This study investigated the neurotoxic effects of aspartame at different concentrations in rats and the protective role of Garcinia kola essential oil. Rats were given aspartame at 15, 35, or 70 mg/kg alone or with Garcinia kola oil at 200 mg/kg for 9 weeks. Aspartame caused oxidative damage and reduced antioxidant enzyme activity in the brain in a dose-dependent manner. Co-administration of Garcinia kola oil protected the brain against aspartame-induced toxicity by improving antioxidant status. The results suggest that Garcinia kola oil has potential as a therapy against aspartame neurotoxicity.

1. ASPARTAME, A DIETRY SWEETNER, CONCENTRATION-DEPENDENTLY
INDUCES NEUROTOXICITY AND OXIDATIVE DAMAGE IN THE BRAIN
OF MALE WISTAR RATS: PROTECTIVE ROLE OF ESSENTIAL OIL OF
Garcinia kola
By
OLUWATAYO, DAMILOLA JOHN
MATRIC. NO.: 188814
PR0JECT SEMINAR
MOLECULAR DRUG METABOLISM AND TOXICOLOGY UNIT
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF BASIC MEDICAL SCIENCES
COLLEGE OF MEDICINE
UNIVERSITY OF IBADAN
SUPERVISOR: DR. O.A. ADARAMOYE
6/17/2020 1

2. OUTLINE
 INTRODUCTION
 RATIONALE OF THE STUDY
 MATERIALS AND METHODS
 RESULTS
 CONCLUSION
6/17/2020 2

3. INTRODUCTION
 Neurotoxicity 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).
 Aspartame is associated with significant dose-dependent neurotoxicity and oxidative
stress despite its widespread use. There is therefore a need for the development of
safer therapy which can be derived from plant sources.
 Garcinia kola is a herb grown in Nigeria with a characteristic astringent and bitter
taste. It is used in ethnomedicine in the treatment of laryngitis, cough, liver disease
and dementia. The plant has shown anti-inflammatory, antimicrobial and antiviral
properties. (Iwu, 1993).
6/17/2020 3

4. STRUCTURE OF ASPARTAME
6/17/2020
4
Figure 1: Structure of Aspartame (Ager et al., 1998)

5. RATIONALE OF THE STUDY
6/17/2020 5

6. RATIONALE OF STUDY
6/17/2020
6
 The effects of aspartame on the brain have been the subject of several
studies that have generated arrays 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 evidences 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 are also studies supporting (Goerss et al., 2000; Beck et al., 2002),
or refuting (Reilyet et al., 1990, Reily and Lajtha, 1995) the occurrence of
aspartame-induced alterations in brain neurotransmitter activities

7. OBJECTIVES
6/17/2020
7
This study was designed:
To determine the neurotoxic effect of Aspartame at different
concentrations in the serum and brain of male wistar rats.
To assess the protective role of essential oil of Garcinia kola against
aspartame-induced toxicity in the serum and brain of male wistar
rats.

8. MATERIALS AND METHODS
EXPERIMENTAL DESIGN
Forty two adult male Wistar rats were randomly assigned into seven groups of six rats each.
 Group 1 (6 animals): The negative control group.
 Group 2 (6 animlas): ASP 15mg/kg
 Group 3 (6 animals): ASP 35mg/kg
 Group 4 (6 animals): ASP 70mg/kg
 Group 5 (6 animals): ASP 35mg/kg + EOGK 200mg/kg
 Group 6 (6 animals): ASP 70mg/kg + EOGK 200mg/kg
 Group 7 (6 animlas): EOGK 200mg/kg
Aspartame and EOGK were administered once daily by oral gavage for consecutive 9 weeks.
Chemicals used for this studies were procured from sigma company and all assays were performed
using current standard procedure.
6/17/2020 8

9. 6/17/2020
ANTIOXIDANTS AND ORGAN FUNCTION MARKERS
 Superoxide dismutase(SOD):Method by Mistra and Fridovich,1972.
 Catalase(CAT): Method by Claiborne, 1985.
 Lipid Peroxidation(LPO): Method by Rice-Evans et al, 1986.
 Reduced Glutathione (GSH): Method by Beutler et al., 1963.
 Glutathione Peroxidase (GPx): Method by Rotruck et al., 1973.
 Glutathione-S-transferase (GST): Method by Habig, Pabst and Jacoby, 1974.
 Acetylcholine Esterase (AChE): Method by Ellman et al., 1961.
 Myeloperoxidase (MPO): Method by Trush et al., 1994.
 Total Nitric Oxide (NO): Method by Green et al., 1982.
9

