The contents and effects of polyphenols in chocolate [Elektronische Ressource] : qualitative and quantitative analyses of polyphenols in chocolate and chocolate raw products as well as evaluation of potential implications of chocolate consumption in human health / submitted by Jan Wollgast
349 Pages
English
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The contents and effects of polyphenols in chocolate [Elektronische Ressource] : qualitative and quantitative analyses of polyphenols in chocolate and chocolate raw products as well as evaluation of potential implications of chocolate consumption in human health / submitted by Jan Wollgast

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Learn all about the services we offer
349 Pages
English

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JUSTUS LIEBIG UNIVERSITY GIESSEN Faculty of Agricultural and Nutritional Sciences, Home Economics, and Environmental Management Institute of Nutritional Sciences The contents and effects of polyphenols in chocolate Qualitative and quantitative analyses of polyphenols in chocolate and chocolate raw products as well as evaluation of potential implications of chocolate consumption in human health Dissertation for obtaining the degree of doctor at the faculty of Agricultural and Nutritional Sciences, Home Economics, and Environmental Management at the University of Gießen, Germany submitted by Dipl. oec. troph. Jan Wollgast Gießen, 2004 Dissertation at the Faculty 09 (Agricultural and Nutritional Sciences, Home Economics and Environmental Management) of the Justus-Liebig-University Giessen (Germany). Disputation: 10 June 2005 Examinations committee: Chairperson: Prof. Dr. K. Becker-Brandenburg 1. Evaluator: Prof. Dr. C. Kunz 2. Evaluator: Herr PD Dr. G. Harzer 3. Evaluator and examiner: Prof. Dr. B. Honermeier Examiner: Prof. Dr. I. Bitsch Table of contents Table of contents 0 INTRODUCTION AND OBJECTIVES 1 1 LITERATURE SURVEY 7 1.1 Theobroma cacao and chocolate 7 1.2 Polyphenols – chemistry and biosynthesis 13 1.3 Polyphenols in cocoa 20 1.4 Analytical methodologies for the analysis of polyphenols in chocolate and chocolate raw products 29 1.

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Published 01 January 2005
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JUSTUS LIEBIG UNIVERSITY GIESSEN

Faculty of Agricultural and Nutritional Sciences,
Home Economics, and Environmental Management
Institute of Nutritional Sciences






The contents and effects of polyphenols in chocolate

Qualitative and quantitative analyses of polyphenols in chocolate and
chocolate raw products as well as evaluation of potential implications
of chocolate consumption in human health






Dissertation
for obtaining the degree of doctor
at the faculty of Agricultural and Nutritional Sciences,
Home Economics, and Environmental Management
at the University of Gießen, Germany




submitted by
Dipl. oec. troph. Jan Wollgast







Gießen, 2004
Dissertation at the Faculty 09 (Agricultural and Nutritional Sciences, Home Economics
and Environmental Management) of the Justus-Liebig-University Giessen (Germany).

Disputation: 10 June 2005

Examinations committee:
Chairperson: Prof. Dr. K. Becker-Brandenburg
1. Evaluator: Prof. Dr. C. Kunz
2. Evaluator: Herr PD Dr. G. Harzer
3. Evaluator and examiner: Prof. Dr. B. Honermeier
Examiner: Prof. Dr. I. Bitsch Table of contents
Table of contents

