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COST-927 Action

THERMALLY PROCESSED FOODS: Possible health implications
Building Skills on the Determination of the Overall Antioxidant Capacity of Thermally Processed Foods

Olsztyn, 26 - 30 January 2009 Institute of Animal Reproduction and Food Research, Department of Food Technology in Olsztyn, Poland

Dr. Dolores del Castillo

Antioxidant activity by the Oxygen Radical Absorbance Capacity assay

ORAC assay

Dr. Dolores del Castillo delcastillo@ifi.csic.es

STARTING POINT

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dolores del Castillo delcastillo@ifi.es .Dr.csic.

A SCREEN FOR BIOLOGICALLY RELEVANT PROTECTIVE AGENTS Author(s): DELANGE RJ. GLAZER AN Source: ANALYTICAL BIOCHEMISTRY Volume: 177 Issue: 2 Pages: 300-306 Published: MAR 1989 Times Cited: 99 Dr.es .DID EXIST ANY PREVIOUS JOB? Title: PHYCOERYTHRIN FLUORESCENCE-BASED ASSAY FOR PEROXY-RADICALS . Dolores del Castillo delcastillo@ifi.csic.

csic.es . IT IS LIMITATED ONLY TO SCREENING THE RADICAL SCAVENING CAPACITY OF THE SAMPLE Dr. IT IS NOT CAPABLE OF QUANTITATING RESULTS 2. Dolores del Castillo delcastillo@ifi.WHAT ARE THE MAIN DISADVANTAGES OF THAT METHOD ? 1.

CHEMICALLY. Dolores del Castillo delcastillo@ifi. AN ANTIOXIDANT CAN EFFICIENTLY REDUCE A PRO-OXIDANT Dr.Basic definitions for easy understanding of the PRINCIPLES of different analytical methods for assessing total antioxidant capacity REDUCTION= A GAIN OF ELECTRONS OXIDATION= A LOSS OF ELECTRONS REDUCTANT OR REDUCING AGENT= A SUBSTANCE THAT DONATES ELECTRONS AND CAUSES TO ANOTHER REACTANT TO BE REDUCED OXIDANT OR OXIDATING AGENT= A SUBSTANCE THAT ACCEPTS ELECTRONS AND CAUSES ANOTHER REACTANT TO BE OXIDIZED ANTIOXIDANT= SUBSTANCE THAT WHEN PRESENT AT LOW CONCENTRATIONS COMPARED WITH THOSE OF AN OXIDIZABLE SUBSTRATE. IT IS AN OXIDANT.es . PROTEINS AND NUECLEIC ACIDS. IT SYNONYM FOR REACTIVE SPECIES.csic. SIGNIFICANTLY PREVENTS OR DELAYS A PRO-OXIDANT INITIATED OXIDATION OF THE SUBSTRATE PRO-OXIDANT= SUBSTANCE THAT CAN CAUSE OXIDATIVE DAMAGE TO LIPIDS.

REACTIVE SPECIES IN BIOLOGICAL SYSTMES: HYDROXYL RADICALS (•OH) SUPEROXIDE ANIONS (O2-) SINGLET OXYGEN (1O2) HYDROGEN PEROXIDES (H2O2) ORGANIC PEROXIDES (R-OOH) NITRIC OXIDE PEROXYNITRITE Dr. Dolores del Castillo delcastillo@ifi.csic.es .

Dolores del Castillo delcastillo@ifi.DEFINITION OF ANTIOXIDANT ACTIVITY ABILITY OF A COMPOUND TO REDUCE PROOXIDANTS OR REACTIVE SPECIES OF PATHOLOGIC SIGNIFICANCE Dr.es .csic.

AN ANTIOXIDANT IS A REDUCTANT. BUT A REDUCTANT IS NOT NECESSARILY AN ANTIOXIDANT Na. Dolores del Castillo delcastillo@ifi.csic. GLUCOSE. ETHANOL AND MANY OTHER REDUCTANT ARE NOT ANTIOXIDANTS Dr.es .

Dolores del Castillo delcastillo@ifi.es . SMALL PEPTIDES PHYTOCHEMICALS (THOUNDS IN FOOD SUPPLY) Dr. Zn.DIETARY ANTIOXIDANTS ESSENTIAL VITAMINE E (TOCOPHEROL) VITAMINE C (ASCORBIC ACID) VITAMIN A (RETINOL AND CAROTENOIDS) NUMEROUS MINERALS (Cu.csic. Se. Fe) NON-ESSENTIAL GLUTATHIONE. Mn.

