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

BERRIES: AN INNOVATIVE HEALTHY CHOICE Relevant biological and nutritional qualities

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 of view

STRAWBERRY (Fragaria x ananassa, Duch.) Vitamin C Flavanols/PACs Anthocyanins Tannins HBA Folate HCA Flavonols Vitamin E

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

STRAWBERRY QUALITY a 3.5 a,b a 3 b,c c,d c,d 2.5 c,d Total content of phenols, mg/g FW d 2 1.5 flavonoids and a 1 a,b a b b a,b b,c b,c c b,c d b,c c,d c anthocyanins e 0.5 f 0 TPC Flavo ACY a 40 35 a a,b a,b 30 a,b a,b 25 Total Antioxidant mmol TEq/g FW b b 20 capacity 15 a a b c,d c c c 10 d 5 0 Irma Patty Adria Sveva AN99.7851 Alba AN94.414.52 AN00.239.55 TEAC FRAP

IN VITRO studies Human dermal fibroblast - HDF mAU 2000 HPLC-DAD-MS 1750  High content of 1500 pelargonidin-3-glucoside analysis 1250 1000  Relevant total antioxidant 750 capacity Pelargonidina-3- 500 glucoside  High content of Vitamin C 250 0 10 20 30 40 50 60 min UV-A Incubation for 24 treatment hours with different 275 kJ/m 2 concentrations of Cell viability the extracts (0.5 – 0.25 – 0.05 mg/ml) 50 DNA damage * 120 DNA damage * 45 100 40 35 80 % Tail Intensity % live cells 30 0.5 mg/ml 60 25 0.25 mg/ml * 20 * 40 0.05 mg/ml 15 ctrl 20 10 5 0 0 0 5 10 15 20 25 30 ctrl 0.05 mg/ml 0.25 mg/ml 0.5 mg/ml min

IN VITRO studies H 2 O 2 treatment / 1h Human dermal fibroblast - HDF Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 – 0.05 mg/ml) Cells Viability

IN VITRO studies H 2 O 2 treatment / 1h Human dermal fibroblast - HDF Intracellular ROS 300 300 30 min 120 min 250 250 200 200 F.U. F.U. 150 * 150 * * * ** ** 100 100 50 50 0 0 0 2 4 6 8 10 0 0.5 1 2.5 5 10 0 2 4 6 8 10 0 0.5 1 2.5 5 10 mM H 2 O 2 mM H 2 O 2 ctrl 0.5 mg/ml ctrl 0.5 mg/ml

IN VITRO studies H 2 O 2 treatment / 1h Human dermal fibroblast - HDF 12 Lipid 10 8 peroxidation G/R * 6 ** ** 4 2 0 0 0,37 0,74 0 5 50 500 µM H 2 O 2 ctrl 0.5 mg/ml 100 75 * * % T.I. 50 DNA * damage 25 0 0 µM 60 µM 80 µM 100 µM µM H 2 O 2 control 0.5 mg/ml extract

IN VITRO studies H 2 O 2 treatment / 1h Human dermal fibroblast - HDF Seahorse XF-24: Mitochondrial respiration (OCR) Mitochondrial respiration Oligomycin 2,4-DNP Rotenone 160 140 OCR (Pmoles/min) 120 100 80 60 40 20 0 0 10 20 30 40 50 60 70 80 minutes Control 0.5 mg/ml 20.000 HuDe; stress agent H 2 O 2 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

IN VITRO studies H 2 O 2 treatment / 1h Human dermal fibroblast - HDF Seahorse XF-24: Glycolysis (ECAR) Glycolysis Rotenone Glucose 2 D-Glucose 20 ECAR (mPh/min) 15 10 5 0 0 10 20 30 40 50 60 70 80 minutes control 0.5 mg/ml 20.000 HuDe; stress agent H 2 O 2 5 mM; strawberry methanolic extract applied 0.5 mg/ml; inhibitor concentrations: Rotenone 3 µg/ml, Glucose 10 mM, 2-DeoxyGlucose (2-DG) 100mM

