed intake and body weight gain of all the rats were recorded during experimental period of 7 days. Furthermore, fecal pellets from each rat were collected on daily basis at 09:00 hours throughout the duration of the experiment and their total number, weight and water content determined. The water content was computed as the difference between the wet and dry weights of the pellet. Gastrointestinal transit (GIT) ratio GIT ratio was determined according to the procedure described by Nakagura et al. (1996). Briefly, on the 7th day of extract administration, 1 mL of carmine (3 g of carmine suspended in 50 mL of 0.5% carboxymethyl- cellulose) was orally administered to the rats. An hour after the administration of the marker (carmine), the animals were humanely sacrificed and the small intes- tines quickly and carefully removed. The distance covered by the carmine and the total length of the small intestine were measured. The GIT ratio was expressed as percentage of the distance travelled by the carmine relative to the total length of the small intestine. Bangladesh J Pharmacol 2012; 7: 42-46 43 Statistical analysis Data were expressed as means ± SEM of six replicates and were subjected to one-way analysis of variance (ANOVA) followed by Duncan multiple range test for statistical difference. Values were considered statisti- cally significant at p<0.05. Results Compared with the unconstipated normal saline trea- ted animals, the loperamide significantly (p<0.05) reduced the feed and water intake by 48 and 53% respectively (Table I). In addition, the fecal parameters (number, water content and weight) reduced signifi- cantly in the loperamide treated animals (Table I). The feed intake, water intake, number of fecal pellets, water content of pellets and weight of fecal pellets were significantly reduced in the loperamide 200 mg/ kg body weight treated animals (Table II). In contrast, the 50 mg/kg body weight of the extract increased these parameters of constipation. The extract at 100 mg/kg body weight produced values of feed intake, water intake, number of fecal pellets, water content of fecal pellets and weight of fecal pellets that compared favorably (p>0.05) with the non-constipated normal saline and senokot treated positive control animals (Table II). Administration of senokot, 50 and 100 mg/ kg body weight of the extract did not significantly alter the body weight of the animals when compared with the normal saline administered animals whereas the body weight of the constipated animals increased significantly. The 200 mg/kg body weight of the extract reduced the body weight of the animals significantly (Table II). Treatment with loperamide decreased the GIT ratio of the animals (Figure 1). This pattern of decrease was extended to the animals administered with 200 mg/kg body weight of the extract. In contrast, the 50 and 100 mg/kg body weight of the extract produced GIT ratio that compared well with both the normal saline and senokot treated animals (Figure 1). Table I Effect of administration of loperamide on feed intake, water intake, and fecal parameters of rats Parameters Normal control Loperamide-induced constipated animals Feed intake (g) 16.8 ± 1.2a 8.8 ± 1.0b Water intake (g) 20.9 ± 1.3a 9.8 ± 0.8b Number of fecal pellets 71.1 ± 3.8a 26.4 ± 1.0b Water content of fecal pellets (g) 1.7 ± 0.2a 0.6 ± 0.1b Weight of fecal pellets (g) 7.0 ± 0.5a 3.2 ± 0.1b n = 6 ± SEM; Values for each parameter carrying superscript different from the non-constipated control are significantly different (p<0.05); Values in brackets are the computed percentage decrease Table II Effect of ethanolic seed extract of C. penduliflorus on some indices of constipation Parameters Constipat- ed control Senekot (5 mg/ kg b/w) Constipated animals plus Extract (mg/kg body weight) Normal control 50 100 200 Feed intake (g) 25.3 ± 1.6a 14.7 ± 1.4b 23.1 ± 1.2a 44.0 ± 2.0c 24.3 ± 1.1a 15.3 ± 0.2b Water intake (mL) 53.8 ± 2.6a 42.8 ± 2.9b 53.4 ± 2.9a 40.8 ± 4.1b 51.4 ± 5.6a 39.8 ± 5.2b Number of fecal pellets 77.8 ± 2.6a 36.8 ± 4.9b 78.0 ± 3.5a 139.1 ± 7.9c 76.6 ± 3.2a 54.7 ± 3.1d Water content of fecal pellets (g) 2.1 ± 0.1a 1.0 ± 0.04b 2.1 ± 0.1a 5.1 ± 0.1c 2.0 ± 0.05a 1.7 ± 0.1d Weight of fecal pellets (g) 8.8 ± 0.6a 3.9 ± 0.3b 9.0 ± 0.2a 15.7± 1.1c 