0 INTRODUCTION AND OBJECTIVES 1
1 LITERATURE SURVEY 7
1.1 Theobroma cacao and chocolate 7
1.2 Polyphenols – chemistry and biosynthesis 13
1.3 Polyphenols in cocoa 20
1.4 Analytical methodologies for the analysis of polyphenols in
chocolate and chocolate raw products 29
1.5 Cocoa polyphenols – potential health implications 44
1.6 Scientific substantiation of health claims – the biomarker
approach 82
2 MATERIALS AND METHODS 92
2.1 Methods applied for the analysis of polyphenols in chocolate and
chocolate raw products 92
2.2 Isolation, purification and characterisation of phenolic
compounds from cocoa powder 100
2.3 Quantitative analysis of polyphenols in chocolate 105
2.4 Polyphenol content in chocolate and chocolate raw products –
identification of important process parameters 109
2.5 Excursus 1: Bioavailability of polyphenols from chocolate in
humans 112
2.6 Excursus 2: Measurement of urinary markers for oxidative DNA-
damage and lipid peroxidation in humans 114
2.7 Statistical analyses 119
3 RESULTS AND DISCUSSION 121
3.1 Evaluation of available methods for the analysis of polyphenols in
chocolate and chocolate raw-products 121
i Table of contents
3.2 Identification of phenolic compounds in chocolate 141
3.3 Set-up and validation of methodologies for quantitative analysis 210
3.4 Polyphenol content in chocolate & chocolate raw products –
Identification of important process parameters 225
3.5 Excursus A: Bioavailability of polyphenols from chocolate in
humans 263
3.6 Excursus B: In-vivo antioxidant activity of polyphenols from
chocolate in humans – the biomarker approach 274
4 CONCLUSIONS AND OUTLOOK 285
5 SUMMARY 290
6 REFERENCES 293
7 AKNOWLEDGEMENTS 331



