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Presented by
RAMLAGAN Piteesha
MPhil/PhD Student
Centre for Biomedical and Biomaterials Research
University of Mauritius
Mauritius
Rencontres de l’Agro-alimentaire en Océan Indien 28th Nov -2nd Dec 2016 Saint Pierre, La Réunion
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Type II diabetes
Worldwide clinical disorder
Increasing deaths due to diabetic complications
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Diabetic complications
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Diabetic complications
AGE receptor
AGE
Target genes transcription
Pro-inflammatory cytokines Vascular adhesion molecules
Oxidative stress
ROS
NFκB activation
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CD36: an AGE receptor
3D representation of CD36
receptor on cellular membrane
(Collot-Teixeira et al., 2007)
Overexpressed in presence of
AGEs
Atherosclerotic
Implicated in insulin
resistance, dyslipidemia
Down-regulates leptin expression
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Diabetes Statistics
Estimated number of people with diabetes worldwide (IDF Diabetes Atlas, 2015)
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Diabetes Statistics
Leading causes of death worldwide (IDF Diabetes Atlas, 2015)
Diabetes estimates (20-79 years)
Mauritius Reunion Madagascar Comoros South Africa
Prevalence, % 22.3 15.8 4.0 9.9 7.6
% of diabetic population
18.3 13.0 1.5 3.6 4.2
Diabetes related deaths
2931.2 NA 5580.2 318.7 57 318.6
Mean diabetes related expenditure (USD), per person
934.3 NA 111.4
152.2 1736.1
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Pomegranate
Antioxidant functional foods with anti-diabetic and anti-atherogenic potentials
Non-edible parts bioactive in multi-assay antioxidant systems
Pomegranate mesocarp
Pomegranate pericarp
Ethnopharmo-cological uses
Polyphenolic richness
Antioxidant potentials
Anti-inflammatory
capacities
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Uses of pomegranate fruit
Food Cosmetics
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AGE receptor
AGE
Target genes transcription
Pro-inflammatory cytokines Vascular adhesion molecules
Oxidative stress
ROS
NFκB activation
Our focus
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Biochemical and antioxidant screening
Freeze dried and ground. Exhaustively extracted by 70% methanol for 3 days. Lyophilised.
Biochemical Assays (Phenolic, Flavonoid, Proanthocyanidin, hydrolyzable tannin)
Antioxidant Assays (FRAP, ORAC, Iron (II) chelating, Superoxide, nitric oxide, hydroxyl, ABTS, DPPH scavenging)
Pomegranate extract (PME)
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Rich in polyphenolics
Phenolic content: 416.1 ± 11.4 mg GAE/g lyophilised powder (LP)
Flavonoid content: 310.6 ± 9.1 mg QE/g LP
Hydrolysable tannin content: 699.4 ± 16.5 mg TAE/g LP
Proanthocyanidin content: 1.6 ± 0.1 mg CCE/g LP
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High antioxidant capacities
In vitro antioxidant activity IC50 of PME (µg/mL) IC50 of gallic acid (µg/mL)
Nitric oxide radical scavenging 1.16 ± 0.03 a 88.16 ± 2.84 b
ABTS radical scavenging 2.74 ± 0.07 b 0.53 ± 0.02 a
DPPH radical scavenging 7.30 ± 0.26 b 1.45 ± 0.11 a
Superoxide radical scavenging 18.77 ± 0.69 b 6.48 ± 0.24 a
Hydroxyl radical scavenging 28.29 ± 1.11 a 173.89 ± 7.18 b
Iron (II) chelating 34.12 ± 1.66 a 5453.53 ± 191.54 b
In vitro antioxidant activities of PME and standard antioxidant
Antioxidant capacities:
• FRAP value of 12.7 ± 1.1 mmol Fe2+ equivalent/g LP (Gallic acid: 45.1 ± 4.5 mmol)
• ORAC value of 1.1 ± 0.1 mmol Trolox equivalent/g LP (Gallic acid: 17.7 ± 2.6 mmol)
PME Green tea Black Tea Gallic acid
FRAP value (mmol) 12.7 ± 1.1b 6.0 ± 0.5b 4.4 ± 0.3b 45.1 ± 4.5a
ORAC value (mmol) 1.1 ± 0.1b 4.0 ± 0.2b 2.2 ± 0.1b 17.7 ± 2.6a
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High antioxidant capacities
Hydroxyl groups and compound configuration (aromatic rings): major determinants of antioxidant potential
Punicalagin
Gallic acid Ellagic acid Quercetin Catechin
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PME’s effect on 3T3-L1 cell viability
Freeze dried and ground. Exhaustively extracted by 70% methanol for 3 days. Lyophilised.
