Histomorphological study on gastric mucosa of albino wistar rats

Research Article ISSN 2277-3657Available online at www.ijpras.comVolume 2, issue 3 (2013),31-37International Journal ofPharmaceutical Research &Allied Sciences<strong>Histomorphological</strong> Study of the Effect of ChronicConsumption of Abelmoschus Esculentus and PiperGuineense on the Gastric Mucosa of Albino Wistar RatsIdorenyin Umoh, Samson Oyebadejo, Eno-obong Bassey and Noah UdohDepartment of Human Anatomy, Faculty of Basic Medical Sciences, University of Uyo, Akwa- Ibom,Nigeria.E-mail: samsonoyebadejo@yahoo.comSubject: PharmacologyAbstractThe effects of Abelmoschus esculentus (okra) and Piper guineense (black pepper) on thehistomorphology of the gastric mucosa of the fundic stomach were investigated. Twenty adult wistarmale rats weighing (123-207g), divided into four groups 1, II, III &1V, group 1 as control and groupsII, III &IV as experimental groups. The rats in the control group were administered with distilled water,while rats in group II and III were administered with 500mg/kg of Abelmoschus esculentus and20mg/kg of piper guineense respectively. Group IV received a combination of the two extracts. After28days of administration of extracts, animals were sacrificed stomach was extracted and processed toparaffin section, cut at 5micron, stained, and observed histopathologically under light microscope.Result showed marked significance of the effect of the single extract of Abelmoschus esculentus, Piperguineense and the combination of both extracts, while the histopathology findings revealed glandularerosion, mucosal erosion, inflammation, ulceration, degeneration of mucosal lining cells andepithelium, degeneration of chief and parietal cells and granulated eosinophilic cells as compared tocontrol group 1. Statistical value in the weight of the body and stomach showed significant value(p

Available online at www.ijpras.comAbelmoschus esculentus, popularly knownamong the Ibibio people of South-SouthernNigeria as “Etighi”, is used for the treatment ofgastric problem on account of its mucilaginouscontent. It has also been reported that the fruitof this medicinal plant is considered anemollient, demulcent and diuretic withstimulant and vulnerary activities (James.1993). This long green pod vegetable with agreen fury outer skin, thin green flesh andsmall edible white seed is eaten in manydifferent ways like; stir fried, cooked in soup,prickled, cooked in meat stew, with soy sauce,with sugar, with rice, deep fried, and even withfresh salads. The seeds are useful in theproduction of Okra oil (Mays, 2007).Piper guineense, also known as African blackpepper or hot leave is widely consumed insome part of West Africa especially Nigeriaand Ghana on account of its nutritional andMedicinal properties (Negbenebor, 1999).Piper guineense also known as Kale, Guineapepper, Sasema, leulaube, Sorowiso, Guineacubeb, Masoro, false cubeb pepper, Beninpeper. Like other members of the pepperfamily, Piper guineense is a climbing vine thatcan grow up to twenty meters in length, withglossy leaves which are about six inches long(Delziel, 1937). According to Irvine, they arenative to the tropical regions of central andWestern Africa and Semi-cultivated incountries such as Nigeria where the leavesknown as “etinkene, odusa among theEfik/Ibibo and “Uziza” among the Igbo areused as flavouring for stew.The medicinal value of this African speciesranges from the treatment of respiratorydiseases to correction of female infertilityproblems, control of weight uterine contractionfor the expulsion of placenta and other remainsfrom the womb (Udoh, 1999). Theantiparasitic, antimicrobial and antifungalactivities of the leaf and seeds of PiperGuineense have also been reported (Ekanemand Obiekezie, 2000; Ngane, 2003; Ekanem,2010).However, it has been reported that seed ofAbelmoschus esculentus have antioxidanteffect (Ansari, 2009). Antioxidant potential ofthe seeds of Abelmoschus esculentus and thetraditional use of this plant in the treatment ofgastrointestinal disorders has been the rationalefor this <strong>study</strong>.On the other hand, studies have revealed thatpiperine, found in Piper guineense couldprevent gastrointestinal disorders developingfrom stress, hyperacidity and overgrowth ofHelicobacter Pylori (Bai, 2000). However,possible side effects of taking Piper guineensein medicinal amount by mouth may includestomach upset or other gastrointestinal adverseeffects (Mc Namara, 2005),this signify thepurpose in which is why this <strong>study</strong> isundertaken to <strong>study</strong> the chronic consumptionof the plant when taking together to establishthe histopathological effect .2.0 Materials and Methods2.1 Drugs and ChemicalsSodium chloride, formaldehyde, sodiumtrioxocarbonade V, sodium bicarbornate,xylene, 70% alcohol, 90% alcohol, absolutealcohol, haematoxylin, eosin, egg albumin,distilled water, paraffin wax were all procuredfrom BDH Chemicals, England. All otherchemicals were of analytical grade.2.2 Animals20 wistar rats (123-207g) were obtained fromthe University of Uyo animal house. Theywere maintained on standard pellets (guineafeed) and water ad libitum. Permission andapproval for animal studies were obtainedfrom the college of health sciences animalethnics committee, University of Uyo.2.3 Sourcing of Plant materialFreshly fruit of Abelmoschus esculentus andpiper guineense were obtained in July, 2012from Itam market, Uyo, Akwa Ibom State,Nigeria. The plant was identified andauthenticated by the Department of Botany,University of Uyo, Uyo, Nigeria.2.4 Preparation of ExtractAbelmoschus esculentus was chopped and airdried. Piper guineese (seeds) was also air driedand after being dried they weighed 600g forAbelmoschus esculentus and 800g for Piperguineense. They were then macerated in 97%ethanol (SIGMA CO., UK) in a flat bottomflask and were kept for 72hrs at roomtemperature. At the end of 72hrs it wasfiltered. The filtrates were concentrated inwater bath at 45 degree Celsius. Theconcentrated extract was preserved inrefrigerator till commencement of research.The weight of the extracts was 40.25g forAbelmoschus esculentus and 24g for Piperguineense.2.5 Acute Toxicity testing.The acute toxicity of Abelmoschus esculentusand Piper guineense on Wistar Albino ratswere determined in two (2) stages for the twoextracts.For Abelmeschus esculentus, in stage oneanimals received 1000, 2000, 3000, 4000 and32

Available online at www.ijpras.com5000mg/kg body weight while in stage two,animals received 2300, 2400, 2500, 2600,2700mg/kg body weight.In acute toxicity of Piper guineensethe same two stages was observed, in stageone; animals received 10, 50, 100, 200, 300mg/kg body weight. Stage 2; received 85, 90,95, 100, 105 mg/kg body weight.All experimental animals were observed forphysical signs of toxicity such as writhing,gasping, palpitation, decreased respiratory rate,body limb and death within 24hours. Theextract was administered intraperitoneally(i.p). The LD50 was found to be 2500mg/kgfor Albemoschus esculentus and 100mg/kg forPiper guineense .According to the modified lorke’smethod.500mg/kg and 20mg/kg per bodyweight were calculated respectively as middledoses for the Albemoschus esculentus andPiper guineense. Doses were considered asstock solution, they were calculated furtherusing 20mls of distilled water for Albemoschusesculentus and 10 mls of distilled water forPiper guineense to obtain working solution.2.6 Experimental Design/Study designMatured 20 albino wistar male rats weighingbetween 123-207g were obtained from thefaculty of Basic Medical SciencesExperimental Research Animal House of theUniversity of Uyo, Uyo Nigeria. They werefed with standard laboratory diet and water adlibitum. Illumination was 12h light /dark cycleand room temperature