Hydrogen peroxide. ROO•. •OH) ROS (R•. Radiation related hydroxyl radical generator •-. H O . Aqueous soluble b. superoxide generator 3. Lipid soluble 2. Thermal control chemical generator a.Basis of the ORAC assay Reactive Oxygen Species (ROS) generators 1. O2 2 2 ANTIOXIDANTS λExc=540 nm OXIDATION Decreased 565 nm emission fluorescence light βPE INDICATOR PROTEIN Oxidation .

Dolores del Castillo delcastillo@ifi.csic.es .Dr.

Dr. Dolores del Castillo delcastillo@ifi.es .csic.

97 2.50 Values in parentheses indicate secondary absorbance maxima. 3 Value for Phycoerythrin 566 is an estimate.0 7.5 1. Dolores del Castillo delcastillo@ifi. aggregates of both larger and smaller size may occur.50 RPhycoerythrin BPhycoerythrin Y-Phycoerythrin C-Phycocyanin R-Phycocyanin Allophycocyanin Phycoerythrin 566 Phycoerythrocy anin 1 2 8.40 0.es Absorbtivity4 (L/g-cm) Molar absorptivity4 (M-cm)-1(10-6) 1. and various aggregates may occur in aqueous solution.73 0.3 8.54 0. .Properties of Phycobiliproteins Pigment Absorbance maximum1 (nm) 565 (495) 545 ~495 (545) 615 617 (555) 652 566 575 Fluorescence emission2 (nm) 575 575 ~563 647 637 660 617 625 220 100 100 55 100 Molecular weight3 (kda) 240 240 Dr.2 10.csic.00 1.30 0.0 7. Phycobiliproteins are aggregates of subunits.15 0.25 0. Values given are for most common reported aggregates. 4 An approximate relative indicator of the quantum efficiency of the pigment (measured at absorbance and emission maxima).44 0.0 8.70 0.40 Fluorescence: Absorbance5 (relative to RPE) 1.85 0.14 0.0 7.

Absorption maxima for phycoerythrins lie between 490 and 570 nm while absorption maxima for phycocyanins are found between 610 and 665 nm. . the phycoerythrins (red) and the phycocyanins (blue). Nomenclature: Phycobiliproteins are classified on the basis of their color into two large groups.Phycobiliproteins are water soluble fluorescent proteins derived from cyanobacteria and eukaryotic algae. These large groups have been subdivided to reflect variation among the proteins in the exact location of the absorbance maximum and the specific shape of the absorbance spectrum.

Dr.The B-phycoerythrin (B-PE) protein is the major light-harvesting pigment of red microalgae Porphyridium cruentum. highly complex steps.csic. In spite of its numerous uses. however. The protein&apos. Dolores del Castillo delcastillo@ifi.s purification from algal extracts involves a number of often-crude. B-PE is used in the pharmaceutical industry as a fluorescent probe and reagent. It is also utilised as a natural dye in the food and cosmetics industries due to its high fluorescence efficiency. its large-scale purification remains problematic and expensive. and also in the food and cosmetics industries. many of which are non-scaleable and result in unsatisfactory yields. B-PE finds application mainly in the pharmaceutical sector as fluorescent probe in biomedical analysis.es .

Dr. Dolores del Castillo delcastillo@ifi.csic.es .

Dolores del Castillo delcastillo@ifi. B-PE´S DISTINCT EXCITATION AND EMISSION WAVELENGTHS 2. WATER SOLUBILITY Dr.csic. HIGH FLUORESCENCE YIELD 3. SENSITIVITY TO ROS 4.WHY DID THE AUTHORS SELECT B-PE AS FLUORESCENCE PORBE? 1.es .

Dr.es . Dolores del Castillo delcastillo@ifi.csic.

Dolores del Castillo delcastillo@ifi.csic.How to classify ORAC assay ? INHIBITION METHOD Involve a pro-oxidant and oxidizable substrate Antioxidant capacity of the antioxidant The pro-oxidant induces oxidative damage to the substrate which is inhibited in the presence of an antioxidant ORAC ASSAY MEASURES THE CAPICITY OF THE ANTIOXIDANT TO DIRECTLY QUENCH FREE RADICALS Very high molar ratio of AAPH to antioxidants (mora than 2000) Dr.es .