Pomegranate extract (PME)
Cell viability assays
3T3-L1 preadipocytes: mimicking diabetes-like oxidative stress
(Castan-laurell et al., 2012)
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PME’s effect on 3T3-L1 cell viability
0 5 10 15 200
50
100
150
**
MTT metabolic activity
Crystal Violet
**
LDH release
*
Concentration (g/mL)
Assa
y
(% a
s c
om
pa
red
to
co
ntr
ol)
0 5 10 15 2020
25
30
35
**
Concentration (g/mL)
Ce
ll N
um
be
r (1
* 1
04)
A B
Effect of PME on preadipocyte viability. Cytotoxicity assessed by (A) MTT metabolic activity, crystal violet and LDH release and (B) Trypan Blue exclusion methods.
Gallic acid increased 3T3-L1 cell death in dose dependent manner (Hsu et al., 2006)
Proportional conc. of flavonoids in lime juice induced apoptosis of human
pancreatic cells (Patil et al., 2009)
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PME’s protective effect against oxidative stress
Pretreatment with PME for 24h
Freeze dried and ground. Exhaustively extracted by 70% methanol for 3 days. Lyophilised.
Pomegranate extract (PME)
3T3-L1 preadipocytes: mimicking diabetes-like oxidative stress
BSA + MGO => BSAMGO (AGEs)
H2O2
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(Barnes et al., 2011)
PME decreases ROS production A (i)
Contr
ol
PME
G0
BSA
+ P
ME
G0
BSA
MG
O
BSA + P
ME
MG
O
BSA
2 O2H + P
ME
2 O2H
0
40
80
120
****** ***
# # #
# # ## #
1 h treatment
***
***
# # #
RO
S p
rod
uc
tio
n
(% a
s c
om
pa
red
to
co
ntr
ol)
Contr
ol
PME
G0
BSA
+ P
ME
G0
BSA
MG
O
BSA + P
ME
MG
O
BSA
2O2H
+ PM
E
2 O2H
0
40
80
120
***
******
***
24 h treatment
******
***
# # #
# # # # # #
# # #
RO
S p
rod
uc
tio
n
(% a
s c
om
pa
red
to
co
ntr
ol)
G0
BSA P
ME
MGO
BSA +
PM
E
MGO
BSA
G0
BSA P
ME
MGO
BSA +
PM
E
MGO
BSA
0
50
100
150
200
250
300
# # #
# # #
§§§
§§§
§§§
1 h treatment 24 h treatment
NO
X1
ge
ne
ex
pre
ss
ion
(% r
ela
tiv
e t
o B
SA
G0)
(ii)
B
Effect of PME on (A) intracellular ROS production at (i) 1 h and (ii) 24 h treatment; (B) mRNA expression NOX1.
