Bordeaux, 27th March 2014 BERRIES: AN INNOVATIVE HEALTHY CHOICE - - PowerPoint PPT Presentation

Luca Mazzoni, PhD student

Università Politecnica delle Marche Dipartimento di Scienze Cliniche, Specialistiche ed Odontostomatologiche

Bordeaux, 27th March 2014

Relevant biological and nutritional qualities

BERRIES: AN INNOVATIVE HEALTHY CHOICE

STRAWBERRY (Fragaria X Ananassa, Duch.)

One of the most commonly consumed berries, both in fresh and processed forms; Relevant source of bioactive compounds due to its high level of Vitamin C, folate and phenolic constituents which express relevant antioxidant capacities; The most studied berry from the agronomic, genomic and nutritional point

• f view

Anthocyanins Vitamin C Flavanols/PACs HBA HCA Flavonols Tannins Folate Vitamin E

STRAWBERRY (Fragaria x ananassa, Duch.)

Pelargonidin-3-glucoside (25-40% TAC)

STRAWBERRY QUALITY

b,c d c,d c,d a c,d a a,b b,c c b,c b a b,c a,b b,c c e d c,d f b a,b a 0.5 1 1.5 2 2.5 3 3.5 mg/g FW

TPC Flavo ACY

Total content of phenols, flavonoids and anthocyanins

a,b b a,b a,b a b a a,b c d c c a c,d a b 5 10 15 20 25 30 35 40 Alba Irma Patty Adria Sveva AN99.7851 AN94.414.52 AN00.239.55 mmol TEq/g FW

TEAC FRAP

STRAWBERRY QUALITY

Total Antioxidant capacity

• High content of

pelargonidin-3-glucoside

• Relevant total antioxidant

capacity

• High content of Vitamin C

min 10 20 30 40 50 60 mAU 250 500 750 1000 1250 1500 1750 2000

Pelargonidina-3- glucoside

UV-A treatment 275 kJ/m2 HPLC-DAD-MS analysis

Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 – 0.05 mg/ml) DNA damage

20 40 60 80 100 120 5 10 15 20 25 30 % live cells min 0.5 mg/ml 0.25 mg/ml 0.05 mg/ml ctrl

* *

Cell viability

5 10 15 20 25 30 35 40 45 50 ctrl 0.05 mg/ml 0.25 mg/ml 0.5 mg/ml % Tail Intensity

* *

IN VITRO studies

Human dermal fibroblast - HDF

DNA damage

Cells Viability

IN VITRO studies

Human dermal fibroblast - HDF

Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 – 0.05 mg/ml)

H2O2 treatment / 1h

H2O2 treatment / 1h

Intracellular ROS

IN VITRO studies

Human dermal fibroblast - HDF

50 100 150 200 250 300 2 4 6 8 10 F.U. mM H2O2

120 min

ctrl 0.5 mg/ml

* * **

0 0.5 1 2.5 5 10

50 100 150 200 250 300 2 4 6 8 10 F.U. mM H2O2

30 min

ctrl 0.5 mg/ml

* * **

0 0.5 1 2.5 5 10

Lipid peroxidation DNA damage

25 50 75 100 0 µM 60 µM 80 µM 100 µM % T.I. µM H2O2 control 0.5 mg/ml extract

* * *

2 4 6 8 10 12 0,37 0,74 G/R µM H2O2 ctrl 0.5 mg/ml

*

5 50 500

** **

H2O2 treatment / 1h

IN VITRO studies

Human dermal fibroblast - HDF

20.000 HuDe; stress agent H2O2 5 mM; strawberry methanolic extract applied 0.5 mg/ml; inhibitors concentration: Oligomycin 1 µg/ml, 2,4-Dinitrophenol (2-DNP) 100 µM, Rotenone 1µM

20 40 60 80 100 120 140 160 10 20 30 40 50 60 70 80 OCR (Pmoles/min) minutes

Mitochondrial respiration

Control 0.5 mg/ml

Oligomycin Rotenone 2,4-DNP

Seahorse XF-24: Mitochondrial respiration (OCR)

H2O2 treatment / 1h

IN VITRO studies

Human dermal fibroblast - HDF

Seahorse XF-24: Glycolysis (ECAR)

5 10 15 20 10 20 30 40 50 60 70 80 ECAR (mPh/min) minutes

Glycolysis

control 0.5 mg/ml

Rotenone 2 D-Glucose Glucose

20.000 HuDe; stress agent H2O2 5 mM; strawberry methanolic extract applied 0.5 mg/ml; inhibitor concentrations: Rotenone 3 µg/ml, Glucose 10 mM, 2-DeoxyGlucose (2-DG) 100mM H2O2 treatment / 1h

IN VITRO studies

Human dermal fibroblast - HDF

Cells Viability

IN VITRO studies

Human dermal fibroblast - HDF

Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 – 0.05 mg/ml)

AAPH treatment / 1h

10 20 30 40 50 60 70 80 90 2.5 5 7.5 10 12.5 15 % live cells mM AAPH Ctrl 0.5 mg/ml

** ** ** *

Anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to oxidant agent.