ADVANTAGES COMPARED TO PREVIOUS METHODS 1.csic. Dolores del Castillo delcastillo@ifi. 2. Dr. An improve in quantitation is achieved in ORAC assay by: a) Taking the reaction between substrate and free radicals to completation b) Using an area-under-curve technique for quantitation compared to the assay that measure the lag phase. The use of peroxyl and hydroxyl radicals as pro-oxidants in the ORAC assay makes its different and unique from the assays that involve oxidants that are necessarily prooxidants.es .

Dolores del Castillo delcastillo@ifi. 1999 .csic.es Involving oxidants that are not necessarily pro-oxidants Involving oxidants that are prooxidants TRAP assay FRAP assay TEAC assay Cyclic voltammetry assay Luminol-based assays Dichlorofluorescin-diacetate (DCFH-DA) based assay Crocin based assay Phycoerythrin (PE) based assay ORAC assay Prior and Cao. Free Radical Biology & Medicine 27 (11/12).ANTIOXIDANT ASSAYS Dr. 1173-1181.

csic.Title: OXYGEN-RADICAL ABSORBENCY CAPACITY ASSAY FOR ANTIOXIDANTS Author(s): CAO GH. ALESSIO HM.es . CUTLER RG Source: FREE RADICAL BIOLOGY AND MEDICINE Volume: 14 Issue: 3 Pages: 303311 Published: MAR 1993 Times Cited: 441 Dr. Dolores del Castillo delcastillo@ifi.

Wu AH. 1995. CP.THE AMOUNT OF SAMPLE CAN BE SUSTANTIALLY REDUCED AND THE SIMULTANEOUS KINETIC ANALYSIS OF MANY SAMPLES IS MADE POSSIBLE BY USSING FLUORIMETRY MICROPLATE READER USING A 96-WELL PLATE Cao. G. 1738-1744. Dr.es . Dolores del Castillo delcastillo@ifi. Verdon. Wang H and Prior RL.csic. Clinical Chemistry 41. Automated assay of oxigen radical absorbance capacity with COBAS FARA II.

Source: CLINICAL CHEMISTRY Volume: 41 Issue: 12 Pages: 17381744 Part: Part 1 Published: DEC 1995 Times Cited: 218 Dr. WU AHB. VERDON CP. Dolores del Castillo delcastillo@ifi.csic. et al.es .Title: AUTOMATED-ASSAY OF OXYGEN RADICAL ABSORBENCY CAPACITY WITH THE COBAS FARA-II Author(s): CAO G.

Hampsch-Woodill M.csic. Dolores del Castillo delcastillo@ifi.es . Prior RL Source: JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY Volume: 49 Issue: 10 Pages: 4619-4626 Published: OCT 2001 Times Cited: 216 Dr.Title: Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe Author(s): Ou BX.

es . Dolores del Castillo delcastillo@ifi.WHAT DID THEY PROPOSE THIS TIME ? Dr.csic.

Dolores del Castillo delcastillo@ifi.csic.es .Dr.

csic. Dolores del Castillo delcastillo@ifi.es .QUANTITATION Dr.

Dolores del Castillo delcastillo@ifi.Dr.es .csic.

Dolores del Castillo delcastillo@ifi.SCHEMATIC ILUSTRATION OF THE PRINCIPLE OF THE ORACFL ASSAY Dr.csic.es .

Dolores del Castillo delcastillo@ifi.csic.HOW DOES THE CALIBRATION CURVE LOOK? Dr.es .

4 4. Excitation occurs at 494 nm and emission at 521. Ionization equilibrium leads to pHdependent absorption and emission over the range of 5 to 9 5.306 g/mol Chemical and physical properties 1. 3. Fluorescence lifetimes of the protonated and deprotonated forms of fluorescein are approximately 3 and 4 ns Dr.csic. Dolores del Castillo delcastillo@ifi.Molecular formula Molar mass C20H12O5 332. Very high fluorescence 2. pKa of 6.es .