24 h treatment
0 100 200 30060
80
100
120
r = 0.97p < 0.001
NOX1 level
RO
Sle
ve
l
1 h treatment
0 50 100 15060
80
100
120
r = 0.85p < 0.001
NOX1 level
RO
Sle
ve
l
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PME reduces oxidatively modified proteins
Contr
ol
PME
G0
BSA
+ P
ME
G0
BSA
MGO
BSA + P
ME
MGO
BSA
2O2H
+ PM
E
2 O2H
0
50
100
150
*
**
24 h AGEs Treatment
***
# # ## # ## #
Pro
tein
Carb
on
yl P
rod
ucti
on
(% a
s c
om
pare
d t
o c
on
tro
l)
Contr
ol
PM
E G0
BSA
+ P
ME
G0
BSA
MGO
BSA +
PME
MGO
BSA
2 O2H +
PME
2 O2H
0.00
0.05
0.10
0.15
0.20
*
*
* *
**
## # #
AO
PP
le
ve
l
( M
ch
lora
min
e-T
eq
uiv
ale
nt
/
g
to
tal
pro
tein
s)
A
Effect of PME on (A) protein carbonyls, (B) AOPP accumulations and (c) linear regression plots and Pearson’s correlation coefficients between ROS level and (i) protein carbonyl and (ii) AOPP levels
0 50 100 15080
100
120
r = 0.72p < 0.001
ROS level
Pro
tein
Ca
rbo
nyl
Le
ve
l
0 50 100 1500.00
0.05
0.10
0.15
0.20
r = 0.55p < 0.01
ROS level
AO
PP
le
ve
l
C (i)
B
C (ii)
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Intrinsic antioxidant enzymes: expression & activity
SOD1
β actin
CAT
β actin
Contr
ol
PME
G0
BSA
+ P
ME
G0
BSA
MGO
BSA
+ P
ME
MGO
BSA
2O2H
+ P
ME
2O2H
0
10
20
30
****
# # #
# # # #
SO
D a
cti
vit
y
(U/m
g t
ota
l p
rote
in)
Contr
ol
PME
G0
BSA
+ P
ME
G0
BSA
MGO
BSA
+ P
ME
MGO
BSA
2O2H
+ P
ME
2O2H
0
10
20
30
40
*
**
# # #
#
#
CA
T a
cti
vit
y
(U/m
g t
ota
l p
rote
in)
1 1 1 0.8 0.6 1 1.5 1.2 1 0.8 0.7 0.7 0.7 0.9 1.2 0.9
Effect of PME (A) SOD, (B) CAT and (C) GPx enzymatic activities and expressions at 24h treatment. (Densitometry values are expressed relative to control and normalized against β-actin.
A B
Contr
ol
PM
E G0
BSA
+ P
ME
G0
BSA
MGO
BSA
+ P
ME
MGO
BSA
2O2H
+ P
ME
2O2H
0
50
100
150
200
***
# # # # #
GP
x a
cti
vit
y
(%/m
g t
ota
l p
rote
in)
GPx1/2
β actin
1 1.3 1.4 1.7 1.4 1 1.2 1.3
C
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BS
AG
0 +
PM
E
PME down-regulates CD36 expression
G0
BSA P
ME
MGO
BSA +
PM
E
MGO
BSA
G0
BSA P
ME
MGO
BSA +
PM
E
MGO
BSA
0
50
100
150
# # #
§§§
§§§
# # #§
1 h treatment 24 h treatment
CD
36 g
en
e e
xp
res
sio
n
(% r
ela
tiv
e t
o B
SA
G0)
Co
ntr
ol
CD36
88 kDa
β-actin
42 kDa
1 h treatment
1 0.3 1 0.3 0.9 0.2 0.8 0.4
CD36
88 kDa
β-actin
42 kDa
24 h treatment
1 0.4 0.9 0.3 0.5 0.1 0.6 0.3
Effect of PME on (A) mRNA expression of CD36 at 1 h and 24 h; (B) CD36 protein expression at (i) 1h and (ii) 24h(Densitometry values are expressed relative to control and normalized against β-actin.
(A)
B (i) (ii)
H2O
2
PM
E
BS
AG
0
BS
AM
GO +
PM
E
BS
AM
GO
H2O
2 +
PM
E
Co
ntr
ol
PM
E
BS
AG
0
BS
AG
0 +
PM
E
BS
AM
GO
BS
AM
GO +
PM
E
H2O
2
H2O
2 +
PM
E
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Conclusion
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PME: Propensity to mitigate obesity-related disorders
Diabetes estimates (20-79 years)
Mauritius Reunion Madagascar Comoros South Africa
% of diabetic population
18.3 13.0 1.5 3.6 4.2
Mean diabetes related expenditure (USD), per person
934.3 NA 111.4
152.2 1736.1
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Acknowledgement
Dr Philippe Rondeau
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