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