IN VITRO studies Submitted Anthocyanin-rich strawberry extract protects against oxidative Human dermal fibroblast - HDF 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 Cells Viability Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 90 * – 0.05 mg/ml) ** 80 ** 70 60 ** % live cells 50 40 30 20 10 AAPH 0 0 2.5 5 7.5 10 12.5 15 treatment / 1h mM AAPH Ctrl 0.5 mg/ml

IN VITRO studies Submitted Anthocyanin-rich strawberry extract protects against oxidative Human dermal fibroblast - HDF 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 Lipid peroxidation Incubation for 24 hours with different concentrations of the extracts (0.5 – 0.25 – 0.05 mg/ml) AAPH treatment / 1h

IN VITRO studies Submitted Anthocyanin-rich strawberry extract protects against oxidative Human dermal fibroblast - HDF 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, AAPH Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino treatment / 1h Live/dead/apoptosis cells by TALI Without strawberry With strawberry 90 90 80 80 70 70 60 60 50 50 % cells Live % cells Live 40 40 Dead Dead 30 30 Apoptotic Apoptotic 20 20 10 10 0 0 Ctr 2,5 5 10 15 Ctr 2,5 5 10 15 mM AAPH mM AAPH

IN VITRO studies Submitted Anthocyanin-rich strawberry extract protects against oxidative Human dermal fibroblast - HDF 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, AAPH Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino treatment / 1h Intracellular ROS

IN VITRO studies Submitted Anthocyanin-rich strawberry extract protects against oxidative Human dermal fibroblast - HDF 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, AAPH Massimiliano Gasparrini, Ana M. Gonzàlez-Paramàs, Celestino Santos-Buelga, Josè L. Quiles, Bruno Mezzetti, and Maurizio Battino treatment / 1h Mithocondrial respiration Oligomyci 2-DNP Rotenon 200 Antimycin A 140 Oligomycin 2-DNP Rotenon Antimycin A 180 120 160 100 140 pmol O2/min pmol O2/min 120 80 100 60 80 60 40 40 20 20 0 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 5 mM AAPH 10 mM AAPH Ctrl 0.5 mg/ml Ctrl 0.5 mg/ml 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

ON ANIMALS studies Strawberry polyphenolic extract ETHANOL ULCER INDEX • Widespread gastric lesion HPLC-DAD- • Oxidative Stress MS analysis A B C D E The rats daily received 40 mg/kg body weight of strawberry crude extract intragastrically using a gavage for A B 10 days A B C D E A B C D E D C E SOD ACTIVITY CAT ACTIVITY

ON ANIMALS studies SVEVA ACY TAC ADRIA ACY TAC Adria cultivar Sveva cultivar Peak Tentative identification (mg/kg) (mg/kg) (Epi)afzelechin- (4→8)Pg -3-glucoside 1 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

ON ANIMALS studies 16 weeks supplementation Doxorubicin injection Diet with 10% strawberry ADRIA SVEVA 0.9 α -Tocoferol Retinol a 0.8 14 n.s. 0.7 12 0.6 b b 10 0.5 mg/ml c 8 mg/ml 0.4 6 0.3 0.2 4 0.1 2 0 0 C group C-DOX group S-DOX group A-DOX group C group C-DOX group S-DOX group A-DOX group

ON ANIMALS studies 16 weeks supplementation Doxorubicin injection Diet with 10% strawberry ADRIA SVEVA GSH a Protein carbonyl content a TBARS a Hydroperoxides a 164.39 ± 2.54 a 7.65 ± 0.23 b 0.46 ± 0.16 b 11.42 ± 0.04 c C group 96.13 ± 1.27 c 24.33 ± 2.54 a 0.90 ± 0.15 a 20.4 ± 0.28 a C-DOX group 139.85 ± 1.04 b 8.74 ± 0.93 b 0.37 ± 0.06 b 12.22 ± 0.37 bc A-DOX group 132.67 ± 0.34 b 5.71 ± 1.15 b 0.42 ± 0.01 b 12.96 ± 0.05 b S-DOX group