Garcinia kola prevents scopolamine-induced memory impairment in mice: Role of oxidative and nitrosative stress

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1 West Afrian Journal of Pharmay (2017) 28 (1) Garinia kola prevents sopolamine-indued memory impairment in mie: Role of oxidative and nitrosative stress Ismail O. Ishola, Wahab O. Okunowo, Gbenga O. Afolayan, Azeezat A. Awoyemi 1 Department of Pharmaology, Therapeutis and Toxiology, Faulty of Basi Medial Sienes, College of Mediine, University of Lagos, PMB 12003, Idi-araba, Lagos, Nigeria. 2 Department of Biohemistry, Faulty of Basi Medial Sienes, College of Mediine, University of Lagos, PMB 12003, Idi-araba, Lagos, Nigeria. Corresponding author : Ismail Ishola add: oishola@mul.edu.ng Phone : ABSTRACT Bakground: The high and inreasing inidene of Alzheimer Disease (AD) worldwide is a major global onern. Oxidative stress has been onsidered to be linked to the aetiology of many diseases, inluding neurodegenerative diseases (NDDs) suh as AD. Garinia kola Hekel (Clusiaeae) has important antioxidant funtions that an help protet against AD. Objetive: This study sought to investigate the protetive effet of ethanol seed extrat of Garinia kola (GCK) on sopolamine-indued memory impairment in rodents. Methods: GCK (25, 50 and 100 mg/kg, p. o.) or tarine (5 mg/kg, i. p.) was administered for 3 onseutive days. Sopolamine (3 mg/kg, i. p.) was given, 1 h after last-treatment on day 3, followed by estimation of memory funtions using the Y-maze and elevated plus maze (EPM) tasks in mie as well as Morris water maze test (MWM) in rats. The biohemial markers of oxidative stress were determined in the prefrontal ortex, hippoampus and striatum after the MWM test on day 8. Results: Sopolamine indued signifiant derease in spontaneous alternation behaviour in Y-maze and inreased transfer lateny in EPM in mie whih was prevented by pretreatment of mie with GCK. In the MWM experiment, GCK proteted rats against spatial learning defiit indued by sopolamine, evidened in session dependent derease in esape lateny. Furthermore, sopolamine indued inreased oxidative and nitrosative stress status in the prefrontal ortex, striatum and hippoampus as ompared with vehile-treated ontrol was ameliorated with GCK administration whih was similar to the effet of tarine. Conlusion: G. kola prevented sopolamine-indued ognitive impairment through attenuation of oxidative/nitrosative stress status. Thus, ould be a potential phytotherapeuti agent in the treatment of dementia. Keywords: Antioxidant system; hippoampus; Morris water maze; sopolamine; Y-maze 35 West Afrian Journal of Pharmay (2017) 28 (1)

2 West Afrian Journal of Pharmay (2017) 28 (1) La Garinia kola empêhe l'affaiblissement de la mémoire induite par la sopolamine hez la souris: rôle du stress oxydatif et nitrosant Auteur de orrespondane : Ismail Ishola oishola@mul.edu.ng Téléphone : RÉSUMÉ Contexte: L'inidene élevée et roissante de la maladie d'alzheimer (MA) dans le monde est une préoupation majeure à l'éhelle mondiale. Le stress oxydatif a été onsidéré omme étant lié à l'étiologie de plusieurs maladies, y ompris les maladies neuro-dégénératives (MND) telles que la MA. Garinia kola Hekel (Clusiaeae) a des fontions anti-oxydantes importantes qui peuvent aider à protéger ontre la MA. Objetif: Cette étude a herhé à étudier l'effet proteteur de l'extrait de graine d'éthanol de Garinia kola (GCK) sur l'altération de la mémoire induite par la sopolamine hez les rongeurs. Méthodes: On a administré du GCK (25, 50 et 100 mg/kg, p. o.) ou de la tarine (5 mg/kg, i. p.) pendant 3 jours onséutifs. La sopolamine (3 mg/kg, i. p.) a été administrée, 1 h après le dernier traitement le jour 3, suivie de l'estimation des fontions de mémoire à l'aide des tâhes du labyrinthe Y et du labyrinthe surélevé (EPM) hez la souris ainsi que du test de labyrinthe de l'eau de Morris (MWM) hez le rat. Les jalons biohimiques du stress oxydatif ont été déterminés dans le ortex préfrontal, l'hippoampe et le striatum après le test MWM le jour 8. Résultats: La sopolamine a induit une diminution signifiative du omportement d'alternane spontanée dans le labyrinthe Y et une augmentation de la latene de transfert dans EPM hez la souris, e qui a été empêhé par prétraitement de souris ave GCK. Dans l'expériene de MWM, les rats protégés par GCK ontre le défiit d'apprentissage spatial induit par la sopolamine, mis en évidene dans la diminution dépendante de la session de latene d'éhappement. En outre, la sopolamine a induit une élévation du niveau de stress oxydatif et nitrosant dans le ortex préfrontal, le striatum et l'hippoampe omparativement au témoin traité au véhiule a été améliorée ave l'administration de GCK qui était similaire à l'effet de la tarine. Conlusion: G. kola a permis d'éviter l'affaiblissement ognitif induit par la sopolamine par atténuation du niveau de stress oxydatif/nitrosant. Ainsi, ela pourrait être un agent phyto-thérapeutique potentiel dans le traitement de la démene. Mots-lés: Système antioxydant; Hippoampe; labyrinthe eau de Morris; La sopolamine; labyrinthe Y- 36 West Afrian Journal of Pharmay (2017) 28 (1)