10. RESULT and discussion
6/17/2020 10

11. 11
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
6/17/2020
Figure 1: Graph showing plot for LC 50 determination (1577ug/ml)
y = -0.022x + 84.695
R² = 0.9412
-20
0
20
40
60
80
100
120
0 500 1000 1500 2000 2500 3000 3500 4000 4500
Series1
Linear (Series1)
DETERMINATION OF LC 50 OF ESSENTIAL OIL OF Garcinia kola USING
BRINE SHRIMPASSAY
CONCENTRATION (ug/ml)
L
I
F
E
/
D
E
A
D
R
A
T
I
O

12. 12
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
6/17/2020
Table 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.
Treatment
Body Weight (g)
Weight of
organ (g)
Organo-somatic
weight (% body
weight)
Initial Final Weight gain Brain Brain
Control 119.53 ± 2.86 214.85 ± 41.35 95.33 ± 41.12 1.78 ± 0.17 0.84 ± 0.10
Asp 15mg/kg 130.07 ± 4.11 209.78 ± 15.68 79.45 ± 11.75 1.55 ± 0.23 0.74 ± 0.13
Asp 35mg/kg 137.97 ± 9.50 215.70 ± 20.06 79.90 ± 11.05 1.77 ± 0.27 0.81 ± 0.06
Asp 70mg/kg 141.83 ± 10.63 203.43 ± 22.89 67.42 ± 13.17a 1.60 ± 0.34 0.78 ± 0.22
Asp 35mg/kg + GK
200mg/kg
140.78 ± 8.65 210.80 ± 27.15 55.88 ± 19.28a 1.53 ± 0.12 0.79 ± 0.08
Asp 70mg/kg + GK
200mg/kg
150.55 ± 9.02 252.90 ± 17.70 92.65 ± 9.73b 1.55 ± 0.27 0.64 ± 0.09
GK 200mg/kg 141.43 ± 8.48 242.63 ± 26.05 90.35 ± 18.13 1.57 ± 0.14 0.68 ± 0.56

13. 13
6/17/2020
Table 2: Effect of Essential Oil of Garcinia kola on Glutathione, Glutathione peroxidase and
Catalase activity in the brain of wistar rat treated with graded doses of Aspartame.
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
TREATMENT Protein
(Serum)
Protein
(Brain)
GSH
(mg/g)
GPx (mg/g) CAT
(U/mg
protein)
CONTROL 5.22 ± 0.66 13.66 ± 0.21 1.85 ± 0.10 15.62 ± 0.30 1.01 ± 0.02
ASP 15 mg/kg 4.68 ± 0.24 13.32 ± 0.45 1.82 ± 0.07 16.06 ± 0.45 1.32 ± 0.32
ASP 35 mg/kg 4.35 ± 0.65 13.02 ± 0.99 1.89 ± 0.01 15.47 ± 1.07 2.41 ± 2.06
ASP 70 mg/kg 3.07 ± 0.33 13.51 ± 0.26 1.79 ± 0.13 15.24 ± 1.04 1.44 ± 0.38
ASP 35 mg/kg +
EOGK 200 mg/kg
4.84 ± 0.08 13.66 ± 0.47 1.84 ± 0.11 15.82 ± 0.62 1.91 ± 0.28
ASP 70 mg/kg +
EOGK 200 mg/kg
5.10 ± 0.92 13.49 ± 0.57 1.84 ± 0.07 16.14 ± 0.15 2.56 ± 0.54
EOGK 200 mg/kg 5.17 ± 3.80 14.40 ± 0.42 1.81 ± 0.04 14.87 ± 0.13 2.07 ± 0.39