Francesca Giampieri, José M. Alvarez-Suarez, Luca Mazzoni , Tamara Y. Forbes-Hernandez, Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino

Submitted

Lipid peroxidation

IN VITRO studies

Human dermal fibroblast - HDF

Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 – 0.05 mg/ml)

AAPH treatment / 1h

Anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to oxidant agent.

Francesca Giampieri, José M. Alvarez-Suarez, Luca Mazzoni , Tamara Y. Forbes-Hernandez, Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino

Submitted

IN VITRO studies

Human dermal fibroblast - HDF

Anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to oxidant agent.

Francesca Giampieri, José M. Alvarez-Suarez, Luca Mazzoni , Tamara Y. Forbes-Hernandez, Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino

Submitted

Live/dead/apoptosis cells by TALI

10 20 30 40 50 60 70 80 90 Ctr 2,5 5 10 15 % cells mM AAPH Live Dead Apoptotic 10 20 30 40 50 60 70 80 90 Ctr 2,5 5 10 15 % cells mM AAPH Live Dead Apoptotic

Without strawberry With strawberry

AAPH treatment / 1h

IN VITRO studies

Human dermal fibroblast - HDF

Anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to oxidant agent.

Francesca Giampieri, José M. Alvarez-Suarez, Luca Mazzoni , Tamara Y. Forbes-Hernandez, Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino

Submitted

Intracellular ROS

AAPH treatment / 1h

IN VITRO studies

Human dermal fibroblast - HDF

Anthocyanin-rich strawberry extract protects against oxidative stress damage and improves mitochondrial functionality in human dermal fibroblasts exposed to oxidant agent.

Francesca Giampieri, José M. Alvarez-Suarez, Luca Mazzoni , Tamara Y. Forbes-Hernandez, Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino

Submitted

Mithocondrial respiration

AAPH treatment / 1h

20 40 60 80 100 120 140 160 180 200 5 10 15 20 25 30 pmol O2/min 5 mM AAPH Ctrl 0.5 mg/ml

Oligomyci 2-DNP Rotenon Antimycin A

20 40 60 80 100 120 140 5 10 15 20 25 30 pmol O2/min 10 mM AAPH Ctrl 0.5 mg/ml

Oligomycin 2-DNP Rotenon Antimycin A

20.000 HuDe; stress agent AAPH; strawberry methanolic extract applied 0.5 mg/ml; inhibitors concentration: Oligomycin 1 µg/ml, 2,4- Dinitrophenol (2-DNP) 100 µM, Rotenone 1µM, Antimycin A 1µM

IN VITRO VERSUS IN VIVO EVIDENCE: HOW TO BYPASS THE GAP?

In vitro effects In vivo effects

Few literature data on strawberry intake and antioxidant status

Strawberry polyphenolic extract

ETHANOL

• Widespread gastric lesion
• Oxidative Stress

ULCER INDEX SOD ACTIVITY CAT ACTIVITY

The rats daily received 40 mg/kg body weight of strawberry crude extract intragastrically using a gavage for 10 days

A B C D E A B C D E A B C D E A B C D E

HPLC-DAD- MS analysis

ON ANIMALS studies

Peak Tentative identification Adria cultivar (mg/kg) Sveva cultivar (mg/kg) 1 (Epi)afzelechin-(4→8)Pg-3-glucoside 3.798 ± 0.4 1.754 ± 0.1 2 Cy-3-glucoside 32.232 ± 2.1 25.974 ± 2.2 3 Pg 3,5-diglucoside 0.588 ± 0.0 0 ± 0.0 4 Pg 3-glucoside 1157.376 ± 12.0 790.234 ± 7.0 5 Pg 3-rutinoside 97.848 ± 4.0 26.176 ± 2.0 6 Pg 3-malonylglucoside 32.562 ± 3.0 45.62 ± 2.2 7 Pg 3-acetylglucoside 7.114 ± 4.0 3.284 ± 1.0 Total Anthocyanin Content 1331.518 ± 4.6 893.042 ± 2.3