3 Nootropi effet of Garinia kola seed extrat INTRODUCTION Alzheimer's disease (AD) is the most ommon ageassoiated neurodegenerative disease haraterized by progressive memory impairment and deterioration of 1 other ognitive funtions. Neuropathologial hallmarks of AD inlude senile plaques and neurofibrillary tangles in neoortial and limbi brain lesions, whih are oupled with neuronal loss or 2 dysfuntion. Another important phenomenon in AD, is the dysfuntion of the holinergi system by amyloid plaques in several brain areas suh as the basal forebrain, ortial regions, and the hippoampus. The loss of holinergi neurons in the brain is losely assoiated with the redution of aetylholine 3 synthesis. Previous studies have shown that the disruption in holinergi neurotransmission is one of the earliest neuropathologial hanges in prelinial AD and may be assoiated with abnormal beta-amyloid 4 (Aâ) aumulation. Therefore, disruption of holinergi neurotransmission with sopolamine may unmask otherwise undetetable ognitive defiits in prelinial AD. Thus, the use of a holinergi neurotransmission enhaner may be an effetive way of identifying novel agents for the prevention or treatment of dementia of Alzheimer's type. In Southwest, Nigeria, Garinia kola seed is hewed for 5,6 its anti-aging and memory enhaning ativities. Garinia kola Hekel (Clusiaeae) also known as bitter kola (English), orogbo'' (Yoruba-Southwestern, Nigeria), ida goro'' (Hausa-Northern, Nigeria), Ugugolu'' (Igbo-Eastern, Nigera), and Garushinia Kora (Japanese) is a medium-sized tree of about m high and a girth of 1.80 m grown in the ostal rainforest in West and Central Afria. The tree is highly valued for its 7 edible nuts. The seeds as a bitter astringent taste when hewed resembling that of raw offee bean followed by 8 a slight sweetness, thus onsumed as a stimulant. The seeds are used in traditional Afrian Mediine for the treatment of several ailments inluding; liver disorders, gonorrhoea, bronhitis, laryngitis, diarrhoea, diabetes, 9,10 aphrodisia and neurologial disorders. Moreover, hepatoprotetive and aphrodisia effets of G. kola has 11,12 been reported. The present study was arried out to investigate effet of G. kola seed extrat on memory impairment indued by sopolamine in mie and rats. In addition, examine the effet of the extrat on sopolamine-indued oxidative and nitrosative stress biomarkers in disrete brain regions of rats. MATERIALS AND METHODS Laboratory animals Male Sprague-Dawley rats ( g) and Swiss albino mie (18-22 g) used in this study were obtained from the Laboratory Animal Centre, College of Mediine, University of Lagos, Lagos, Nigeria. The animals were kept in well ventilated environment, housed in standard ages and were fed on standard pellets (Livestok Feeds, Lagos, Nigeria) and tap water ad libitum. The animals were alimatized for 7 days before the ommenement of the experiment. The experimental proedures adopted in this study were in ompliane with the ethial standards of the Researh Grant and Animal Experimentation Committee of the College of Mediine, University of Lagos, Nigeria and in aordane with the United States National Institutes of Health Guidelines for Care and Use of Laboratory Animals in Biomedial Researh (2011). Plant materials Garinia kola seeds were purhased from Mushin herbal market, Lagos state, Nigeria. Botanial identifiation and authentiation was done by Mr. T.K. Odewo (a forestry expert) in the Department of Botany, University of Lagos, Akoka, Lagos State, Nigeria. A vouher speimen with herbarium number LUH 6138 was deposited in the herbarium of same Department for referene purpose. Preparation of extrat One hundred and twenty grams of peeled seeds were slied and air dried at room temperature. The air dried seeds were pulverized with a Warring ommerial blender. The powdered seed was soaked in 1.4 L of ethanol for 72 h at room temperature with intermittent agitation (12 hourly; manually). After extration, the sample was filtered and the filterate evaporated to dryness under redued pressure by Heidolph rotary evaporator to yield 22 g (18.33% w/w; brownish extrat). The extrat was suspended in 2% arboxymethylellulose in orn oil as vehile. Drugs and hemials Ethanol, tarine, glaial aeti aid, Folin-Cioalteu reagent, sopolamine hydrobromide, thiobarbituri aid, sodium hloride, sodium hydroxide, potassium f e r r i y a n i d e, t r i h l o r o a e t i a i d, napthylethylenediamine dihydrohloride, ferri hloride, DPPH solution, dithio-bis-nitrobenzoi aid (DTNB), and bovine serum albumin (Sigma Aldrih, St. Louis MO, USA), normal saline (Unique Pharmaeutial Ltd, Lagos, Nigeria). 37 West Afrian Journal of Pharmay (2017) 28 (1)