was 25±2 o C. Theanimals were divided into four groups, onecontrol (1) and three experimental groups (II,III and IV), which consisted of 5 normal abinowistar rats per group. The control group wasgiven distilled water while the experimentalgroup II, III and IV were exposed daily to500mg/kg body weight of Abelmoschusesculentus alone, 20 mg/kg of body weightpiper guinenese alone and 500 mg/kg ofAbelmoschus esculentus combined with 20mg/kg of piper gineense respectively by oraladministration for 28 days. In this <strong>study</strong>, all theanimals’ experimentations were carried outfollowing the guidelines for the care and use oflaboratory animals obtained from theinstitutional animal ethics committee.2.7 Sample collection and Histopathologicalanalysis.Twenty four hours after last exposure, theanimal were anesthetized with chloroformvapour and dissected. The harvested stomachwere carefully dissected out, trimmed of all fatand connective tissue blotted dry to removeany blood. The tissues were fixed in 10%formal saline, and then transferred to a gradedseries of ethanol. On day 1, they were placedin 70% alcohol for 7 hours, then transferred to90% alcohol and left in the latter overnight. Onday 2, the tissues were passed through threechanges of absolute alcohol for an hour eachthen cleared in xylene. Once cleared, thetissues were infiltrated in molten paraffin waxin the oven at 58°C. Three changes of moltenparaffin wax at one-hour intervals were made,after which the tissues were embedded in waxand blocked out. Prior to embedding, it wasensured that the mounted sections to be cut bythe rotary microtome were orientatedperpendicularly to the long axis of the kidney,liver and pancreas. The sections weredesignated "vertical sections". Serial sectionsof 5 µm thick were obtained from a solid blockof tissue, fixed on clean albuminized slides toprevent sections coming off the slides and laterstained with Haematoxylin and Eosin stainingtechniques, after which they were passedthrough ascending grade of alcohol, cleared inxylene and mount in DPX mountant, allowedto dry at room temperature and observedHistopathologically under digital lightmicroscope.2.8 Gross morphometrical analysisThe initial and final weight of the rats and theweight of the kidney in each group were takenusing the weighing balance. The values of allthe morphometric analysis were comparedstatistically using SPSS 17 Software.2.9 PhotomicrographyRecords of the Histological and histochemicalresults were obtained by photomicrographyusing digital photomicrographic microscope atthe Gross Anatomy Research Laboratory,Department of Human Anatomy, College ofHealth sciences, University of Uyo,Uyo,Akwa-Ibom, Nigeria as illustrated in Plate 1 to 5.33

Available online at www.ijpras.com3.0 Results3.1 Statistical Analysis result.Table 1: Showing the effect of extracts on initial and final body weights.Groups (n) Drug Administered Initial body weight(g) Final body weight (g)1 Control (no treatment) 137.90±6.16 169.50±6.53*Abelmochus esculentus-500mg/kg-28 167.80±7.81 200.10±11.40**2days3 Piper guineense-20mg/kg -28 days 181.10±8.56 198.10±12.22**Combined A.esculentus +151.78±7.86 205.44±12.73***4 P.guineense-28daysMean ± SEM, *=P

Available online at www.ijpras.comPlate 4 Kidney treated with a Combination ofAbesmochus esculentus and Pipper guineensefor 28 days showed severe cellularabnormalities of cortex and medulla, withnumerous tubular necrosis lined with epithelialdegeneration, degenerated tubules,vacuolization, interstitial odema,inflammation, glomerular inflammation,cellular and vascular degeneration as comparedto the control group.Finally, the result of the microscopicexamination showed histopathological damageto the renal tissues of rats exposed toAbelmoschus esculentus and piper guineense,compared to the tissues from the control rats(plate 1). The kidney of rats in the test groupswere observed to have