ii Table of contents

0 INTRODUCTION AND OBJECTIVES 1
1 LITERATURE SURVEY 7
1.1 Theobroma cacao and chocolate 7
1.1.1 History 7
1.1.2 Cocoa bean 9
1.1.3 Fermentation and drying 9
1.1.4 Chocolate making 10
1.2 Polyphenols – chemistry and biosynthesis 13
1.3 Polyphenols in cocoa 20
1.4 Analytical methodologies for the analysis of polyphenols in
chocolate and chocolate raw products 29
1.4.1 Sample extraction techniques 30
1.4.2 Techniques for isolation and purification of phenolic compounds
from natural products 31
1.4.3 Colorimetric methods for polyphenol analysis 32
1.4.4 Non-extractable procyanidins through hydrolysis with butanol-
hydrochloric acid (proanthocyanidin assay) 34
1.4.5 Normal-phase-high-performance liquid chromatography 36
1.4.6 Liquid chromatography with mass spectrometric detection 37
1.4.7 Liquid chromatography-mass spectrometry for quantitative analysis 39
1.4.8 Capillary electrophoresis as alternative to high performance liquid
chromatography 40
1.5 Cocoa polyphenols – potential health implications 44
1.5.1 Epidemiological evidence 44
1.5.2 Bioavailability 46
1.5.3 Potential health effects of chocolate, cocoa, and cocoa polyphenols –
evidence from in vitro, ex vivo, animal and human intervention
studies 55
1.5.3.1 Antioxidant effects 55
1.5.3.2 Effects related to cancer 59
1.5.3.3 Effects on cardiovascular health 61
1.5.3.4 Effects on immune response and inflammatory diseases 67
iii Table of contents
1.5.4 Nutrients and nutritional value of chocolate 69
1.5.4.1 Lipids (cocoa butter) in chocolate and cardiovascular health 71
1.5.4.2 Free sugars in chocolate 74
1.5.4.3 High energy-density of chocolate 77
1.5.4.4 Conclusions 80
1.6 Scientific substantiation of health claims – the biomarker
approach 82
1.6.1 8-OHdG as a marker of oxidative DNA damage 84
1.6.2 F -isoprostanes as a marker of lipid peroxidation 86 2
2 MATERIALS AND METHODS 92
2.1 Methods applied for the analysis of polyphenols in chocolate and
chocolate raw products 92
2.1.1 Reagents and standards 92
2.1.2 General procedure for the extraction of polyphenols from cocoa
liquor and chocolate 92
2.1.3 Colorimetric methods 93
2.1.3.1 Folin-Ciocalteu assay 93
2.1.3.2 Vanillin-HCl assay 93
2.1.3.3 Prussian-Blue assay 94
2.1.3.4 Non-extractable procyanidins through hydrolysis with a modified
proanthocyanidin assay 94
2.1.3.5 Parameters of method performance for assay comparison 95
2.1.4 Normal phase and reversed phase high-performance liquid
chromatography with ultraviolet and electrochemical detection 95
2.1.4.1 Normal phase high-performance liquid chromatography 96
2.1.4.2 Reversed phase high-performance liquid chromatography 96
2.1.5 Micellar electrokinetic capillary chromatography 97
2.1.6 High-performance liquid chromatography with mass spectrometric
detection applied for the identification of phenolic compounds in
chocolate 97
2.1.6.1 HPLC-MS system 1 98
2.1.6.2 HPLC-MS system 2 99
2.2 Isolation, purification and characterisation of phenolic
compounds from cocoa powder 100
2.2.1 Chemical reagents 101
iv Table of contents
2.2.2 Equipment 102
2.2.3 Procedure for extraction, isolation and purification 102
2.2.4 Structure elucidation of isolated compounds 104
2.2.4.1 Nuclear magnetic resonance 104
2.2.4.2 Simple and collision-induced dissociation mass spectrometry 104
2.2.4.3 Hydrogen/deuterium exchange mass spectrometry 105
2.3 Quantitative analysis of polyphenols in chocolate 105
2.3.1 Chemicals and Reagents 106
2.3.2 Sample preparation 106
2.3.3 High-performance liquid chromatography with mass spectrometric
detection 107
2.3.3.1 MS-System 1 107
2.3.3.2 MS-System 2 107
2.3.4 Other techniques 108
2.3.5 Method validation parameters 108
2.4 Polyphenol content in chocolate and chocolate raw products –
identification of important process parameters 109
2.4.1 Chocolate manufacturing process lines and sampling plan 109
2.5 Excursus 1: Bioavailability of polyphenols from chocolate in
humans 112
2.5.1 Chemicals and Reagents 112
2.5.2 Subjects and study design 112
2.5.3 Sample preparation 113
2.5.4 High-performance liquid chromatography with mass spectrometric
detection 113
2.6 Excursus 2: Measurement of urinary markers for oxidative DNA-
damage and lipid peroxidation in humans 114
2.6.1 Chemicals and Reagents 115
2.6.2 Equipment 115
2.6.3 Sample preparation for 8-hydroxydeoxyguanosine 115
2.6.4 High-performance liquid chromatography-tandem mass spectrometry
for analysis of 8-hydroxydeoxyguanosine 116
2.6.5 Sample preparation for 15-F -isoprostane 116 2t
2.6.6 High-performance liquid chromatography-isotope dilution-tandem
mass spectrometry for analysis of 15-F -isoprostane 117 2t
v Table of contents
2.6.7 Method validation parameters 118
2.7 Statistical analyses 119
3 RESULTS AND DISCUSSION 121
3.1 Evaluation of available methods for the analysis of polyphenols in
chocolate and chocolate raw-products 121
3.1.1 Colorimetric methods for polyphenol analysis 121
3.1.2 Normal-phase versus reversed-phase high-performance liquid
chromatography 129
3.1.3 Micellar electrokinetic capillary chromatography as alternative to
high performance liquid chromatography 132
3.1.4 High-performance liquid chromatography with mass spectrometric
detection 138
3.1.5 Non-extractable procyanidins through hydrolysis with a modified
proanthocyanidin assay 139
3.2 Identification of phenolic compounds in chocolate 141
3.2.1 Identification of the major phenolic cocoa compounds 141
3.2.2 Identification of minor phenolic compounds 158
3.2.2.1 Quercetin and quercetin glycosides 158
3.2.2.2 Hydroxycinnamic acid derivates 164
3.2.2.3 Minor flavan-3-ols (catechins and procyanidins) 169
3.2.3 Isolation, purification and characterisation of phenolic compounds
from cocoa powder 174
3.2.3.1 Structure elucidation by nuclear magnetic resonance spectroscopy 176
3.2.3.1.1 Hydroxycinnamic acid amides (clovamides) 176
3.2.3.1.2 Procyanidins 187
3.2.3.2 Determination of labile protons by liquid phase hydrogen/deuterium
exchange followed by mass spectrometry 193
3.2.4 Phenolic compounds suggested in literature but not confirmed 203
3.2.5 Summary of identified phenolic compounds in cocoa 208
3.3 Set-up and validation of methodologies for quantitative analysis 210
3.3.1 Sample extraction and sample preparation 210
3.3.2 High-performance liquid chromatography coupled to electrospray
ionisation-mass spectrometry as quantitative method of analysis for
polyphenols in chocolate and chocolate raw products 217
3.3.2.1 Selection of best suited mass spectrometric detection technique 217
vi Table of contents
3.3.2.1 Validation of the high-performance liquid chromatography-mass
spectrometry method for the quantitative analysis of polyphenols in
chocolate and chocolate raw products 219
3.3.2.1.1 Linearity 219
3.3.2.1.2 Trueness, precision and range of application 222
3.4 Polyphenol content in chocolate & chocolate raw products –
Identification of important process parameters 225
3.4.1 Sampling 226
3.4.2 Results from the monitoring of the chocolate manufacturing
processes 230
3.4.3 Polyphenol content of fermented cocoa beans of various origins 247
3.4.4 Polyphenol content in various samples of dark and milk chocolate 250
3.4.5 Quality control of analytical results 257
3.4.6 Folin-Ciocalteu assay versus HPLC-MS 258
3.4.7 Analyses of non-extractable polyphenols (NEPP) 261
3.5 Excursus A: Bioavailability of polyphenols from chocolate in
humans 263
3.6 Excursus B: In-vivo antioxidant activity of polyphenols from
chocolate in humans – the biomarker approach 274
3.6.1 High-performance liquid chromatography-tandem mass spectrometry
for the measurement of urinary 8-hydroxydeoxyguanosine 275
3.6.2 High-performance liquid chromatography-tandem mass spectrometry
for the measurement of urinary 15-F -isoprostane 277 2t
3.6.3 Biological variation of urinary levels of 8-OHdG and 15-F -IsoP 281 2t
4 CONCLUSIONS AND OUTLOOK 285
5 SUMMARY 290
6 REFERENCES 293
7 AKNOWLEDGEMENTS 331