8.6 ± 0.5a 8.5 ± 0.5a Body weight gain (g) 13.2 ± 1.0a 31.8 ± 1.0b 13.2 ± 1.1a 12.5 ± 2.1a 12.6 ± 2.0a 9.0 ± 0.1c n = 6 ± SEM; Values for each parameter carrying superscript different from the normal saline control are significantly different (p<0.05) 44 Bangladesh J Pharmacol 2012; 7: 42-46 Discussion Medicinal plants are now sources of direct therapeutic agents or as new raw material base for the synthesis of useful drugs. Greater percentage of the populace now depend on herbal remedies for the treatment of consti- pation since the orthodox laxatives are becoming increasingly expensive, unaffordable, slow-acting with its undesirable toxicological effects such as nausea, flatulence, abnormal cramping, diarrhea, colonic dama- ge and colorectal cancer (Kot and Pettit-Young, 1992; Gattuso and Kamm, 1993; Fasola and Egunyomi, 2005; Obi et al., 2006). The present study has revealed a dose specific pharmacological activity of the ethanolic seed extract of C. penduliflorus. Loperamide-induced constipation is a widely used model for spastic constipation (Takasaki et al., 1994). It causes constipation in animals by inhibiting intestinal water secretion (Hughes et al., 1984) and colonic peris- talsis (Sohji et al., 1978) which consequentially affects the flow entering the colon (Lawrence et al. 1986). These inhibitory activities extend fecal evacuation time and consequently delays intestinal luminal transit (Yamada and Onoda, 1993). In the present study, loperamide induced constipation in the animals as evidenced by the reduction in the feed intake, water intake as well as the fecal parameters. This further corroborates the use of loperamide to induce constipation. Interestingly, the extract demonstrated a dose specific laxative activity since it was only the 100 mg/kg body weight that produced the desired results that could be regarded as laxative. For instance, it was only the 100 mg/kg body weight of the extract that produced simi- lar values of indices of constipation monitored in the present study to that of the reference drug, senokot and the non constipated normal saline treated control. The 50 mg/kg body weight appears to induce diarrhea whereas the reverse was the case for the 200 mg/kg body weight. The possible bowel movement in the loperamide-induced constipated rats by the extract at 100 mg/kg body weight further suggest laxative acti- vity at this dose. The reversion of the feed and water intake in 100 mg/kg body weight treated animals also buttresses the laxative activity in the extract at this dose. It is also worthy of mention that the 100 mg/kg body weight of the extract may not predispose to overweight whereas the constipated rats administered normal saline gained weight probably due to accumu- lated fecal pellets in their body (Wintola et al., 2010) resulting from suppressed colonic movement. The transit process of the entire gastrointestinal tract reflects an overall gastrointestinal motor activity. Therefore, the increase in the GIT ratio by the extract suggests enhanced intestinal motility and colonic peristalsis in the animals. This may be due to the ability of the extract to stimulate the release of fluid which increased the intestinal secretion. Overall, this study has revealed that oral administration of ethanolic seed extract of C. penduliflorus exhibited dose specific laxative activity at 100 mg/kg body weight in loperamide-induced constipated animals. References Adesogan EK. The structure of penduliflaworosin, a new furanoid diterpene from Croton penduliflous. J Chem Soc Perkin Trans. 1981; 1: 1151-53. Adjanohoun E, Ahiyi MRA, Assi LA, Dramane K, Eewude JA. Tradition al medicine and pharmacopoeia: Contribu- tion to ethnobotanical and organization of Africa. Unity/ G IT R a tio ( % ) 100 80 60 40 20 0 Normal saline control Constipated 50 mg/kg body weight extract 100 mg/kg body weight extract 200 mg/kg body weight extract Senekot (5 mg/kg body weight) b a a a a c Figure 1: GIT ratio of C. penduliflorus seed extract-treated constipated rats Bangladesh J Pharmacol 2012; 7: 42-46 45 Author Info Anofi Omotayo Tom Ashafa (Principal contact) e-mail: ashafaaot@qwa.ufs.ac.za Scientific Technical and Research Commission, 1991, p 420. Azuzu JU, Shetly SW, Anika SM. The toxic effect of chronic administration of gut-stimulating principle of Croton penduliflous Hutch seed in mice. Drug Chem Toxicol. 