Justus Liebig University Gießen Faculty of Agricultural Sciences, Nutritional Sciences and Environmental Management Institute of Agronomy and Plant Breeding I Head: Prof. Dr. Bernd Honermeier Phenolic acids and antioxidative capacity on ancient wheat namely einkorn (T. monococcum ssp.), emmer (T. turgidum ssp.) and spelt wheat (T. aestivum ssp. spelta) and on germinated bread wheat (T. aestivum ssp. aestivum) A dissertation submitted for the degree of Doctor of home economics and nutritional science (Doctor oeconomiae trophologiaeque- Dr. oec. troph.) in the Faculty of Agricultural Sciences, Nutritional Sciences, and Environmental Management at Justus Liebig University Gießen Submitted by: Nadine Engert, M. Sc. From Glauchau Gießen, June 24, 2011 Phenolic compounds: Are they healthy? Are they present in wheat? Have they always been present in wheat? Fig. 0-1:Health benefits by whole grain consumption (VITAGLIONE et al., 2008) II Table of Contents Phenolic compounds: II Index of Tables V Index of Figures VII Index of Equations IX Abbreviations X 1 Introduction 1 2 Objectives 6 3 Literature review 8 3.1 Characterization of the genus Triticum 8 3.1.1 Domestication 8 3.1.2 Taxonomy of wheat 10 3.2 Secondary plant metabolites 12 3.2.1 Phenolic acids 14 3.2.2 Oxidative stress 18 3.2.3 Health effecting potentials and nutrition of phenolic compounds 23 3.2.4 Bioavailability 32 3.3 Phenolic acids in wheat 35 3.4 Antioxidative capacity tests 36 4 Material and methods 53 4.1 Characterization of the experiments 53 4.2 Grain quality parameters 55 4.3 Sample preparation for HPLC and antioxidative capacity 55 4.4 HPLC analysis 57 4.4.1 HPLC method 1 57 4.4.2 HPLC method 2 57 4.5 Analysis of antioxidative capacity 58 4.5.1 Folin-Ciocalteu assay 58 4.5.2 ORAC assay 58 4.6 Statistical analysis 59 5 Publications 61 5.1 Experiment 1 “Evaluation of ancient wheat species” 61 5.2 Experiment 2 “Sprouting experiment with bread wheat” 81 III 5.3 Experiment 3 “Evaluation of spelt wheat cultivars” 101 6 Discussion 118 7 Summary 131 8 Zusammenfassung 134 9 Declaration 137 10 Publication list 138 11 Acknowledgements 140 IV Index of Tables Index of Tables Literature review (chapter 3) Tab. 3-1: IUPAC nomenclature of hydroxycinnamic acids and hydroxy- benzoic acids present in wheat (Triticum sp.) 18 Tab. 3-2: Nomenclature of reactive species (HALLIWELL and WHITEMAN, 2004) 19 Tab. 3-3: In vitro Antioxidant capacity Assays (modified by Huang et al., 2005) 25 Experiment 1 “Evaluation of ancient wheat species” (chapter 5.1) Tab. 1: Wheat material of the eight species with corresponding accessions 64 Tab. 2: Concentration of phenolic acids (TPA) [μg GAE/g] in caryopses of different wheat species 69 Tab. 3: Concentration of ferulic acid (FA) [μg GAE/g] in caryopses of different wheat species 70 Tab. 4: Pearsons’s correlation coefficients (r) between phenolic acids (TPA) and antioxidant capacity (TPC, ORAC) of different wheat species 72 Experiment 2 “Sprouting experiment with bread wheat” (chapter 5.2) Tab. 1: Effect of N fertilization and cultivar on thousand grain weight (TGW, [g]), falling number (FN, [s]), gluten index (GI) and crude protein (CP, [% DM]) in wheat samples 87 Tab. 2: Effect of sprouting time on the total phenolic acids (TPA) [µg GAE/g] in wheat samples depending on N fertilization and cultivar 88 Tab. 3: Effect of sprouting time on the total phenolic content (TPC) [µg GAE/g] in wheat samples depending on N fertilization and cultivar 91 Tab. 4: Effect of sprouting time on the antioxidative capacity by the ORAC assay [µmol TE/g] in