vii Table of tables
Table of tables

Table 1.1 Main Classes of Polyphenolic Compounds 14
Table 1.2 Classification of Food Flavonoids 15
Table 1.3 Polyphenol content (mg/g) in coca powder, dark chocolate, and milk
chocolate according to the applied analytical procedure 26
Table 3.1 Method performance characteristics of three commonly used
colorimetric assays for the analysis of total polyphenol content in
cocoa 127
Table 3.2 Scheme for calculation of initial simplex 134
Table 3.3 Main masses in the CID-MS-MS spectrum of deprotonated
-
procyanidin tetramer (m/z 1153 = [M-H] ) 152
1
Table 3.4 H spectra for clovamide (caffeoyl-dihydroxyphenylalanine) 178
13 1Table 3.5 C and H spectra for dideoxyclovamide (p-coumaroyltyrosine) 179
1Table 3.6a H spectra for caffeoyltyrosine (monodeoxyclovamide isomer1) 183
1
Table 3.6b H spectra for p-coumaroyldihydroxyphenylalanine
(monodeoxyclovamide isomer2) 184
13
Table 3.7 C spectra of procyanidin dimer B2 190
13Table 3.8 C spectra of procyanidin trimer C1 192
Table 3.9 Overview on phenolic compounds in chocolate and chocolate raw
products identified and/or confirmed in this study 209

viii