EVALUATION OF ANTIOXIDANT ACTIVITY OF NEW NUTRACEUTICALS: IN VITRO AND CELLULAR ASSAYS. Ana Denicola

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1 EVALUATION OF ANTIOXIDANT ACTIVITY OF NEW NUTRACEUTICALS: IN VITRO AND CELLULAR ASSAYS Ana Denicola Lab. Fisicoquímica Biológica. Instituto Química Biológica. Facultad de Ciencias, UdelaR. Center for Biomedical Research in Free Radicals (CEINBIO) INNOVA 2011

2 NUTRACEUTICALS any substance that may be considered a food or part of a food, and provides medical or health benefits, including the prevention and treatment of disease. Foundation for Innovation in Medicine 1989 a nutrient that not only maintains, supports, and normalizes any physiologic or metabolic function, but can also potentiate, antagonize, or otherwise modify physiologic or metabolic functions Food Res. Int Nutraceuticals may include dietary fibers, polyunsaturated fatty acids, amino acids, proteins, minerals, different types of phenolic compounds and antioxidants

3 NATURAL ANTIOXIDANTS Drugs Contamination Smoking Endogenous stimuli Cancer ROS RNS Protein oxidation Lipoperoxidation Membrane damage DNA damage

4 NO 3 O 2 H 2 O Trx(SH) 2 NADPH Prx TR Prx NO TrxS 2 NADP + ONOOH ONOO O 2 SOD Fe 2+ Fe 3+ H 2 O 2 OH + OH H 2 O 0.3 (OH + NO 2 ) +0.7 NO 3 Catalasa GPx GSH GSSG GR NADPH NADP + CO 2 H 2 O ONOOCO 2 LH R RH O 2 L LOO NO 2 + CO 3 LOOH LH

5 OXIDATIVE STRESS Antioxidantes Oxidantes Aging Cardiovascular and neurodegenerative diseases Cancer

6 ANTIOXIDANTS MECHANISMS OF ACTION Antioxidants Free radical scavenging Signaling Direct reaction of the antioxidant compound with biological oxidants to stop or ameliorate oxidation ROS RNS Antioxidant compound affects redox signaling (i.e. induce expression of antioxidant enzymes) Protein oxidation Lipoperoxidation Membrane damage DNA damage

7 EVALUATION OF ANTIOXIDANT CAPACITY There is no single method to comprehensively determine antioxidant capacity In vitro Cellular assays radical scavenging In vivo antioxidant response Antioxidant: a compound able to reduce the oxidative cellular state Jones, D.P. Redefining oxidative stress. Antioxid. Redox Signal. 2006, 8, 1865-

8 IN VITRO RADICAL SCAVENGING radical generator stable radical (Abs., Fluor) ± antioxidant target ± antioxidant radical left oxidized target detection Direct methods (e.g. DPPH, nitroxides) Competition methods (e.g. ORAC, TRAP, TEAC)

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10 EVALUATION OF ANTIOXIDANT CAPACITY There is no single method to comprehensively determine antioxidant capacity In vitro radical scavenging Competition method like ORAC using reference (Trolox) Reducing capacity (FRAP, Folin) Metal chelating capacity

11 EVALUATION OF ANTIOXIDANT CAPACITY There is no single method to comprehensively determine antioxidant capacity In vitro radical scavenging Competition method like ORAC using reference (Trolox) Reducing capacity (FRAP, Folin) Metal chelating capacity Cellular assays (cellular antioxidant response)

12 URUGUAYAN PROPOLIS Raw propolis Southern region of Uruguay Late Spring/ early Summer Ethanolic extracts, EEP 40 g/l EEP Absorbance at 294 nm

13 Total Polyphenols and Flavonoids EEP [Polyphenols] [Flavonoids] (mg/g propolis) 1 85 ±6 22 ± ±3 11 ± ±2 13 ± ±24 45 ± ±1 4 ± ±3 7 ± ±26 54 ± ±3 22 ± ±14 35 ±3 Similar to argentinean and south Brazil propolis (Isla, J. Ethnopharmacol. 2001) Higher than tropical Brazil propolis (Mendes da Silva, Food Chem. 2006) 3300 fold higher than honey (Gheldof, J. Agric. Food Chem. 2002) 3 fold higher than red wine (Davalos, J. Agric. Food Chem. 2004) ±18 41 ±2

14 Abs a λ maxima [Flavonoides](mg/g propoleos) The UV absorbance is a simple and rapid way to estimate the total content of polyphenols in the propolis extract

15 Polyphenol composition of propolis extracts (RP HPLC) Silva, V. et al. J. Agric. Food Chem. 2011, 59,

16 IN VITRO ASSAYS: ORAC URF [Trolox] 0 3 μm 6 μm 9 μm 12 μm x Time (min) competition method assess peroxyl radical scavenging express as Trolox equivalents AUC 8.0x x x [Trolox](µM)

17 ORAC concentracion Trolox equivalente valor ORAC = [ EEP]( mg/ l) EEP ORAC Value (µmoles Trolox eq/mg propolis) 2.5 ± ± ± ± ± ± ± ± ± ±0.8 EE7 EE10 EE4 EE9 ~ 8000 µmol TE/g EE8 = EE1 = EE5 = EE6 EE2 EE3 500 fold higher than honey (Gheldof, J. Agric. Food Chem. 2002) 150 fold higher than red wine (Davalos, J. Agric. Food Chem. 2004)

18 ORAC value correlates with flavonoid content

19 IN VITRO ASSAYS: inhibition of LDL oxidation LDL = Low Density Lipoprotein Apolipoprotein: ApoB 100 Transport of cholesterol Oxidized LDL associated with atherosclerosis

