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Aspartame, a dietary sweetener, concentration dependently induces neurotoxicity and oxidative damage in the brain of the male wistar rat. Protective role of Garcinia kola

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BY OGBOLE, SAMSON OMATRIC NO: 188819

PROJECT SEMINAR

MOLECULAR DRUG METABOLISM AND TOXICOLOGY UNITSUPERVISOR: DR O.A. ADARAMOYE

Aspartame, a dietary sweetener, concentration-dependently induces neurotoxicity and oxidative damage in the brain of male wistar rat: Protective role of the Oil of Garcinia kola

2/21/2017

OUTLINE2/21/2017INTRODUCTION

RATIONALE AND AIMS OF THE STUDY

MATERIALS AND METHODS

RESULTS

CONCLUSION2

INTRODUCTION2/21/20173

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INTRODUCTION2/21/20174Neurotoxicity is toxicity in the nervous system. It occurs when exposure to natural or artificial toxic substances (neurotoxins), alters the normal activity of the nervous system in such a way as to cause damage to nervous tissue (Robert et al., 1996).

Oxidative stress is a disturbance in the balance between the production of reactive oxygen species (free radicals) and antioxidant defenses (Betteridge , 2000).

Aspartame (L-aspartyl-L-phenylalanine methyl ester), a low-calorie sweetener, is about 180 times sweeter than normal sugar and is used in over 5000 foods and beverages (Shapiro 1988). Approximately 50% of the aspartame molecule is phenylalanine, 40% is aspartic acid and 10% is methanol (Newsome 1986).

Garcinia kola is a herb grown in Nigeria with a characteristic astringent, bitter and resinous taste, used in ethnomedicine in the treatment of laryngitis, cough, liver disease and dementia. The plant has shown anti-inflammatory, antimicrobial and antiviral properties. In addition, the plant possesses anti-diabetic, and anti-hepatotoxic activities (Iwu, 1993)

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STRUCTURE OF ASPARTAME2/21/20175

Figure 1: Structure of Aspartame (Ager et al., 1998)

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RATIONALE OF STUDY2/21/20176Aspartame a dietary sweetener, has been implicated in various ailments such as cancer, seizures, headaches, depression, attention deficit disorder and dizziness.

Aspartame's effects on the brain had been the subject of several studies that had generated an array of contradicting results (Christian et al., 2004; Bergstrom et al., 2007; Abd El-samad, 2010; Abdel-Salam et al., 2012., Ashok et al., 2013). Some studies have reported evidence of neuronal injury in several brain regions (Omar, 2009, Abdel-Samad, 2010 Abu-Taweel et al., 2014), while others have reported no evidences of such (Finklestein et al., 1988, Burdock, 2005).

There however remains a dearth of information on dosage at which Aspartame induces toxicity.

This study was therefore designed to evaluate the toxic effect of Aspartame in the brain of male wistar rats and the protective effect of the oil of Garcinia kola

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OBJECTIVES2/21/20177

Determine the neurotoxic effect of Aspartame at different concentrations in brain and the protective role of the oil of Garcinia kola in aspartame-induced toxicity in the brain of male wistar rats.

Determine the neurotoxic effect of Aspartame at different concentrations on behaviour of the male wistar rat and protective role of the Oil of Garcinia kola

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MATERIALS AND METHOD2/21/20178EXPERIMENTAL DESIGNForty two adult male wistar rats were randomly assigned into seven groups of six wistar rats each.

Group 1: Negative control group (administered with distilled water only)Group 2: Administered with 15mg/kg ASP Group 3: Administered with 35mg/kg ASP Group 4: Administered with 70mg/kg ASPGroup 5: Administered with 35mg/kg ASP + co-treated with 200mg/kg OGKGroup 6: Administered with 70mg/kg ASP + co-treated with 200mg/kg OGKGroup 7: Positive control group (administered with 200mg/kg OGK only)

Aspartame (ASP) and Essential Oil of Garcinia kola (EOGK) were administered once daily by oral gavage Chemicals used for this studies were procured from sigma company and all assays were performed using current standard procedure.

All administration was done through oral gavage. ASP was administered 5 times weekly and OGK administered 3 times a week respectively for 9 weeks.Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals co-treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil of Garcinia kola only

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2/21/2017RESULTS & DISCUSSION9

10Values are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola only. LC = Lethal concentration2/21/2017Figure 2: Graph showing plot for LC 50 determination (1577ug/ml)

DETERMINATION OF LC 50 OF OIL OF Garcinia kola USING BRINE SHRIMP ASSAYConcentration of oil of Garcinia kolaLifeDeadratio

11Values are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)

2/21/2017Table 1.: Effect of aspartame induced toxicity and role of Essential Oil of Garcinia kola on the body weight, brain weight and organo-somatic weight of brain.