80 euros. 500 g 142.90 euros. 100 g 37. λem 514 nm in 0.0 mg/mL (UV)fluorescenceλex 542 nm.2 storage temp.es .1 M phosphate pH7. 5mg 318.07367 Fluka B-Phycoerythrin concentration~10.csic.10 euros. λem 576 nm in 0.00 euros P128 Sigma B-Phycoerythrin from Porphyridium cruentum (lyophilized powder) 1mg 356 euros 46960 Fluka Fluorescein sodium salt Fluorescence λex 490 nm. Dolores del Castillo delcastillo@ifi.2-8°C Prices: 1mg 82.50 euros Dr.1 M Tris pH 8.0 Prices: 25 g 12.

SUCH AS B-PE AND R-PE. DIFFERENT PEs. POSSESS DIFERENT FLUORESCENCE INTENSITY AND REACTIVITY TO PEROXY RADICAL 2.es . A SINGLE PE LOT NUMBER SHOULD BE USED FOR A PLANNED PROJECT Dr.ARE THERE OTHER REASONS FOR CHANGING THE FLUORESCENCE PROBE? 1. DIFFERENCES EXIST IN THE SAME PE WITH DIFFERENT LOTS 3.csic. Dolores del Castillo delcastillo@ifi.

csic.HOW TO EXPLAIN THE INCONSISTENCY OF PE FROM LOT TO LOT? PURIFICATION PROCESS B-PE 30% PURE Dr.es . Dolores del Castillo delcastillo@ifi.

es .MORE? B-PE INTERACT WITH POLYPHENOLS Dr.csic. Dolores del Castillo delcastillo@ifi.

csic.es . Dolores del Castillo delcastillo@ifi.COMPARISON OF FL WITH B-PE ANALYSIS IN ABSENCE OF AAPH B-PE FL STRONG PROTEIN BINDING Dr.

csic. Dolores del Castillo delcastillo@ifi.es .COMPARISON OF FL WITH B-PE B-PE DECLINED MORE RAPIDLY THAN DID FL FLUORESCENCE Dr.

csic. Dolores del Castillo delcastillo@ifi.6-3.COMPARISON OF FL WITH B-PE THE FL YIELDS A CONSISTENTLY HIGHER ORAC VALUE AS COMPARED TO B-PE (~1.es .5 FOLD) Dr.

HOW DO THEY INTERACT?

HYDROPHOBIC INTERACTION

HYDROGEN BONDING

Dr. Dolores del Castillo delcastillo@ifi.csic.es

ORACFL MECHANISM

HAT= HYDROGEN ATOM TRANSFER

Dr. Dolores del Castillo delcastillo@ifi.csic.es

ORACFL MECHANISM

Dr. Dolores del Castillo delcastillo@ifi.csic.es

ORACFL MECHANISM .

es .THE FOLLOWINGS EQUATIONS ILUSTRATES THE STEPWISE PROCESS OF HAT RADICAL INICIATOR ABSTRACTS A HYDROGEN ATOM FROM THE ANTIOXIDANT REACTION BEWTEEN ROO• AND THE TARGET MOLECULE IS INHIBITED Dr. Dolores del Castillo delcastillo@ifi.csic.

Dolores del Castillo delcastillo@ifi.SUMMARY OF IMPROVEMENTS 1. ORACFL ASSAY DIRECTLY ESTIMATES THE CHAINBREAKING ANTIOXIDANT ACTIVITY Dr.csic. ROGGEDNESS 2.es . FL SHOWS EXCELLENTE PHOTOSTABILITY SO THAT THE ORACFL ASSAY CAN BE TRANSFERRED TO A 96WELL PLATE READER. FL DOES NOT INTERACT WITH ANTIOXIDANT SAMPLES 3. 4. THE USE OF FL REDUCES THE COST OF THE EXPERIMENT 5.

Dolores del Castillo delcastillo@ifi.WHAT DOES ORACFL ASSAY MEASURE? ORACFL ASSAY MEASURES HYDROPHYLIC ANTIOXIDANT ACTIVITY AGAINST PEROXYL RADICAL ORACFL ASSAY CANNOT BE CONSIDERED A “TOTAL ANTIOXIDANT ACTIVITY ASSAY” IT IS IMPOSSIBLE TO MEASURE TOTAL ANTIOXIDANT ACTIVITY USING A SINGLE ASSAY Dr.es .csic.

Dolores del Castillo delcastillo@ifi.es .csic.FOOD APPLICATION Dr.