20 Inhibition of LDL oxidation by propolis Abs 234 nm μg/ml 0.4 μg/ml EEP1 EEP2 EEP3 EEP4 LDL EEP7 EEP8 EEP9 EEP10 LDL Tiempo (min) Tiempo (min) EEP high in polyphenols retarded LDL oxidation (lag or exponential phase) Silva, V. et al. J. Agric. Food Chem. 2011, 59,

21 IN VITRO ASSAYS: inhibition of tyrosine oxidation Tyrosine: target residue for post translational modifications: Phosphorylation: cellular signaling Oxidation: Nitrotyrosine Increased under pathological conditions Antioxidant: A A + NO 2 A + NO 2

22 Nitro tyrosine formation by ONOO NO 2 -Y biological formation is a radical mechanism Peroxynitrite (ONOO-) can form NO 2 -Y: CO 2 ONOO CO 3 + NO 2 Y Y Y NO 2

23 TYROSINE OXIDATION INHIBITION BY PROPOLIS Y-NO 2 EEP 4 EEP4 (high polyphenols) prevented Y NO 2 formation in a dose dependent manner Silva, V. et al. J. Agric. Food Chem. 2011, 59,

24 CELLULAR ASSAYS 3 different responses at the cellular level upon exposure to polyphenols: low dose = cellular antioxidant defense high dose = induces apoptosis very high dose = oxidative damage, necrosis

25 CELLULAR ASSAYS 3 different responses at the cellular level upon exposure to polyphenols: low dose = cellular antioxidant defense high dose = induces apoptosis Natural antioxidant Antiproliferative activity on cancer cells very high dose = oxidative damage, necrosis

26 CELLULAR ASSAYS ENDOTHELIAL CELLS Endothelial function is critical to mantain vascular homeostasis

27 2 important enzymes at the endothelium: enos (Nitric Oxide Synthase) NOX (NADPH Oxidase)

28 Effect of propolis ethanolic extracts on BAEC (bovine aortic endothelial cells) Exposure of BAEC to vehicle (control) and EEP dilutions enos expression, activity NOX expression, activity Confluent culture BAEC (passage 4) 100X

29 Nitric oxide synthase (NOS) NOS 3, enos Constitutive expression in the endothelium NO (from enos) keeps vascular homeostasis

30 Induction of enos expression by propolis EEP 9 (high in polyphenols) significangtly induced enos expression Silva, V. et al. J. Agric. Food Chem. 2011, 59,

31 NADPH oxidase (NOX) Membrane enzyme Activated by assembling of subunits NOX1 and NOX4, vascular isoforms Uses NADPH and O2 to form superoxide radical anion (O2. )

32 INHIBITION OF NOX4 BY PROPOLIS EEP9 decreased NOX activity and mrna NOX4 expression Silva, V. et al. J. Agric. Food Chem. 2011, 59, 6430-

33 Effect of propolis on the endothelium: increase expression of enos inhibit activity of Nox4 increase NO bioavailability (better vasodilation)

34 CELLULAR ASSAYS CANCER CELLS Selective antiproliferative effect on cancer cells Different tumor cell lines can be used Compare with effect on proliferation of normal cells

35 ANTIPROLIFERATIVE ACTIVITY BY PROPOLIS Sample IC 50 (mg/ml) EEP EEP EEP Abs 450 nm Dose dependent cytotoxic effect of EEP against HT 29 colon cancer cells

36 ANTIPROLIFERATIVE ACTIVITY BY PROPOLIS on colon cancer cells (100x)

37 ANTIPROLIFERATIVE ACTIVITY BY PROPOLIS H2 HT_15_Dec_10 G1: R1=5000> (G2: R1 & R6) Region Count % Hist % All Total R , R , R , R , H7 HT_15_Dec_10 G1: R1=5000> (G2: R1 & R6) Region Count % Hist % All Total R , R , R , R , H9 HT_15_Dec_10 G1: R1=5000> (G2: R1 & R6) Region Count % Hist % All Total R , R , R , R , EEP kill HT 29 colon cancer cells mainly by apoptosis

38 Specificity of antiproliferative activity of propolis Fibroblasts HT-29 MCF Cell proliferation (%) 50 0 EEP2 EEP7 EEP9 0,3 mg/ml 0,1 mg/ml 0,1 mg/ml EEP displayed selective antiproliferative activity against cancer cells

39 Conclusions The antioxidant capacity of uruguayan propolis was assayed in vitro and cellular assays ORAC 8000 µmol Trolox eq/g propolis (higher than other natural products) Efficiently inhibits LDL oxidation and tyrosine nitration Flavonoid composition (RP HPLC) indicates poplar tree origin Induces enos expression and NOX4 inhibition (high NO bioavailability in endothelium) Specific antiproliferative activity against breast and colon cancer cells

40 Thanks to Verónica Silva, Gonzalo Genta, Martín Masner, Matías Möller Lab. Fisicoquímica, Facultad de Ciencias, UdelaR, Uruguay. Andrea Medeiros, Natalia Romero, Rafael Radi Depto. Bioquímica, Facultad de Medicina, UdelaR, Uruguay. Instituto Pasteur Montevideo. Horacio Heinzen Lab. Farmacognosia, Facultad de Química, UdelaR, Uruguay. Denise Fernandes, Francisco Laurindo InCor, Facultade Medicina, USP, Brasil. Walter Fierro MSP, Uruguay Comisión Sectorial de Investigación Científica (CSIC) Universidad de la República Sociedad Apícola Uruguaya (SAU) Agencia Nacional de Investigación e Innovación (ANII) Uruguay