4 Ishola et al Qualitative and quantitative phytohemial analysis Preliminary qualitative phytohemial analysis The Preliminary qualitative phytohemial sreening of 13 GCK was arried out using the method of Edeoga et al. Determination of total phenoli ontent Total phenoli ontent of GCK was determined using 14 Folin-Cioalteu reagent as desribed by Turkmen et al. with slight modifiations. Briefly, 0.1 ml of GCK (4 mg /ml) or standard solution at different onentrations, was mixed with 0.75 ml of Folin- Cioalteu's phenol reagent (10-fold diluted with water). The mixture was kept at room temperature for 5 min and 0.75 ml of 6% sodium arbonate was added. After 90 min of reation, its absorbane was read at 725 nm. The standard alibration (0-25 µg/ml) urve was plotted using galli aid. The total phenolis were expressed as mg galli aid equivalent/gram dry weight. Negative ontrol was prepared by adding 0.1 ml of ethanol instead of GCK. In vitro evaluation of antioxidant ativities. Nitri oxide radial (NO ) savenging assay Nitrite generated from sodium nitroprusside (SNP) was 15 measured aording to the method of Maroi et al. Briefly, the reation mixture (5 ml) ontaining SNP (5 mm) in phosphate buffered saline (ph 7.3), with or without the various onentrations GCK ( µg/ml), was inubated at 25 C for 180 min in front of a visible polyhromati light soure (25W tungsten lamp). * The NO radial thus generated interated with oxygen to produe the nitrite ion whih was assayed at 30 min intervals by mixing 1 ml of inubation mixture with an equal amount of Griess reagent (1% sulfanilamide in 5% phosphori aid and 0.1% naphthylethylenediaminedihydrohloride (NAD). The absorbane of the hromophore (purple azo dye) formed during the diazotisation of nitrite ions with sulphanilamide and subsequent oupling with NAD was measured at 546 nm. The nitrite generated in the presene or absene of the GCK was estimated using a standard urve based on sodium nitrite solutions of known onentrations. Eah experiment was arried out at least three times and the data presented as an average of three independent determinations. Galli aid was used as the positive * ontrol. The perentage savenging of NO by GCK and galli aid (standard antioxidant) was alulated aording to the equation: % Inhibition = Absorbane of ontrol Absorbane of sample 100 Absorbane of ontrol 1, 1-Diphenyl-2-pirylhydrazyl (DPPH) free radial savenging ativity The free radial savenging potential of GCK were 16 determined aording to the proedure of Awah et al. with some minor modifiations. Briefly, an aliquot of 50 µl of GCK of various onentrations ( µg/ml) were mixed with 950 µl of ethanoli solution of DPPH (3.4 mg/ 100 ml). The reation mixture was inubated at 37 C for 1 h in the dark. The free radial savenging potential of GCK was expressed as the disappearane of the initial purple olour. The absorbane of the reation mixture was reorded at 517 nm using UV-Visible spetrophotometer (Agilent 8453, Germany). Galli aid was used as the positive ontrol. The perentage inhibition of DPPH free radial was alulated aording to this formula: 38 West Afrian Journal of Pharmay (2017) 28 (1)

5 Nootropi effet of Garinia kola seed extrat The effetive onentration needed to savenge DPPH free radial by 50% (IC 50) was alulated by regression analysis of the dose response urve plotted between perentage inhibition versus onentration of the test samples and the standard. Ferri ion reduing power assay The reduing apaity of GCK may serve as a good indiator of its potential antioxidant property. The reduing power apaity of GCK was investigated using 17 the method of Oyaizu. Various onentration of GCK or standard solution (1.0 ml) were mixed 2.5 ml of potassium buffer (0.2 M, ph 6.6) and 2.5 ml of potassium ferriyanide [K3Fe(CN)6] (1% w/v). After 30 min of inubation at 50 C, 2.5 ml of 10% trihloroaeti aid solution was added to eah test tube and the mixture was entrifuged at 3,000 rpm for 10 min. Then, 2.5 ml of the supernatant solution was withdrawn from the tube and mixed with 2.5 ml of distilled water and 0.5 ml of ferri hloride solution (0.1%, w/v) and the absorbane was measured at 700 nm and galli aid was used as standard. Aute toxiity test The aute toxiity effet of GCK was determined using the fixed dose protool of the Organization of Eonomi Co-operation and Development (OECD) guidelines for 1 8 testing of hemials, TG (2001) for oral administration. 11 female mie were given GCK (250 mg/kg, p.o., n =1; 2000 mg/kg, p.o., n =5; and 4000 mg/kg, p.o., n = 5). Behavioural signs of toxiity and mortality were observed following extrat administration; during the first 30 minutes, then the seond, fourth, sixth hour and one daily for 14 days for delayed toxiity or mortality. Behavioural test Y-maze test The test relies on the innate tendeny of mie to explore a novel environment to assess spatial reognition. Male Swiss albino mie (18-22 g) were randomly divided into 6 groups (n = 6) and treated as follows for 3 days: Group1-vehile (10 ml/kg; p.o.; normal ontrol), Group 2- vehile (10 ml/kg, p.o.; amnesi model), Group 3- tarine (5 mg/kg; i.p.), Group 4-6: GCK (25, 50 and 100 mg/kg; p.o., respetively). Fifteen minutes after the last 19 treatment on day 3, sopolamine (3 mg/kg, i.p.) was administered to mie in Group 2-6. Five minutes postsopolamine injetion, eah mouse was plaed at the entre of the Y-maze faing the south arm 'B' and allowed to explore the maze freely for a period of 5 min. The number and the sequene of arm entries were observed. An arm entry was sored when all four paws were in the arm. Alternation behaviour was defined as onseutive entries into all three arms (i.e., ABC, CAB, or 20 BCA but not BAB). The perentage of spontaneous alternation was measured as an index of working memory by alulating the ratio of the atual number of alternations to the possible number (defined as the total number of arm entries minus two) multiplied by 100, i.e., % Alternation = [(number of alternations) 100 (total number of arm entries x 2)] The total number of arm entries was measured as an index of loomotor ativity. Elevated plus maze test (EPM) The plus maze onsists of two open and two losed arms ( m eah) elevated to a height of 38.5 m. Male Swiss albino mie (18-22 g) were randomly divided into 6 groups (n = 6) and treated as follows for 3 days: Group1-vehile (10 ml/kg; p.o.; normal ontrol), Group 2- vehile (10 ml/kg, p.o., amnesi model), Group 3- tarine (5 mg/kg; i.p.), Group 4-6: GCK (25, 50 and 100 mg/kg; p.o., respetively). Fifteen minutes after the last 19 treatment on day 3, sopolamine (3 mg/kg, i.p.) was administered to mie in Group 2-6. Five minutes postsopolamine injetion, eah mouse was plaed at the end of an open arm, faing away from the entral platform. Transfer lateny (TL) was taken as the time taken by the mouse to move into any one of the losed arms with all its four legs. After eah test, the maze was arefully leaned up with 10% ethanol solution. The ut off time was 90 s. TL was reorded on the first day. Memory retention was examined 24 h after the first day trial. Signifiant redution in TL value of retention 21 indiated improvement in memory. Morris water maze test The aquisition and retention of a spatial navigation task was examined using a Morris water maze (MWM). Male Sprague Dawley rats ( g) were randomly divided into 6 groups (n = 6) and treated as follows for 3 onseutive days: Group1-vehile (10 ml/kg; p.o.; normal ontrol), Group 2- vehile (10 ml/kg, p.o., amnesi model), Group 3- tarine (5 mg/kg; i.p.), Group 4-6: GCK (25, 50 and 100 mg/kg; p.o., respetively). Fifteen minutes after the last treatment on day 3, 19 sopolamine (3 mg/kg, i.p.) was administered to rats in Group 2-6. The MWM onsists of a irular water tank (120 m diameter and 50 m height) filled with water (26±2 C) to a depth of 30 m loated in a darkened test room, 39 West Afrian Journal of Pharmay (2017) 28 (1)