SVEVA ADRIA ACY TAC ACY TAC

ON ANIMALS studies

16 weeks supplementation Diet with 10% strawberry

Doxorubicin

injection

2 4 6 8 10 12 14

C group C-DOX group S-DOX group A-DOX group mg/ml

α-Tocoferol

n.s.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

C group C-DOX group S-DOX group A-DOX group mg/ml

Retinol

a c b b

SVEVA ADRIA

ON ANIMALS studies

16 weeks supplementation Diet with 10% strawberry

Doxorubicin

injection

GSHa Protein carbonyl contenta TBARSa Hydroperoxidesa C group 164.39 ± 2.54a 7.65 ± 0.23b 0.46 ± 0.16b 11.42 ± 0.04c C-DOX group 96.13 ± 1.27c 24.33 ± 2.54a 0.90 ± 0.15a 20.4 ± 0.28a A-DOX group 139.85 ± 1.04b 8.74 ± 0.93b 0.37 ± 0.06b 12.22 ± 0.37bc S-DOX group 132.67 ± 0.34b 5.71 ± 1.15b 0.42 ± 0.01b 12.96 ± 0.05b

SVEVA ADRIA

ON ANIMALS studies

16 weeks supplementation Diet with 10% strawberry

Doxorubicin

injection

GSTa GPxa GRa SODb Catalaseb C group 430.01 ± 3.95a 0.55 ± 0.04a 170.96 ± 5.75a 123.33 ± 1.81a 18.98 ± 1.52a C-DOX group 221.42 ± 27.49b 0.31 ± 0.03b 83.81 ± 4.61b 45.66 ± 0.05c 9.73 ± 0.78b A-DOX group 425.26 ± 33.43a 0.53 ± 0.07a 167.73 ± 6.02a 106.75 ± 2.68ab 19.90 ± 1.78a S-DOX group 424.02 ± 4.37a 0.52 ± 0.03a 164.13 ± 3.41a 89.18 ± 2.54b 19.40 ± 1.01a

SVEVA ADRIA

ON ANIMALS studies

16 weeks supplementation Diet with 10% strawberry

Doxorubicin

injection

2 4 6 8 10 12 14 16 18 C group C-DOX group S-DOX group A-DOX group % DNA damage c a a b

SVEVA ADRIA

ON ANIMALS studies

16 weeks supplementation Diet with 10% strawberry

Doxorubicin

injection

0,5 1 1,5 2 2,5 3 C group C-Dox group S-Dox group A-Dox group nmol DCF/mg protein a a b b

10 20 30 40 50 60 70 80 90 5 10 15 20 25 30 35 40

pmolO2/min/mgprot Time (min)

C group C-DOX group S-DOX group A-DOX group

Succinate Ascorbate + TMPD Antimycin A Rotenone

SVEVA ADRIA

ON ANIMALS studies

32 Old rats (16 month) 32 Young rats (4 month)

Standard diet (AIN93)

AIN93

Standard diet (AIN93)

AIN93

+ Strawberry

I II

Standard diet (AIN93)

AIN93

+ Fragole IV

Standard diet (AIN93)

AIN93

III

Time

Day 57 Day 58

+ Strawberry

Day 60 Sacrifice Experimental design

Doxorubicin injection

ON ANIMALS studies

(Young and old Wistar rats)

Markers Plasma

Proteic oxidation (GSH, carbonyl groups) Lipidic oxidation (TBARS Hydroperoxides) Lipidic Profile Hepatic damage Antioxidant capacity and free radical scavenging

Lymphocytes

ROS intracellular concentration

Liver

Homogenate Proteic Oxidation (GSH, carbonyl groups) Lipidic oxidation (TBARS, Hydroperoxides) Antioxidant enzymes (GPx, GR, GST, Catalase, SOD) Gene expression (Western blot) Hepatocytes ROS Intracellular concentration Mitochondria ROS intracellular concentration Mitochondrial functionability (XF- 24 extracellular flux analyzer) ATP Citrate synthase Hystopathology analysis

ON ANIMALS studies

(Young and old Wistar rats)

ON ANIMALS studies

(Young and old Wistar rats)

PLASMA:  Improvement of protein and lipid oxidation markers;  Improvement of lipidic profile;  Attenuation of hepatic damage markers;  Decrease of ROS levels. LIVER:  Improvement of protein and lipid oxidation markers;  Increase of antioxidant enzymes;  Decrease of ROS levels in hepatocytes. LIVER MITOCHONDRIA:  Increase of ATP and cytrate synthase levels;  Decrease of ROS levels;  Increase of mitochondrial respiration rate;  Increase of vitamin E levels.