TreatmentBody Weight (g)Weight of organ (g)Organo-somatic weight (% body weight)InitialFinalWeight gainBrainBrainControl119.53 2.86214.85 41.3595.33 41.121.78 0.170.84 0.10Asp 15mg/kg130.07 4.11209.78 15.6879.45 11.751.55 0.230.74 0.13Asp 35mg/kg137.97 9.50215.70 20.0679.90 11.051.77 0.270.81 0.06Asp 70mg/kg141.83 10.63203.43 22.8967.42 13.17a1.60 0.340.78 0.22Asp 35mg/kg + GK 200mg/kg140.78 8.65210.80 27.1555.88 19.281.53 0.120.79 0.08Asp 70mg/kg + GK 200mg/kg150.55 9.02252.90 17.7092.65 9.73b1.55 0.270.64 0.09GK 200mg/kg141.43 8.48242.63 26.0590.35 18.131.57 0.140.68 0.56

12Values are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)2/21/2017Figure 3: Effect of Oil of Garcinia kola on Glutathione-S-Transferase activity in the brain of wistar rats treated with graded doses of Aspartame.

ASSESSMENT OF EFFECT OF ASPARTAME ON THE ANTIOXIDANT ACTIVITY AND THE PROTECTIVE EFFECT OF THE OIL OF Garcinia kola ON THE BRAIN OF MALE WISTAR RATS

132/21/2017Figure 4: Effect of Oil of Garcinia kola on Superoxide Dismutase activity on the brain of wistar rats treated with graded doses of Aspartame

ASSESSMENT OF EFFECT OF ASPARTAME ON THE ANTIOXIDANT ACTIVITY AND THE PROTECTIVE EFFECT OF THE OIL OF Garcinia kola ON THE BRAIN OF MALE WISTAR RATS

Values are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)

142/21/2017Figure 5: Effect of Oil of Garcinia kola on Lipid Peroxidation level on the brain of wistar rats treated with graded doses of Aspartame

ASSESSMENT OF EFFECT OF ASPARTAME ON THE ANTIOXIDANT ACTIVITY AND THE PROTECTIVE EFFECT OF THE OIL OF Garcinia kola ON THE BRAIN OF MALE WISTAR RATSValues are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)

152/21/2017Table 2: Effect of Oil of Garcinia kola on Glutathione, Glutathione peroxidase and Catalase activity in the brain of wistar rat treated with graded doses of Aspartame.

TREATMENTProtein (Serum)Protein (Brain)GSH (mg/g) GPx (mg/g) CAT(U/mg protein)CONTROL5.22 0.6613.66 0.211.85 0.1015.62 0.301.01 0.02ASP 15 mg/kg4.68 0.2413.32 0.451.82 0.0716.06 0.451.32 0.32ASP 35 mg/kg4.35 0.6513.02 0.991.89 0.0115.47 1.072.41 2.06ASP 70 mg/kg3.07 0.3313.51 0.261.79 0.1315.24 1.041.44 0.38ASP 35 mg/kg + EOGK 200 mg/kg4.84 0.0813.66 0.471.84 0.1115.82 0.621.91 0.28ASP 70 mg/kg + EOGK 200 mg/kg5.10 0.9213.49 0.571.84 0.0716.14 0.152.56 0.54EOGK 200 mg/kg5.17 3.8014.40 0.421.81 0.0414.87 0.132.07 0.39

Values are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)

162/21/2017Figure 6: Effect of aspartame on myeloperoxidase level and the protective role of the oil of Garcinia kola in the brain of wistar rats treated with graded doses of Aspartame

INFLAMMATORY EFFECT OF ASPARTAME AND THE ASSESSMENT OF THE PROTECTIVE ROLE OF THE OIL OF Garcinia kola IN THE BRAIN OF MALE WISTAR RATSValues are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)

172/21/2017Figure 7: Effect of aspartame on nitric oxide level and the protective effect of the oil of Garcinia kola in the brain of wistar rats treated with graded doses of Aspartame.