developed interstitialodema, nuclear clumping, valcuolization,tubular necrosis, cellular degeneration, tubularatrophy epithelial degeneration and infiltrationboth in females and males albino rats of eachgroup (plate 2-4). This gave an indication thatexposure to Abelmoschus esculentus and piperguineense may induce cellular alterations ofnormal kidneys. Cytoarchitecture, distortingthe functional integrity of the renal tissues. Theobservations made from the tissue microscopicanalysis, indicated the existence ofdisturbances in the filtration function of thegastric mucosa in rats exposed to Abelmoschusesculentus and piper guineense.ALSMAGMBCGgMPCMnBPlate 1. Control fundic stomach at magnification A(x100) & B(x400) stained with H & Etechnique. Note: M-mucosa, SM-Submucosa, Gg-gastric gland, GM-Glandular mucosa, LN-Lymphatic nodules, Mn- mucus neck cells, P-parietal cells, CH- chief cells.CCGeDMSMBMCPIEDLGPlate 2 Fundic stomach treated with ABELMOSCHUS ESCULENTUS (500mg/kg) atmagnification C(x100) & D(x400) stained with H & E technique. Note: M-mucosa, SM-Submucosa,Gg-gastric gland, I-inflammation, CD-Cellular degeneration, Ge-gastric erosion, P-parietal cells, CHchiefcells, BV-Blood vessel, L-lamina propria, ED-Epithelial degeneration.35

Available online at www.ijpras.comESSMEBVVCCDCHPELDFMMCDVCEPCHIPlate 3 Fundic stomach treated with PIPER GUINEENSE (20mg/kg) at magnification E(x100) &F(x400) stained with H & E technique. Note: CD-Cellular degeneration, I-inflammation, M-mucosa,ELD-Epithelial lining cells, Gp-gastric pit, CH-Chief cells, P-Parietal cells, Mn-Mucus neck cells, E-Erosion.GPVCEGEGHCHGCIGEEGpIEVPLATE 4 Fundic stomach treated with ABELMOSCHUS ESCULENTUS (500mg/kg) andPIPER GUINEENSE(20mg/kg) at magnification G(x100) & H(x400) stained with H & Etechnique.Note: E-Erosion, CD-Cellular degeneration, S-Serosa, CH-chief cells, BV-Blood vessel, LN-Lymphatic nodules, ELD-Epithelial lining cells, VC-vascular congestion, P-Parietal cells, Mn-muscusneck cells, GM-glandular mucosa, I-Inflammation, Ge-gastric erosion, IO-Interstitial oedema.4.0 DiscussionThis <strong>study</strong> investigated the Histopathologicaleffect of chronic consumption of combinationAbelmoschus esculentus and piper guineenseon the fundic stomach in rat model. Inassessing the gastric effects of these extracts,the gross morphology of the stomach wereexamined, <strong>study</strong> showed a significant increasein weight of animals and a significant decreasein organ weight in each group. The decrease inthe weight of the stomach of the experimentalgroups may have occurred as a result of gastricnecrosis and erosion and cellular degenerationof fundic stomach. Significant distortions inthe architectural integrity of the mucosal,laminal propria and submucosa were observed.Specifically, Inflammation of the gastricmucosa and epithelial lining degeneration,infiltration of polymorphonuclear cells into thepits and degeneration of the parietal and chiefcells are also noted. The observations madefrom this <strong>study</strong> indicated a condition of gastriccellular abnormalities and correlated theprevious report on the gastric ulcerationeffects of cayenne pepper, another specie ofpepper which contains capsaicin a knownactive ingredient in piper guineense, (Mbongueet al., 2005). The results of this <strong>study</strong> thereforeprovide a clear indication that Abelmoschusesculentus and piper guineense contain somechemical substances with that is capable ofinducing Glandular atrophy, intestinalmetaplasia, superficial gastritis, gastricerosion, erosive gastritis and gastric ulcer. Thespecific chemical constituents and mechanismsresponsible for these effect reported in this36