14. 14
6/17/2020
Figure 1: Effect of Essential Oil of Garcinia kola on Lipid Peroxidation level on the brain
of wistar rats treated with graded doses of Aspartame
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
SERUM BRAIN
0.0
0.5
1.0
Control
ASP 15 mg/kg
ASP 35 mg/kg
ASP 70 mg/kg
ASP 35 mg/kg + EOGK 200 mg/kg
ASP 70 mg/kg + EOGK 200 mg/kg
EOGK 200 mg/kg
b b
a,b
TREATMENTS
LPOActivity
(molMDA/mgprotein)
ASSESSMENT OF EFFECT OF ASPARTAME ON THE LEVEL OF LIPID
PEROXIDATION AND THE PROTECTIVE ROLE OF THE ESSENTIAL OIL OF
GARCINIA KOLA ON THE BRAIN OF MALE WISTAR RATS

15. 15
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
6/17/2020
Figure 2: Effect of Essential 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 GLUTATHIONE-S-
TRANSFERASE ACTIVITY AND THE PROTECTIVE ROLE OF THE ESSENTIAL
OIL OF GARCINIA KOLA ON THE BRAIN OF MALE WISTAR RATS
0.00
0.02
0.04
0.06
b ASP 15 mg/kg
ASP 35 mg/kg
ASP 70 mg/kg
ASP 35 mg/kg + EOGK 200
mg/kg
ASP 70 mg/kg + EOGK 200
mg/kg
EOGK 200mg/kg
a
Control
TREATMENTS
GSTActivity
(mol/mgprotein/min)

16. 16
6/17/2020
Figure 3: Effect of Essential Oil of Garcinia kola on Superoxide Dismutase activity on the
brain of wistar rats treated with graded doses of Aspartame
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
ASSESSMENT OF EFFECT OF ASPARTAME ON THE SUPEROXIDE DISMUTASE
ACTIVITY AND THE PROTECTIVE ROLE OF THE ESSENTIAL OIL OF
GARCINIA KOLA ON THE BRAIN OF MALE WISTAR RATS
0.0
0.5
1.0
1.5
a
a,b
a,b
ASP 15 mg/kg
ASP 35 mg/kg
ASP 70 mg/kg
ASP 35 mg/kg + EOGK 200
mg/kg
ASP 70 mg/kg + EOGK 200
mg/kg
EOGK 200mg/kg
Control
TREATMENTS
SODActivity
(U/mgprotein)

17. 17
6/17/2020
Figure 4: Effect of aspartame on myeloperoxidase level and the protective role of the essential
oil of Garcinia kola in the brain of wistar rats treated with graded doses of Aspartame
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
0
5
10
15
20
25
a
ASP 15 mg/kg
ASP 35 mg/kg
ASP 70 mg/kg
ASP 35 mg/kg + EOGK 200
mg/kg
ASP 70 mg/kg + EOGK 200
mg/kg
EOGK 200mg/kg
Control
TREATMENTS
MPOActivity
(U/mgprotein)
ASSESSMENT OF THE EFFECT OF ASPARTAME ON THE MYELOPEROXIDASE
LEVEL AND THE PROTECTIVE ROLE OF THE ESSENTIAL OIL OF GARCINIA
KOLA IN THE BRAIN OF MALE WISTAR RATS

18. 18
6/17/2020
Figure 5: Effect of aspartame on total nitric oxide level and the protective role of the essential
oil of Garcinia kola in the brain of wistar rats treated with graded doses of Aspartame.
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
ASSESSMENT OF THE EFFECT OF ASPARTAME ON THE TOTAL NITRIC OXIDE
LEVEL AND THE PROTECTIVE ROLE OF THE ESSENTIAL OIL OF GARCINIA
KOLA IN THE BRAIN OF MALE WISTAR RATS
SERUM BRAIN
0
20
40
60
80
100
Control
ASP 15 mg/kg
ASP 35 mg/kg
ASP 70 mg/kg
ASP 35 mg/kg + EOGK 200 mg/kg
ASP 70 mg/kg + EOGK 200 mg/kg
EOGK 200 mg/kg
a
aa
a
a
b
a
a
a bb
TREATMENTS
TotalNOActivity
(U/mgprotein)