6 Ishola et al Four equally spaed points around the edge of the pool were designed as N (North), E (East), S (South) and W (West). A blak oloured round platform height of 28 m, and diameter of 10 m was plaed 2 m below the surfae of water in a onstant position in the middle of the NE quadrant in the pool. The time taken for the rat to esape from the water onto the platform was measured. The position of platform was kept unaltered throughout the trials. Five minutes post-sopolamine injetion on day 3, the animal was gently released into the pool from the SW quadrant in all the trials (day 1 of training). The animal was given 60 s (ut-off time) to find the hidden platform and allowed to stay on it for 10 s. The time taken for the rat to loate the esape platform was reorded using a stop wath. In the event that the animal was unable to loate the hidden platform within 60 s, it was gently guided to it and was allowed to stay on it for 10 s. Eah animal was subjeted to a daily session of 3 trials per day for 5 days onseutively (i.e. days 3 to 7). Esape lateny time (ELT) to loate the hidden platform in water maze was noted as an index of 19 learning. [The hoie of rats for the Morris water maze paradigm was due to the fat that, it will easier to isolate the hippoampus and striatum for biohemial assays from rats ompared to mie due to their brain size]. Brain tissue preparation The rats were deapitated, 45 min after the MWM task on day 7 under hloral hydrate (300 mg/kg, i.p.) anaesthesia. The skull was ut open and the brain was exposed from its dorsal side. The whole brain was quikly removed and the prefrontal ortex (PFC), striatum (STR) and hippoampus (HIP) were isolated on ie bar plate. The isolated brain areas were homogenized with an Ultra-Turrax T25 (USA) homogenizer at a speed of 9500 rpm in 0.03 M sodium phosphate buffer, ph-7.4. The homogenate was used to measure MDA, GSH, SOD and nitrite. Determination of lipid peroxidation Malondialdehyde (MDA), whih is an indiator of lipid peroxidation, was spetrophotometrially measured by using the thiobarbituri aid assay as previously 22 desribed by Ohkawa et al. Two hundred mirolitre of the supernatant was added and briefly mixed with 1 ml of 50% trihloroaeti aid in 0.1 M HCl and 1 ml of 26 mm thiobarbituri aid. After vortex mixing, samples were maintained at 95 C for 20 min. Afterwards, samples were entrifuged at 960 g for 10 min and supernatants were read at 532 nm. A alibration urve was onstruted using MDA as standard and the results were expressed as U/mg protein. Determination of redued glutathione (GSH) level GSH was determined by its reation with 5, 5'-dithiobis (2-nitrobenzoi aid) (Ellman's reagent) to yield a yellow hromophore whih was measured 23 spetrophotometrially. To measure the GSH level, the tissue homogenate (in 0.1 M phosphate buffer ph 7.4) was taken. The brain homogenate was mixed with an equal amount of 10% trihloroaeti aid (TCA) to preipitate the proteins. The mixture was kept for 5 min prior to entrifugation (Remi old entrifuge) at 2000 g for 10 min at 4 C. The supernatant (100 µl) of proessed tissue sample mixed with 0.5 ml of Ellman's reagent (5, 5'-dithio bis -2-nitrobenzoi aid) (0.1 mm) (DTNB)) prepared in 2 ml of phosphate buffer (ph 8.4) and 0.4 ml of double-distilled water and the reation mixture was shaken vigorously on vortex. The absorbane was read at 412 nm within 15 min. Determination of atalase ativity Catalase (CAT, EC ) ativity was assayed 24 following the method of Sinha. The reation mixture onsisted of 150 ìl phosphate buffer (0.01 M, ph 7.0), 100 ìl supernatant. Reation was started by adding 250 ìl H2O M, inubated at 37 C for 1 min and reation was stopped by addition of 1.0 ml of dihromate: aeti aid reagent. The tubes were immediately kept in a boiling water bath for 15 min and the green olour developed during the reation was read at 570 nm on a spetrophotometer. Control tubes, devoid of enzyme, were also proessed in parallel. Results were expressed as U/mg protein. Determination of superoxide dismutase (SOD) ativity The ativity of superoxide dismutase (SOD, EC ) was assayed by monitoring its ability to inhibit the photohemial redution of nitroblue tetrazolium (NBT). Eah 1.5 ml reation mixture ontained 100 mm TRIS/HCl (ph 7.8), 75 mm NBT, 2 ìm riboflavin, 6 mm EDTA, and 200 ìl of supernatant. Monitoring the inrease in absorbane at 560 nm followed the prodution of blue formazan. One unit of SOD is defined as the quantity required to inhibit the rate of NBT redution by 50% as previously desribed by 25 Winterbourn et al. The enzyme ativity is expressed as units/mg protein. Determination of nitrite level Nitrite was estimated in the rats brain using the Greiss reagent and served as an indiator of nitri oxide prodution. One hundred miroliter of Greiss reagent (1:1 solution of 1% sulphanilamide in 5% phosphori 40 West Afrian Journal of Pharmay (2017) 28 (1)