Dolores del Castillo delcastillo@ifi.csic.FOOD APPLICATION Dr.es .

csic. Dolores del Castillo delcastillo@ifi.es .WHAT DID THE AUTHORS CONCLUDE? Dr.

csic.es . Dolores del Castillo delcastillo@ifi.FURTHER IMPROVEMENTS Times Cited: 103 Dr.

csic.ADVANTAGES 1.es . THE USE OF THE ROBOTIC LIQUID HANDLING AND PLATE READER TECHNOLOGIES HAS NOT COMPROMISED THE QUALITY OF DATA OBTAINED Dr. Dolores del Castillo delcastillo@ifi. THE EFFICIENCY IS IMPROVED WITH AT LEAST A 10 FOLD INCREASE IN SAMPLE PREPARATION AND IN INSTRUMENT UTILIZATION (130 SAMPLES/DAY) 2.

Dr.es .csic. Dolores del Castillo delcastillo@ifi.

csic. Dolores del Castillo delcastillo@ifi.es .Dr.

es .Dr.csic. Dolores del Castillo delcastillo@ifi.

Dr.csic. Dolores del Castillo delcastillo@ifi.es .

es .Dr.csic. Dolores del Castillo delcastillo@ifi.

Dr.es . Dolores del Castillo delcastillo@ifi.csic.

es .csic. Dolores del Castillo delcastillo@ifi.NEXT STEP Volume: 50 Issue: 7 Pages: 1815-1821 Published: MAR 27 2002 Times Cited: 90 Dr.

WHAT DID THEY DO? Dr. Dolores del Castillo delcastillo@ifi.csic.es .

ORACFL-LIPO ASSAY Dr.csic. Dolores del Castillo delcastillo@ifi.es .

es .csic. Dolores del Castillo delcastillo@ifi.Dr.

MECHANISMS FOR OXIDATION Dr. Dolores del Castillo delcastillo@ifi.es .csic.

MECHANISMS FOR OXIDATION Dr.es . Dolores del Castillo delcastillo@ifi.csic.

es .Dr.csic. Dolores del Castillo delcastillo@ifi.

es . Dolores del Castillo delcastillo@ifi.csic.FOOD APPLICATION Dr.

Dr. Dolores del Castillo delcastillo@ifi.csic.es .

es . Dolores del Castillo delcastillo@ifi.Dr.csic.

csic.Dr. Dolores del Castillo delcastillo@ifi.es .

Dolores del Castillo delcastillo@ifi.csic.es .Dr.

es .MAIN IMPROVEMENT Dr.csic. Dolores del Castillo delcastillo@ifi.

es .S.csic. Dolores del Castillo delcastillo@ifi. MARKETS Dr.FURTHER DATA ON FOOD DATA ON LIPOPHILIC AND HYDROPHILIC ORACFLVALUES FOR OVER 100 COMMON FOODS IN U.

es .FURTHER DATA ON FOOD Dr. Dolores del Castillo delcastillo@ifi.csic.

Dr.es .csic. Dolores del Castillo delcastillo@ifi.

csic. Dolores del Castillo delcastillo@ifi.es .WHAT ABOUT CONCLUSIONS? Dr.

Arkansas.es .usda.gov/Services/docs.htm?docid=15866 This database contains ORAC data for 277 food items and represents a collaboration between the Nutrient Data Laboratory. Dr. Dolores del Castillo delcastillo@ifi.ars.2007 http://www.csic.Oxygen Radical Absorbance Capacity (ORAC) of Selected Foods . Beltsville Human Nutrition Research Center and the Arkansas Children's Nutrition Center in Little Rock.

C. Dolores del Castillo http://www.es . But these natural foods also contain other compounds. that may contribute to health. many fruits and vegetables are known to be good sources of antioxidant vitamins.usda. Dr. For example.The database will be used by scientists to help guide ongoing research into how antioxidants may correlate to health benefits. collectively known as phytonutrients. and beta carotene.ars.htm delcastillo@ifi.csic. a form of vitamin A.gov/is/pr/2007/071106. such as E.

es .ars. Go to: http://www.gov/nutrientdata/ORAC Dr.csic.usda.How to access to the databse?. Dolores del Castillo delcastillo@ifi.

es CAN FOOD PROCESSING GIVE RISE NEOANTIOXIDANTS? SEVERAL CHEMICAL EVENTS CONTRIBUTE TO THE OVERALL ANTIOXIDANT PROPERTIES OF PROCESSED FOODS CAN BE MEASURED THE NEOANTIOXIDANTS BY ORAC ASSAY? .csic.delcastillo@ifi.