ON HUMANS studies

Subjects started a 30-day supplementation period with the consumption of 500 g of fresh strawberries per day

An improved resistance to hemolysis was observed after 30 days of strawberry supplementation and 15 days after the end of the study

AFTER STRAWBERRY INTAKE

** * * * * * * ** ** ** *

ON HUMANS studies

Subjects started a 2-week supplementation period with the consumption of 500 g of fresh strawberries per day

Significant reduction in DNA damage (Comet assay) of lymphocytes exposed to oxidative stress, after strawberry consumption

AFTER STRAWBERRY INTAKE

ON HUMANS studies

Subjects started a 30-day supplementation period with the consumption of 500 g of fresh strawberries per day AFTER STRAWBERRY INTAKE

A) Resting platelet B) Central clustering platelets C) Degranulated platelets

FRAP ORAC Vit C Uric Acid MDA 8-OHdG Isoprostane LDL-C Total Cholesterol Triglycerides

ON HUMANS studies

Subjects started a 30-day supplementation period with the consumption of 500 g of fresh strawberries per day

Until today, berries in general were considered to be a rich- natural source of bioactive compounds (mainly polyphenols) with antioxidant properties.. …..polyphenols don’t have

• nly antioxidant properties.

They also display other effects, like pro-apoptotic or pro-oxidant effects, that may seem negative but in hyperproliferative cells can have, in turn, positive effects

BUT

IN VITRO studies

N202/1E N202/1A

Treated with different concentrations of strawberries methanolic extracts

Murine mammary tumoral cell lines Cancer cells

No detectable HER 2/neu

• ncoprotein expression

High levels of HER 2/neu

• ncoprotein expression

Cell viability

IN VITRO studies

Apoptosis rate and intracellular ROS concentration after 48 hours of incubation with strawberry methanolic extracts.

Cancer cells Murine mammary tumoral cell lines

IN VITRO studies Cancer cells

Seahorse XF-24 : Glycolysis at different concentrations

• f strawberry extracts
• N202/1E respond to glycolysis inhibitors better than N202/1A;
• Strawberry treatment (for example 3 µg/ml of anthocyanins) is capable to

increase the glycolysis in N202/1E, but decreases glycolysis in N202/1A.

Murine mammary tumoral cell lines

IN VITRO studies Cancer cells Murine line FVB/N 233 neu-NT

Standard diet (AIN93)

AIN93

Standard diet (AIN93)

AIN93

+ Strawberry

I II Sacrifice Time Experimental design

2 months

• ld

6 months

• ld

Day 120 Murine line FVB/N 233 neu-NT

• Kinetics of breast cancer development
• Lung metastases formation

IN VITRO studies Cancer cells Murine line FVB/N 233 neu-NT

Control Strawberr y Control Strawberry Control Strawberr y

Tumor number Tumor volume Tumor free mice

Kinetics of cancer development

IN VITRO studies Cancer cells Murine line FVB/N 233 neu-NT

Lung metastases formation:

• Metastases number
• % mice with metastases
• Metastases size

Could strawberries protect against oxidative damage through direct or Indirect activation of the SIRT 1/PGC 1α/NRF 2 Pathway? Strawberry bioactive compounds

NRF2 nucleus

ARE

SIRT1 Gpx, Gclc, Cat

ROS NFkB?

Inflammation Development of age-related diseases and aging Apoptosis Cellular energetics Cellular function

AMPK

Mitochondrial biogenesis

SIRT1

PGC- 1α

NO NADPH oxidase

TNFα? eNOS ? ?

• f DNA damage

OGG1

Evidences for strawberry health benefits

• Take home messages -

Characterization of the main bioactive compounds and of their antioxidant capacity Strawberry treatment In vitro

• Protection against UV

irradiation in human fibroblasts;

• Improvement of

mitochondrial respiration and glycolysis after H2O2- and AAPH-induced stress in human fibroblasts. In vivo

• Improved resistance
• f erythrocytes to

hemolysis;

• Improvement of
• xidative stress

biomarkers and lipid profile in plasma;

• Reduced DNA

damage in lymphocytes after H2O2-induced stress.

• Inhibition of viability,

increase of apoptosis and intracellular ROS production and impairment of mitochondrial functionality in murine breast cancer cells.

• Protection against

ethanol-induced gastric lesions;

• Reduction of breast

tumor number and volume and lung metastases;

• Improvement of

antioxidant status and mitochondrial functionality against DOX-induced stress.

SOCIAL IMPACT

Many thanks to:

• Prof. Josè L.Quiles
• Prof. Bruno Mezzetti
• Dr. Franco Capocasa
• Dr. Jacopo Diamanti
• Dr. Stefano Bompadre
• Dr. Stefania Romandini
• Dr. Josè M. Alvarez Suarez
• Dr. Francesca Giampieri
• Dr. Massimiliano Gasparrini
• Dr. Tamara Y. Forbes-Hernandez
• Dr. Sara Tulipani

Prof .Celestino Santos Buelga

• Dr. Ana Paramas

Prof .Dragana Dekanski

• Dr. Slavica Ristic
• Dr. Nevena V. Radanjic
• Dr. Natasa D. Petronijevic