wheat samples depending on N fertilization and cultivar 92 Experiment 3 “Evaluation of spelt wheat cultivars” (chapter 5.3) Tab. 1: Morphological and quality parameters of spelt wheat grain (Triticum aestivum ssp. spelta) dependent on cultivar: TKW (thousand kernel weight, [g]), HLW (hektoliter weight ([kg/hl]), SDSS (sodium dodecyl sulphate sedimentation) test, FN (falling number, [s]), CP (crude protein, [%])) gluten index (GI, [%]) and gluten [%] (mean, α=0.05) 105 V Index of Tables Tab. 2: Relative parts [%] of total phenolic acids (TPA), antioxidative capacity by TPC and ORAC assay of three fractions in sum of all analyzed cultivars: fraction 1 (soluble free phenolic acids), fraction 2 (soluble conjugated phenolic acids) and fraction 3 (bound phenolic acids) 106 Tab. 3: Concentration of total phenolic acids (TPA) [µg GAE/g] of five spelt wheat cultivars in three extracted fractions (mean±SD) 106 Tab. 4: Total phenolic content (TPC) [µg GAE/g] of five spelt wheat cultivars in three extracted fractions and in sum of all three fractions (mean±SD) 107 Tab. 5: Oxygen radical absorbance capacity (ORAC) values [µmol TE/g] of five spelt wheat cultivars in three extracted fractions and in sum of all three fractions (mean±SD) 108 Tab. 6: Pearson correlation coefficient r between total phenolic acids (TPA). total phenolic content (TPC) and oxygen radical absorbance capacity (ORAC) 108 Discussion (chapter 6) Tab. 6-1: TPA and FA concentrations in µg GAE/g whole wheat flour and relation (%) of FA to TPA for all three experiments (exp.) (experiment 1 chapter 5.1, experiment 2 chapter 5.2, experiment 3 chapter 5.3) and different sprouting times (mean) 120 Tab. 6-2: Total phenolic compounds (TPC) [µg GAE/g] and ORAC values [µmol TE/g] of wheat species in the three executed investigations (exp.) (experiment 1 chapter 5.1, experiment 2 chapter 5.2, experiment 3 chapter 5.3) (mean) 125 VI Index of Figures Index of Figures Fig. 0-1:Health benefits by whole grain consumption (VITAGLIONE et al., 2008) II Literature review (chapter 3) Fig. 3-1: Origin of ancient wheat (NESBIT, 2001) 8 Fig. 3-2: Evolutionary relationship of wheat (modified by FELDMAN, 2001) 10 Fig. 3-3: Classification of dietary phytochemicals (modified by LIU, 2004) 13 Fig. 3-4: Basic structure of phenolic compounds: phenol 14 Fig. 3-5: Hydroxybenzoic acids (C6-C1) 15 Fig. 3-6: Hydroxycinnamic acids (C6-C3) 15 Fig. 3-7: Phenylpropanoid pathway (KULL, 2000) 16 Fig. 3-8: Mutual association between free radicals and their reactive metabolites (DURACKOVA, 2010) 20 Fig. 3-9: Schematic representation of the pathways leading to the generation of reactive oxygen species (ROS) and their selective dismutation (HADDAD, 2004) 21 Fig. 3-10: A general schematic showing the regulation of cell death and signal transduction pathways in response to reactive oxygen species (HADDAD, 2004) 21 Fig. 3-11: Mutual association between oxidants and antioxidants. NOS- NO- synthase, ROS- reactive oxygen species, CAT- catales, SOD- superoxide dismutase, GPx- Glutathione peroxidase, GST- glutathione S-transferase, UA- uric acid, GSH- glutathione reduced (modified by DURACKOVA, 2010) 22 Fig. 3-12: Bioactivities of dietary polyphenols (modified by HAN et al., 2007) 23 Fig. 3-13: Cancer risk factors (modified by ANAND et al., 2008) 27 Fig. 3-14: Intervention of antioxidants as blocking agents and suppressing agents during carcinogenesis (neoplasia) (JOHNSON, 2007) 28 Material and method (chapter 4) Fig. 4-1: Procedure of the preparation of the used wheat grain samples 56 Experiment 1 “Evaluation of ancient wheat species” (chapter 5.1) Fig. 1: HPLC chromatogram of a wheat sample with standards. Peak 1 vanillic acid (VA), 2 syringic acid (SYA), 3 caffeic acid (CA), 4 p- coumaric acid (PCA), 5 ferulic acid (FA), 6 sinapic acid (SIA) 66 VII Index of Figures Fig. 2: Germination rate [%], and thousand grain weight (TGW) [g] of different wheat species (mean ± SD, α=0.05) 68 Fig. 3: Falling number (FN) [s], crude protein content [%], and sedimentation test of different wheat species (mean ± SD, α=0.05) 68 Fig. 4: Total phenolic acids (TPA_total) and total ferulic acid (FA_total) values [µg GAE/g] in different wheat species (mean ± SD, α=0.05) 70 Fig. 5: Total phenolic content (TPC) [mg GAE/g] in different wheat species (mean ± SD, α=0.05) 71 Fig. 6: Antioxidant capacity (ORAC) [µmol TE/g] in different wheat species for fraction 1, 2 and 3 (mean ± SD, α=0.05) 72 Experiment 2 “Sprouting experiment with bread wheat” (chapter 5.2) Fig. 1: Different sprouting levels of wheat: A- presprouted grain after 24 h; B- fully sprouted grain after 48 h of sprouting 84 Fig. 2: Interaction effect of N fertilization and cultivar on sedimentation by Zeleny in wheat samples (mean ± SD, α=0.05) 87 Fig. 3: Effect of sprouting time on the composition of the extracted fractions (TPA1, TPA2, TPA3) of total phenolic acids (TPA) in wheat samples 89 Fig. 4: Effect of sprouting time on the composition of phenolic acids (TPA) in wheat grain 89 Fig. 5: Effect of sprouting time on the composition of the extracted fractions (TPC1, TPC2, TPC3) of total phenolic contents (TPC) in wheat samples 90 Fig. 6: Effect of sprouting time on the composition of the extracted fractions (ORAC1, ORAC2, ORAC3) of the antioxidative capacity by the ORAC assay in wheat samples 92 Experiment 3 “Evaluation of spelt wheat cultivars” (chapter 5.3) Fig. 1: Concentration of total phenolic acids (TPA) [µg GAE/g] and ferulic acid (FA) [µg GAE/g] of five spelt wheat cultivars in sum of all three extracted fractions (mean±SD, α=0.05) 107 Discussion (chapter 6) Fig. 6-1: Effects of sprouting time on FA concentrations [µg GAE/g] in wheat samples of experiment 2 (chapter 5.2) (mean, α=0.05) 122 Fig. 6-2: Effects of sprouting time on TPA concentrations [µg GAE/g] in wheat samples of experiment 2 (chapter 5.2) (mean, α=0.05) 122 VIII Index of Equations Index of Equations Literature review (chapter 3) Eq. 3-1: Spending an hydrogen atom (KOLTOVER, 2010) 24 Eq. 3-2: Radical-radical coupling (modified by CHIMI et al., 1991) 24 Eq. 3-3: HAT kinetics (HUANG et al., 2005, PRIOR et al., 2005) 37 Eq. 3-4: ET reaction (HUANG et al., 2005, PRIOR et al., 2005) 39 IX Abbreviations Abbreviations AAPH 2,2’-azobis(2-methylpropionamidine)dihydrochloride ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid AUC Area under curve CA Caffeic acid CCC Chlorcholin chloride CHD Coronary heart disease CRC Colorectal cancer CVD Cardio vascular disease DAD Diode array detector DPPH Diphenyl-1-picrylhydrazyl DSC Differential scanning calorimetry ET Electron transfer FA Ferulic acid FAE Ferulic acid equivalent FCR Folin-Ciocalteu reagent FN Falling number FRAP Ferric ion reducing antioxidant parameter GAE Gallic acid equivalent GI Gluten index GIT Gastro intestinal tract GSH Glutathione HAT Hydrogen atom transfer HLW Hectoliter weight HPLC High performance liquid chromatography KIHD Kuopio Ischaemic Heart Disease LA Linoleic acid MDA Malondialdehyde OIT Oxidation induction temperature ORAC Oxygen radical absorbance capacity PA Phenolic acid PAL Phenylalanine ammonia lyase PC Phenolic compound X