7 Nootropi effet of Garinia kola seed extrat aid and 0.1% napthaylamine diamine dihydrohlori aid in water) was added to 100ìl of supernatant and 26 absorbane was measured at 542nm. Nitrite onentration was alulated using a standard urve for sodium nitrite. Protein estimation Protein was measured in all brain samples by the 27 method of Lowry et al. Bovine serum albumin (BSA) (1 mg/ml) was used as standard and measured in the range of mg/ml. Statistial analysis Results obtained are expressed as mean ± SEM (n=6). Level of signifiane was analysed using one or two way ANOVA (whihever is appliable) followed by Tukey post ho multiple omparison test using Graphpad prism version 6 (Graphpad prism In, CA, USA). Results Aute toxiity test Oral administration of GCK up to 4000 mg/kg neither indued toxi behaviours nor mortality throughout the period of 14 days observation. Preliminary phytohemial analysis and total phenoli ontents The preliminary phytohemial analysis revealed the presene of saponins, alkaloid, flavonoid, tannin, terpenoids, and steroid. Quantitative estimation of GCK, showed that the total phenoli ontent is 36.58±0.06 mg GAE/g of dry GCK. Effet of GCK on free radial generation in vitro The results of the in vitro antioxidant assay showed the ability of GCK to savenge free radials indued by DPPH, nitri oxide and ferri ion as shown in Table 1. The results show that the hydrogen donating ability of GCK was lower than that of galli aid. Similarly, the NO savenging ability of GCK was similar to that of galli aid. Moreover, the ferri ion reduing power assay measures the eletron donating apaity of an antioxidant. An inreased absorbane is indiative of higher reduing power. The results showed that the reduing power of GCK and the standard, galli aid inreased progressively (Table 1). Table 1: The in vitro inhibitory onentration of G. kola and galli aid against free radial generation Parameters GCK IC50 µg/ml Galli aid IC50 µg/ml DPPH Nitrite FRAP IC - median inhibitory onentration 50 DPPH - 1, 1-Diphenyl-2-pirylhydrazyl FRAP- ferri ion reduing apaity assay Effet of GCK on sopolamine-indued memory defiit in the Y-maze test Sopolamine (3 mg /kg) markedly impaired spontaneous alternation behavior (Fig. 1). GCK signifiantly [F(5, 30) = 15.27, P < 0.001] attenuated the impairment of spontaneous alternation behavior indued by sopolamine. However, sopolamine produed no signifiant effet on the total number of arm entries when ompared with vehile-treated ontrol. Also, GCK [F (5, 30) = 1.535, P=0.2087] had no signifiant effet on the total number of arm entries (Fig. 2). 41 West Afrian Journal of Pharmay (2017) 28 (1)