Available online at www.ijpras.com<strong>study</strong> are not clear. It may be assumed that thereactive metabolites of Abelmoschusesculentus and piper guineense constituentscould have interacted with the gastric mucosaand intestinal glands to cause derangementsingastric structures and functions.The combination of the two extracts showedsevere ulcerative effect of the gastric mucosa,indicating that in combination their effect tothe fundic stomach potentiates each other. Theinteraction of these metabolites with the fundicstomach may be responsible for cellular injuryand subsequent damage to the tissues. Thefunctionality of the stomach may becompromised due to damage of the gastrointestinaltract.5.0 ConclusionResults obtained in this <strong>study</strong> show thatchronic exposure to Abelmoschus esculentusand piper guineense induced adverse anddetrimental effects on the gastric mucosafunction in rat model. These observationsindicated that exposure to Abelmoschusesculentus at doses as high as 500mg/kg bodyweight and piper guineense at doses of20mg/kg body weight and above is a riskfactor for gstric function impairment and theassociated disorders. This work showedGlandular atrophy, intestinal metaplasia,superficial gastritis, gastric erosion, erosivegastritis and gastric ulcer in all experimentalgroups. Further <strong>study</strong> is recommended withisolated components of these extracts and withlower concentration of extract of Abelmoschusesulentus and piper guineense to confirm theunderlying mechanism and active constituentsresponsible for the observed activitydocumented by the results of this <strong>study</strong>.AcknowledgementsWe wish to acknowledge Mr Ajayi Oyewunmi,Histoscientist from the university of Uyoteaching who assisted in the preparation of thehistopathological slides, Miss Osibajo KehindeAdefowope, computer scientist for her help inthe computer typesetting of the works andMiss Akaninyene Attah at the Animal House,University of Uyo for her help in the care ofthe animals used for this research work.“Cite this article”Umoh I.,Oyebadejo S., Bassey E.,Udoh N.“<strong>Histomorphological</strong> Study of the Effect ofChronic Consumption of AbelmoschusEsculentus and Piper Guineense on theGastric Mucosa of Albino Wistar Rats” Int. J.of Pharm. Res. & All. Sci.2013; Volume 2,Issue 3,31-37ReferencesBai, Y. E. Xult (2000): “Protective actin ofpiperine against experimental gastriculcer”.Acta Pharmacol Sin. 21 (4): 357 – 9.Dalziel, J., (1937): The useful plants of WestTropical Africa. Crown Agents, LondonEkanem, A., Obiekezie, A. (2000).Antiparasitic effects of leaf extract of piperguineense on the jurveules of hetorobranchuslongitilis (cuvier and valencieness). Afr. J.Fisher Aguacult, 8:74.Ekanem, F. V., Udoh and Oku, (2010): Effectsof ethanol Extract of Piper guineense seed onthe conception of mice. Cameroon. Fitoter.69:5-34.James, A., Duka, Judith, L. Ducellier (1993):CRC Handbook of alternative cash Crops CRCpress LLC Boca Raton, pg. 1-3.Leung, A. & Foster, S. (2011): Encyclopediaof common National Ingredient use in food,drugs and cosmetics, 2 nd edition. John Wileyand Sons, New York. P. 457 – 457.Lorke D. A new approach to practical acutetoxicity testing. Arch Toxicol 1983, 54, 275.McNamara, F. N. Randall, A. Gunthorpe, M. J.(2005): Effect of piperine; the PungentComponent of black peper, at the humanvanilloid reciphor (TRPRI). Br. J. pharmacol.144(6). 781 – 790.McWhorter, John, H. (2000): The missingSpanish creoles. Recolaring the Birth ofPlantation contact. Languages. University ofCaliforma press. P. 77 ISBN 6-520 – 219997 –6. Retrieved 2008-1129.Murray, M.J. (2002). Disease of the stomach,In: Manual of Equine Gastroenterology, Mair,T.,Divers, T. & Ducharme, N., pp.241-248,W.B. Saunders, ISBN: 0-7020-2486-4,Chatham, UK.Neghenebor, C., Godiga, A., Lgene, S., (1999):Evaluation of Clarias anguillarns treated withspice (Piper guineense) for washed mice andkama book type product. Food compos, Anal.2: 12 – 315.Ngane, A., Biyiti L., Bouchet, P., Nkegfact,A., Zolo P., (2003): Antifungal activity ofpiper guineense of Cameroon. Fitoter. 4(5):464 – 468.37