INFLAMMATORY EFFECT OF ASPARTAME AND THE ASSESSMENT OF THE PROTECTIVE ROLE OF THE OIL OF Garcinia kola IN THE BRAIN OF MALE WISTAR RATS

Values are mean S.D. of 6 animals per group; Negative Control = Animals treated with distilled water onlyAsp = Animals treated with aspartame only; Asp + OGK = Animals treated with aspartame + Oil of Garcinia kola; Positive Control = Animals treated with Oil Garcinia kola onlya = significantly different from control group (p < 0.05)b = significantly different from ASP at all doses (p < 0.05)

182/21/2017Figure 8: Effect of Oil of Garcinia Kola on (a)Rearing, (b) Grooming

A. RearingB. GroomingBEHAVIOURAL RESULTPROTECTIVE ROLE OF OIL OF Garcinia kola ON TOXIC EFFECT OF ASPARTAME IN MALE WISTAR RATS ACTIVITIES IN AN OPEN FIELD TEST

192/21/2017Figure 9: Effect of Oil of Garcinia Kola on Percentage of alteration in Hole-Board Test

BEHAVIOURAL RESULTPROTECTIVE ROLE OF OIL OF Garcinia kola ON TOXIC EFFECT OF ASPARTAME IN MALE WISTAR RATS ACTIVITIES IN HOLE-BOARD TEST

202/21/2017Figure 10: Effect of Oil of Garcinia Kola on Percentage of alteration in Y Maze

BEHAVIOURAL RESULTPROTECTIVE ROLE OF OIL OF Garcinia kola ON TOXIC EFFECT OF ASPARTAME IN MALE WISTAR RATS ACTIVITIES IN Y MAZE

212/21/2017Table 3: Photomicrograph showing protective effect of Oil of Garcinia kola in the brain of wistar rats treated with graded doses of Aspartame (Haematoxylin and Eosin staining)

TreatmentsHippocampal regionCerebral cortexNeuronal cellsGlia cellsControlNormalNormalNormalNormalASP 15mg/kgNormalDead cells, hypocellularityDead cells, chromatolysisNecrosisASP 35mg/kgShrunk cells, Choroid plexus congestionShrunk cells, necrosisNecrosis, mild hemorrhageNecrosisASP 70 mg/kgLoss of neuronal cellsShrunk cells, necrosisNecrosisNecrosisASP 35mg/kg + OGK 200mg/kgNormalFusion of layersVery few shrunk cellsMost are normalASP 70mg/kg + OGK 200mg/kgNormal well defined laminaeSome chromalolyzed cellsVery few shrunk cells, mineralized necrosisNormalOGK 200mg/kgNormalNormalNormalNormal with mildly thickened wall

1: Control5. 35mg/kg Aspartame + 200mg/kg Oil of Garcinia kola2. 15mg/kg Aspartame6. 70 mg/kg Aspartame + 200mg/kg Oil of Garcinia kola3. 35mg/kg Aspartame7. 200mg/kg Oil of Garcinia kola4. 70mg/kg Aspartame

222/21/2017Figure 12:Photomicropraph of effect of ASP and the protective effect of OGK in the brain of wistar rats treated with graded doses of Aspartame using H & E.1: Control1. Green (Glia cells), Black (Neuronal cells)ASP: Aspartame2. 15mg/kg Asp2. Black (shrunken cerebral cortex)OGK: Oil of Garcinia kola3. 35mg/kg Asp3. Red (Shrunken necrotized cells Neuronal/Glia)4. 70mg/kg Asp4. Black (necrotized Glia and Neuronal Cells)5. 35mg/kg Asp + 200mg/kg EOGK5. Neuronal and Glia cells appearing normal6. 70 mg/kg Asp + 200mg/kg EOGK6. Normal neuronal cells7. 200mg/kg EOGK7. Normal neuronal cells

PHOTOMICROGRAPH ASSESSMENT OF EFFECT OF ASPARTAME AND THE PROTECTIVE ROLE OF OIL OF Garcinia kola ON THE BRAIN OF MALE WISTAR RATS USING HAEMATOXYLIN AND EOSIN STAIN (Mag. X 400)7654321

CONCLUSION2/21/2017

The results obtained from the present study showed that the administration of Aspartame to male wistar rats induced oxidative stress, depression and memory loss especially at 70mg/kg.

However, the treatment of aspartame-induced male wistar rats with Oil of Garcinia kola potentiates protective effect against tissue damage in the brain.

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REFERENCES2/21/201724

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2/21/2017THANKS

FOR

LISTENING25

SOD 480 CAT 240 LPO 532 GSH 412 GPx GST 340 AChE MPO 460 Protein 540/595 TNO 550

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