ramlagan piteesha mphil/phd student centre for ......presented by ramlagan piteesha mphil/phd...
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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
Type II diabetes
Worldwide clinical disorder
Increasing deaths due to diabetic complications
Diabetic complications
Diabetic complications
AGE receptor
AGE
Target genes transcription
Pro-inflammatory cytokines Vascular adhesion molecules
Oxidative stress
ROS
NFκB activation
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
Diabetes Statistics
Estimated number of people with diabetes worldwide (IDF Diabetes Atlas, 2015)
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
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
Uses of pomegranate fruit
Food Cosmetics
AGE receptor
AGE
Target genes transcription
Pro-inflammatory cytokines Vascular adhesion molecules
Oxidative stress
ROS
NFκB activation
Our focus
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)
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
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
High antioxidant capacities
Hydroxyl groups and compound configuration (aromatic rings): major determinants of antioxidant potential
Punicalagin
Gallic acid Ellagic acid Quercetin Catechin
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)
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)
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
(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
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)
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
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
Conclusion
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
Acknowledgement
Dr Philippe Rondeau