Dolores del Castillo delcastillo@ifi.csic.es .KEY CHEMICAL EVENTS INDUCING FORMATION OF NEW ANTIOXIDANTS DURING THERMAL FOOD PROCESSING Dr.

Dr.es .csic. Dolores del Castillo delcastillo@ifi.

Dolores del Castillo delcastillo@ifi.es .Dr.csic.

csic. Dolores del Castillo delcastillo@ifi.PAY ATENTION TO SAMPLES PREPARATION Dr.es .

PAY ATENTION TO SAMPLES PREPARATION Dr.es .csic. Dolores del Castillo delcastillo@ifi.

DO YOU HAVE ANY FURTHER QUESTION? Dr.csic. Dolores del Castillo delcastillo@ifi.es .

Dolores del Castillo delcastillo@ifi.csic.Dr.es .

PAY ATENTION TO SAMPLES PREPARATION

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dr. Dolores del Castillo delcastillo@ifi.csic.es

HMW MRPs contain reductone and amino reductone structures, which provide a potential for reducing and metal chelation activities, both of which are relevant to the antioxidant mechanisms noted forMRPs. In the present study, all three sugar-lysine HMW MRPs displayed a similar, significant capacity to scavenge DDPH radicals, which was also found for earlier products of the MR generated from heated histidine-xylose reactants. Measuring total antioxidant activity of MRPs by the ORAC method provided an excellent basis for assessing MRP scavenging capacity for oxygen radicals between different sugar reactant sources. It is of interest that the ORAC activity of all three HMW MRPs was not only similar when expressed in Trolox equivalents, but also agree well with the DPPH measurements. Moreover, the Trolox equivalent for all three MRP HMW mixtures far exceeds the total antioxidant activity determined previously for ORAC measures of different soft fruits, such as strawberries (approx. 150 μmol Trolox/g dry matter [dm]), blueberries (approx. 200 μmol Trolox/g dm), and raspberries (approx.100 μmol Trolox/g dm). The potential for a large contribution from HMW MRPs to total antioxidant intake, in terms of ORAC values, suggests that the estimated 1.2–1.7 mmol Trolox/day value proposed for US consumers may be underestimated if MRPs derived from processed foods are considered in the ORAC range for total antioxidant intake.

Dr. Dolores del Castillo delcastillo@ifi.csic.es

Dolores del Castillo delcastillo@ifi.es .csic.WHAT OTHER REACTIONS MAY CONTRIBUTE TO THE OVERALL ANTIOXIDANT ACTIVITY OF PROCESSED FOODS? Dr.

es . Dolores del Castillo delcastillo@ifi.csic.WHAT DID WE DO? Dr.

es .csic. Dolores del Castillo delcastillo@ifi.Dr.

csic. Dolores del Castillo delcastillo@ifi.DOES THE METHOD NEED FURTHER IMPROVEMENTS? Dr.es .

ORAC values are extremely dependent upon the employed target molecule.WHAT DO THEY THINK ABOUT THAT? Oxygen radical absorbance capacity (ORAC) values have been obtained for a series of teas and herbal infusions employing 2. for extracts with comparable amounts of phenols. while if pyrogallol red is employed as target molecule green tea appears as nearly nine times more efficient. The amounts of phenols in the extracts were evaluated by Folin’s methodology. Even more. Dr. and fluorescein and pyrogallol red as target molecules. Extreme care must then be taken for conclusions obtained from ORAC values estimated by employing a single target molecule. ORAC-fluorescein value of Aloysia citriodora is larger than that of green tea.csic. while opposite conclusions are obtained if ORAC-pyrogallol red values are considered. relative ORAC values measured for different infusions depend upon the employed methodology.20-azobis (2-amidinopropane) as free radical source. Dolores del Castillo delcastillo@ifi. For example. Similarly.es . herbal infusions are more efficient than teas by ORAC fluorescein.

csic.es . THE DISCUSSION SECTION IS OFICIALLY OPENED Dr.IS THERE MORE WORK LEFT TO BE DONE? WHAT ELSE CAN WE DO? FEEL FREE YOURSELF TO MAKE PROPOSALS. Dolores del Castillo delcastillo@ifi.