8 Ishola et al % Spontaneous alternation behavior Vehile Sop 3 GCK 25 *** * # GCK 50 GCK 100 Sopolamine 3 mg/kg,i.p. ** Tarine 5 Figure 1: Effet of GCK on spontaneous alternation behaviour in sopolamine-indued memory defiit test in Y-maze # * ** test in mie. Values are expressed as mean±sem (n =6), p<0.05 versus vehile only treated; p<0.05, p<0.01, *** p<0.001 versus vehile + sopolamine treated; p<0.001 versus GCK 25 mg/kg treated. Statistial level of signifiane analysis by one way ANOVA followed by Tukey post ho multiple omparison test. 30 Total number of entries Vehile Sop 3 GCK 25 GCK 50 GCK 100 Tarine 5 Sopolamine 3 mg/kg, i.p. Figure 2: Effet of GCK on total number of arm entries in sopolamine-indued memory defiit test in Y-maze task. Values are expressed as mean±sem (n =6). Statistial level of signifiane analysis by one way ANOVA followed by Tukey post ho multiple omparison test. Effet of GCK on sopolamine-indued memory defiit in the EPM test In the EPM, no signifiant differene was found in the mean transfer lateny in aquisition trial. One way ANOVA revealed no signifiant effet of GCK treatment [F(5,24)=2.481,P=0.0602] during aquisition trial in the EPM test in mie. A signifiant differene was found in the mean retention trial TL between the groups; [F(5,48)=6.195, P< 0.01]. 3 days pre-treatment of mie with GCK reversed the memory impairment indued by sopolamine. Post ho analysis showed that sopolamine (3 mg/kg) signifiantly inreased transfer lateny when ompared with vehile-treated ontrol during retention trial on day 2 indiating amnesia. GCK (50 mg/kg) treatment produed signifiant (P<0.01) redution in transfer lateny in retention trial when ompared with aquisition trial (Fig. 3). 42 West Afrian Journal of Pharmay (2017) 28 (1)

9 Nootropi effet of Garinia kola seed extrat 60 Aquisition Retention Transfer lateny (s) ## ## # 0 Vehile Sop GCK 25 GCK 50 GCK 100 Tarine 5 Sopolamine 3 mg/kg, i.p. Figure 3: Effet of GCK on transfer lateny in sopolamine-indued memory defiit in EPM test in mie. Values are # ## expressed as mean±sem (n =6). P<0.05, P<0.01 versus aquisition trial. Statistial level of signifiane analysis by one way (aquisition trial) or two way (retention trial) ANOVA followed by Tukey post ho multiple omparison test. Effet of GCK on spatial learning in Morris water maze test Vehile only treated rats quikly aquired the spatial task as observed by a gradual and session-dependent derease in esape lateny time (ELT) [F (4, 20) =57.02, p<0.001]. Intraperitoneal administration of sopolamine indued spatial memory impairment as indiated by no signifiant hange [F (4, 20) =2.52, nd th P=0.0665] in ELT from 2 to 5 sessions when ompared with the first session. However, pre-treatment of rats with tarine before sopolamine injetion produed a session dependent and signifiant derease [F (4, 20) nd th =17.28, P<0.001] in ELT from the 2 to 5 session when ompared with the first session. Similarly, oral administration of GCK prevented the spatial-learning defiit indued by sopolamine in rats. GCK (25 or 100 mg/kg) signifiantly dereased mean ELT from third s e s s i o n [ F ( 4, 2 0 ) = , P < ], [F(4,20)=9.17,P<0.001], respetively, when ompared to first session, while GCK 50 mg/kg redued the mean ELT signifiantly from the seond to the fifth session [F (4, 20)=9.98,P<0.001]. The derease in ELT produed by GCK was omparatively similar.to that of tarine-treated groups (Fig.4). 43 West Afrian Journal of Pharmay (2017) 28 (1)

10 Ishola et al 80 Esape lateny time (S) a b b a b 0 Vehile Sop 3 Ta 5 GCK 25 GCK 50 GCK Sop 3 mg/kg, i.p. Figure 4: Effet of GCK on spatial learning task following sopolamine-indued memory impairment in Morris water a b maze test in rats. Values are expressed as mean ELT (se)±s.e.m (n = 6). p<0.05, p<0.01, p<0.001 versus session 1. Statistial level of signifiane analysis by two way ANOVA followed by Tukey post ho multiple omparison test. Effet of GCK on malondialdehyde level The effet of GCK on the levels of MDA in the prefrontal ortex, striatum and hippoampus of rats after the MWM, was examined to evaluate its effet on sopolamine-indued lipid peroxidation. Sopolamine administration indued signifiant inrease in the levels of MDA in disrete brain regions as ompared with vehile-treated ontrol group. However, pretreatment of rats with GCK (25, 50 and 100 mg/kg) or tarine (5 mg/kg) signifiantly (P<0.001) redued the MDA levels in the prefrontal ortex, striatum and hippoampus. Two way ANOVA revealed signifiant effet of GCK or tarine treatment (F(5,72)=43.61, P<0.001), sopolamine pretreatment (F(2,72)=4.18, P=0.019) and pretreatment treatment interation (F(10,72)=7.915, P<0.001) (Fig. 5). 44 West Afrian Journal of Pharmay (2017) 28 (1)

11 Nootropi effet of Garinia kola seed extrat MDA (U/mg protein) PFC STR HIPPO 0.00 Veh Sop TAC 5 GCK 25 GCK 50 GCK 100 Sop 3 mg/kg, i.p. Figure 5: The effet of GCK on malondialdehyde ativity within the prefrontal ortex, striatum and hippoampus of ã sopolamine-indued amnesi rats. Values are expressed as mean±sem (n=6). P<0.001 versus vehile-treated, ontrol; P<0.001 versus vehile + sopolamine treated. Statistial level of signifiane analysis by two way ANOVA followed by Tukey post ho multiple omparison test. Effet of GCK on glutathione ativity Administration of sopolamine resulted in a signifiant derease in redued glutathione level (F(2,72)=0.4744, P=0.6242) in the prefrontal ortex, striatum and hippoampus when ompared with vehile-treated ontrol. The redution in GSH level indued by sopolamine was reversed by pretreatment of rats with GCK or tarine in the prefrontal ortex, striatum and hippoampus. Moreover, two ANOVA revealed the main effet of GCK (25, 50 and 100 mg/kg) treatment (F(5,72)=54.43,P<0.001) and pretreatment treatment interation (F(10,72)=14.84, P<0.01) (Fig. 6). 45 West Afrian Journal of Pharmay (2017) 28 (1)