19. 19
6/17/2020
Figure 6:Effect of aspartame on acetylcholinesterase activity and the protective role of the essential oil of
Garcinia kola in the brain of wistar rats treated with graded doses of Aspartame.
Values are mean ± S.D. of 6 animals per group;
Negative Control = Animals treated with distilled water only
Asp = Animals treated with aspartame only; Asp + EOGK = Animals treated with aspartame + Essential Oil of Garcinia kola;
Positive Control = Animals treated with Essential Oil Garcinia kola only
a = significantly different from control group (p < 0.05)
b = significantly different from ASP at all doses (p < 0.05)
ASSESSMENT OF EFFECT OF ASPARTAME ON ACETYLCHOLINESTERASE
ACTIVITY AND THE PROTECTIVE ROLE OF THE ESSENTIAL OIL OF
GARCINIA KOLA ON THE BRAIN OF MALE WISTAR RATS
0.00
0.05
0.10
0.15
a
ASP 15 mg/kg
ASP 35 mg/kg
ASP 70 mg/kg
ASP 35 mg/kg + EOGK 200 mg/kg
ASP 70 mg/kg + EOGK 200 mg/kg
EOGK 200mg/kg
Control
TREATMENTS
AChEActivity
U/ml

20. HISTOPATHOLOGICAL ASSESSMENT OF THE EFFECT OF ASPARTAME AND THE
PROTECTIVE ROLE OF ESSENTIAL OIL OF GARCINIA KOLA ON THE BRAIN OF
MALE WISTAR RATS USING CRESYL FAST VIOLET (M. X400)
20
1 2
7654
3
1: Control 5. ASP 35 mg/kg + 200mg/kg EOGK
2. ASP 15 mg/kg 6. ASP 70 mg/kg + 200mg/kg EOGK
3. ASP 35 mg/kg 7. 200mg/kg EOGK
4. ASP 70 mg/kg
ASP = Aspartame EOGK = Essential Oil of Garcinia kola
Figure 8:Histology of effect of ASP and the protective role of EOGK in the brain of wistar rats treated with graded doses of
Aspartame using CFV.

21. CONCLUSION
6/17/2020
• The metabolism of aspartame may induce oxidative stress due
to free radicals generated. Also the increase in the level of
Nitric Oxide and Myeloperoxidase in the groups treated with
aspartame (at all doses) suggests inflammation in the brain.
• Essential oil of Garcinia kola however was able to ameliorate
the neurotoxic effect of aspartame and plays a protective role
against neurotoxicity and oxidative damage in the brain.
• Further studies will be needed to closely establish the
mechanism(s) by which essential oil of Garcinia kola protects
against neurotoxicity and oxidative damage.
21

22. REFERENCES
6/17/2020
Camfield PR, Camfield CS, Dooley JM, Gordon K, Jollymore S and Weaver DF (1992).
Aspartame exacerbates EEG spike wave discharge in children with generalised absence epilepsy: a
double blind controlled study. Neurology 42: 1000-1003.
Edmeads J (1988). Aspartame and headache. Headache 28: 64-65.
Janssen PJ, and van der Heijden CA (1988). Aspartame: review of recent experimental and
observational data. Toxicology 50: 1-26.
Lipton RB, Newman LC, Cohen JS and Solomon S (1989) Aspartame as a dietary trigger of
headache. Headache 29: 90-92. Koehler SM and Glaros A (1988). The effect of aspartame on
migraine headache. Headache 28: 10-14.
Saravis S, Schachar R, Zlotkin S, Leiter LA and Anderson GH (1990). Aspartame: effects on
learning, behavior, and mood. Pediatrics 86: 75-83.
Schiffman SS, Buckley CE, Sampson HA, Massey EW, Baraniuk JN, Follett JV, and Warwick ZS
(1987). Aspartame and susceptibility to headache. N Engl J Med 317: 1181-1185.
Van den Eeden SK, Koepsell TD, Longstreth WT Jr, van Belle G, Daling JR and McKnight B
(1994). Aspartame ingestion and headaches: a randomized crossover trial. Neurology 44: 1787-
1793.
Walton RG, Hudak R and Green-Waite RJ (1993). Adverse reactions to aspartame: double-blind
challenge in patients from a vulnerable population. Biol Psychiatry 34: 13-17.
Wolraich ML, Lindgren SD, Stumbo PJ, Stegink LD, Appelbaum MI and Kiritsy MC (1994).
Effects of diets high in sucrose or aspartame on the behavior and cognitive performance of
children. N Engl J Med 330: 301-307.
22

23. Thanks
for
listening
6/17/2020 23