1989; 12: 85-93. Azuzu IU, Chineme CN. Acute toxicity and gastrointestinal irritant effect of Croton penduliflorus seed oil in mice. Phytoter Res. 1988; 2: 46-50. Azuzu IU, Gray AI, Waterman PG. The extraction, isolation and identification of the purgative component of Croton penduliflorus seed oil. J Ethnopharmacol. 1988; 23: 267-71. Babalola AK. An examination of the usage of herbal contraceptives and abortifacients in Lagos State, Nigeria. Ethnobot Leaflets. 2009; 13: 146-46. Bengtsson M, Ohlsson B. Psychological well-being and symptoms in women with chronic constipation treated with sodium picosulphate. Gastroenterol Nurs. 2005; 28: 3-12. Bustos D, Ogawa K, Pons S, Soriano E, Banji JC, Bustos FL. Effect of loperamide and bisacodyl on intestinal transit time, fecal weight and short chain fatty acid excretion in the rat. Acta Gastroenterol Latinoam. 1991; 21: 3-9. Fasola TR, Egunyomi A. Nigerian usage of bark in phyto- medicine. Ethno Res Appli. 2005; 3: 73-77. Gattuso JM, Kamm MA. The management of constipation in adults. Alimen Pharmacol Ther. 1993; 7: 487-500 Higgins PD, Johanson JF: Epidemiology of constipation in North America: A systematic review. Am J Gastroenterol. 2004; 99: 750-59. Hughes JR, Hatsukami DK, Pickens RW, Krahn D, Malin, S, Luknic A. Effect of nicotine on the tobacco withdrawal syndrome. Psychopharmacology 1984; 83: 82-87. Johanson JF, Kralstein J: Chronic constipation: A survey of the patient perspective. Aliment Pharmacol Ther. 2007; 25: 599- 608. Kot TV, Pettit-Young NA. Lactulose in the management of constipation: A current review. Ann Pharmacother. 1992; 26: 1277-82. Lawrence RS, Carol ASA, Stephen GM, John SF. Mechanism of the antidiarrheal effect of loperamide. Gastroenterology 1984; 86: 1475-80. Meite S, Bahi C, Yeo D, Datte JY,Djaman JA, N’guessan DJ. Laxative activities of Mareya micrantha (Benth.) Mull. Arg. (Euphorbiaceae) leaf aqueous extract in rats. BMC Compl Alter Med. 2010; 10: 7. Muller-Lissner S: The patholophysiology, diagnosis and treatment of constipation. Dtsch rtztebl Int. 2009; 106: 424-32. Obi RK, Iroagba II, Ojiako OA. Virucidal potential of some edible Nigerian vegetables. Afr J Biotechnol. 2006; 5: 1785-88. Odesanmi OS, Saibu GM, Ojokuku SA, Faremi AT, Bakare RI, Banjo AF. Comparative metabolic and histophatological effects of Croton penduliflorus (a herbal abortifacient) and depo provera in non-pregnant and pregnant Dutch white rabbits. In: Traditional and modern health systems in Nigeria. Falola T, Heaton M (eds). Eritrea, Africa World Press, 2006, pp 130-46. Odugbemi T. A textbook of medicinal plants from Nigeria. Nigeria, University of Lagos Press, 2008, p 588. Ojokuku SA, Odesanmi OS, Magbagbeola OA. The effects of oral administration of Croton penduliflorus seed oil and medroxyprogesterone acetate on fasting blood sugar, lipid and haematology of pregnant rabbits. Intl J Trop Med. 2011; 6: 35-38. Ojokuku SA, Odesanmi OS, Magbagbeola OA. The effects of oral administration of Croton penduliflorus seed oil and depo provera on liver and kidney functions of pregnant Dutch white rabbits. Int J Bio Chem Sci. 2010; 4: 424-31. Nagakura Y, Naitoh Y, Kamato T, Yamano M, Miyata K: Compounds processing 5-HT3 receptor antagonistic acti- vity inhibit intestinal propulsion in mice. Eur J Pharmacol. 1996; 311: 67-72. Salatino A, Salatino MLF, Negri G. Traditional uses, chemistry and pharmacology of croton espécies (Euphorbiaceae). J Braz Chem Soc. 2007; 18: 11-33. Shoji Y, Kawashima K, Shimizu M. Pharmacological studies of loperamide, an anti-diarrhea agent. Folia Pharmacol Jpn. 1978; 74: 155-63. Takasaki K, Kishibayashi N, Ishii A, Karasawa A. Effects of KW-5092, a novel gastroprokinetic agent, on the delayed colonic propulsion in rats. Japan J Pharmacol. 1994; 65: 67-71. Wintola OA, Sunmonu TO, Afolayan AJ. The effect of Aloe ferox Mill. in the treatment of loperamide-induced constipa- tion in Wistar rats. BMC Gastroenterol. 2010; 10: 95. Yamada K, Onoda Y. Comparison of the effects of T- 1850, yohimbine and naloxone on mouse colonic propulsion. J Smooth Muscle Res. 1993; 29: 47-53. 46 Bangladesh J Pharmacol 2012; 7: 42-46 DatePrinted: This article was downloaded by you on: Nov 22, 2017