12 Ishola et al GSH (U/mg protein) 1.5 PFC STR HIPPO Veh Sop TAC 5 GCK 25 GCK 50 GCK 100 Sop 3 mg/kg, i.p. Figure 6: The effet of GCK on glutathione ativity within the prefrontal ortex, striatum and hippoampus of ä sopolamine-indued amnesi rats. Values are expressed as mean±sem (n=6). P<0.001 versus vehile-treated, ontrol, P<0.001 versus vehile + sopolamine treated. Statistial level of signifiane analysis by two way ANOVA followed by Tukey post ho multiple omparison test. Effet of GCK on superoxide dismutase level Administration of sopolamine indued signifiant (F(2,72)=98.53, P<0.001) redution in superoxide dismutase in the prefrontal ortex, striatum and hippoampus when ompared with vehile-treated ontrol. However, 3 days pretreatment of rats with GCK or tarine prevented the derease in superoxide dismutase level in the prefrontal ortex, striatum and hippoampus. Two way ANOVA revealed signifiant t r e a t m e n t e f f e t o f G C K o r t a r i n e (F(5,72)=126.60,P<0.001) and pretreatment treatment interation (F(10,72)=22.38, P<0.01) (Fig. 7). 46 West Afrian Journal of Pharmay (2017) 28 (1)

13 Nootropi effet of Garinia kola seed extrat SOD (U/mg protein) PFC STR HIPPO 0 Veh Sop TAC 5 GCK 25 GCK 50 GCK 100 Sop 3 mg/kg, i.p. Figure 7: The effet of GCK on superoxide dismutase ativity within the prefrontal ortex, striatum and hippoampus ä of sopolamine-indued amnesi rats. Values are expressed as mean±sem (n=6). P<0.001 versus vehile-treated, ontrol; P<0.001 versus vehile + sopolamine treated. Statistial level of signifiane analysis by two way ANOVA followed by Tukey post ho multiple omparison test. Effet of GCK on nitrite generation Sopolamine administration indued a signifiant inrease in the level of nitri oxide in the prefrontal ortex, striatum and hippoampus of rats (F(2,72)=59.30, P<0.001) when ompared with vehiletreated ontrol. However, 3 days pretreatment of rats with GCK or tarine prevented (P<0.001) the sopolamine indued inrease in nitrite generation in the prefrontal ortex, striatum and hippoampus. Two way ANOVA revealed signifiant treatment effet of GCK or tarine (F(5,72)=53.15,P<0.001) and pretreatment treatment interation (F(10,72)=18.32, P<0.01) (Fig. 8). 47 West Afrian Journal of Pharmay (2017) 28 (1)

14 Ishola et al Nitrite (U/mg protein) PFC STR HIPPO 0 Veh Sop TAC 5 GCK 25 GCK 50 GCK 100 Sop 3 mg/kg, i.p. Figure 8: The effet of GCK on nitrite level within the prefrontal ortex, striatum and hippoampus of sopolamineã indued amnesi rats. Values are expressed as mean±sem (n=6). P<0.001 versus vehile-treated, ontrol; P<0.001 versus vehile + sopolamine treated. Statistial level of signifiane analysis by two way ANOVA followed by Tukey post ho multiple omparison test. DISCUSSION Findings obtained from this study showed that GCK possesses memory enhaning effet evidened in its ability to prevent sopolamine-indued working memory and spatial learning defiits in rodents as well as its ability to savenge oxidative and nitrosative stresses in disrete brain regions. Sopolamine, a musarini aetylholine reeptor antagonist, is used as the gold standard for induing ognitive defiits in man 3,28 and animals. The sopolamine model is still used extensively for prelinial testing of new substanes 29,30 designed to treat ognitive impairment. It is well known that holinergi neuronal systems play an important role in the ognitive defiits assoiated with 3 AD, ageing and neurodegenerative diseases. The spontaneous alternation behaviour in the Y- maze, as an index of short-term memory is a highly useful method to sreen ompounds against amnesi rodent 20,31 model.. The GCK-indued inrease in alternation behavior did not affet the number of arm entries thus, ruling out psyhostimulant ativity. The EPM test on the other hand was originally developed to estimate 32 anxiety in rodents. However, it was modified to evaluate spatial learning and memory in rodents. The parameters measured are not the same: the number of entries into the open and losed arms, the time spent in the open arms for anxiety, but transfer lateny (TL), whih reflets the time the mie took to move from the open arm to either of the enlosed arms for the 3 3, 3 4 memory proesses. In the present study, sopolamine indued spatial learning defiit whih was ameliorated by GCK treatment. The derease in transfer lateny on 2nd day (i.e., 24?h after the first trial) when ompared with aquisition trial indiated improvement 34,35 of memory. Morris water maze test evaluates spatial memory and 36 detets hanges in the entral holinergi system. In this study, sopolamine administration inreased the esape lateny indiating spatial learning and long term memory defiits at a dose of 3 mg/kg, this dose has been reported to have no effet on swimming ability and appears to be dissoiated from drug-indued 36 hyperativity. The vehile-treated ontrol group rapidly aquire spatial learning in the MWM task. Similarly, pretreatment of rats with G. kola signifiantly shortened esape lateny time from day 3. The session dependent derease in esape lateny produed by G. 37 kola represents long-term memory. These results suggest that the extrat improves long-term memory in amnesi rat model indued by sopolamine treatment. Oxidative and nitrosative stress our in biologial systems due to the dysregulation of the redox balane, aused by a defiieny of antioxidants and/or the 38 overprodution of free radials. It has been proposed 48 West Afrian Journal of Pharmay (2017) 28 (1)

15 Nootropi effet of Garinia kola seed extrat that the progressive inrease in ROS and onsequent oxidative damage play the major role in neurodegenerative disorders. Ciobia et al showed that the levels of SOD and GPX derease in rats by sopolamine and the level of malondialdehyde inrease in same rats, ompared with saline-treated rats. Several researh works validate the harmful influene of sopolamine on ortex and hippoampus-dependent learning and memory. Jeong et al. showed that memory impairment in the sopolamine-indued animal model is assoiated with inreased oxidative and nitrosative stress within the brain. This improvement was mainly shown by the inreased levels of GSH, SOD, and atalase as well as the dereased intraellular reative oxygen speies, MDA and nitrite onentrations in the aforementioned entral areas. Previous studies identified some ative ompounds in G. kola whih inlude Garinia biflavonoid (GB) 1, GB2, 43 kolaviron, kolaflavone and kolaflavanone. The phytohemial sreening of G. kola showed that it ontains high quantities of alkaloids, and moderate quantities of saponins, tannins, flavonoids, yanogeni glyosides, sterols and phenols. In addition, these results indiated that the different onstituents of G. kola ould be potent free radial savenger, as they savenged the DPPH radial, hydroxyl radial and NO radial in vitro system. G. kola reated with DPPH radial, and onverted it to 1, 1-diphenyl-2- pirylhydrazine, and the degree of disoloration (indiated by derease in absorption) indiated the 44 savenging ativity. The strong savenging apaity of the extrat on DPPH was possibly due to the hydrogen donating ability of the polyphenoli ompounds. Nitri oxide is onsidered as relatively less reative, its metaboli produt suh as peroxynitrite, formed after reating with oxygen, is extremely reative and an indue toxi reations, inluding thiol group oxidation, protein tyrosine nitration, lipid peroxidation, and DNA 45 modifiation. The nitrite savenging ability of extrats further expands the role of this plant as a potent antioxidant. The reduing power apaity of the extrat may provide a signifiant indiation about the potential antioxidant apaity of the seeds. However, further study is ongoing to investigate possible amelioration of sopolamine-indued ognitive impairment by kolaviron in rodents. CONCLUSION Findings from this study demonstrated memory enhaning properties of Garinia kola through enhanement of antioxidant systems in the prefrontal ortex, striatum and hippoampus. In addition, Garinia kola might offer a useful therapeuti hoie in either the prevention or the treatment of dementia. ACKNOWLEDGEMENT The authors are grateful to Mr. C. Miah of the Department of Pharmaology, Therapeutis and Toxiology, and Mr. S.A. Adenekan of the Department of Biohemistry, College of Mediine of the University for their tehnial assistane. REFERENCES 1. Van Der Flier WM, Van Den Heuvel DM, Weverling- Rijnsburger AW, Spilt A, Bollen EL, Westendorp RG, Middelkoop HA, and Van Buhem MA. (2002). Cognitive deline in AD and mild ognitive impairment is assoiated with global brain damage. Neurology, 59(6): Braak, H., Braak, E., Grundke-Iqbal, I., and Iqbal, K. (1986). Ourrene of neuropil threads in the senile human brain and in Alzheimer's disease: a third loation of paired helial filaments outside of neurofibrillary tangles and neuriti plaques. Neurosiene Letter, 65, Bartus RT, Dean RL, Beer B, and Lippa AS (1982). The holinergi hypothesis of geriatri memory dysfuntion. Siene, 217: Lim YY, Maruff P, Shindler R, Ott BR, Salloway S, Yoo DC, Noto RB, Santos CY, and Snyder PJ. (2015) Disruption of holinergi neurotransmission exaerbates Aâ-related ognitive impairment in prelinial Alzheimer' disease. Neurobiology of Aging, pii: S (15) Cyril-Olutayo CM, Adekunle TO, and Taiwo OE (2012). Ethnobotanial survey of plants used as memory enhaner and anti-ageing in Ondo state, Nigeria. International Journal of Pharmay, 2; Elufioye OT, Obuotor, EM, Sennuga, AT., Agbedahunsi, JM., and Adesanya SA. (2009). Aetylholinesterase and butyrylholinesterase inhibitory ativity of some seleted Nigerian mediinal plants. Brazilian Journal of Pharmaognosy, 20(4): Farombi EO, Adedara IA, Oyenihi AB, Ekakitie E, and Kehinde S. (2013). Hepati, testiular and spermatozoa antioxidant status in rats hronially treated with Garinia kola seed. Journal of Ethnopharmaology, 146(2): Atawodi SE, Mende P, Pfundstein B, Preussmann R, and Spiegelhalder B. (1995). Nitrosatable amines 49 West Afrian Journal of Pharmay (2017) 28 (1)

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