Justus Liebig University Giessen Institute of Agronomy and Plant Breeding I Department of Agronomy Prof. Dr. Bernd Honermeier Investigations on the effect of light reduction on yield, growth, and secondary metabolites of lemon balm (Melissa officinalis L.) Dissertation for a Doctorate Degree in Nutritional Sciences (Dr. troph.) in the Faculty of Agricultural Sciences, Nutritional Sciences and Environmental Management Justus Liebig University Giessen Submitted by Marco Russo from Uelzen, Germany Giessen, 2019 Mit Genehmigung des Fachbereichs Agrarwissenschaften, Ökotrophologie und Umweltmanagement der Justus-Liebig-Universität Gießen Examination committee: 1. Supervisor: Prof. Dr. Bernd Honermeier 2. Supervisor: Prof. Dr. Gertrud Morlock Examiner: Prof. Dr. Silvia Rudloff Examiner: Prof. Dr. Gunter P. Eckert Chairperson: Prof. Dr. Joachim Aurbacher Date of defense: November 27, 2019 Table of contents I Table of contents Table of contents .............................................................................................................. I Abbreviations ................................................................................................................. IV List of figures ................................................................................................................ VII List of tables.................................................................................................................. XII List of figures in appendix .......................................................................................... XIII List of tables in appendix ............................................................................................. XV 1 Introduction ........................................................................................................ 1 2 Literature review ................................................................................................ 5 2.1 Botanical characterization of lemon balm (Melissa officinalis L.) .......................... 5 2.2 Cultivation of lemon balm ..................................................................................... 7 2.3 Yield characteristics of lemon balm .................................................................... 10 2.4 Requirements of the European Pharmacopoeia ................................................ 11 2.5 Secondary metabolites of lemon balm ............................................................... 12 2.5.1 Phenolic compounds ......................................................................................... 13 2.5.2 Essential oil ....................................................................................................... 18 2.6 Antioxidants ....................................................................................................... 23 2.7 Light and its influence on plant growth and secondary metabolites .................... 29 2.7.1 Light and plant growth ....................................................................................... 29 2.7.2 Light and selected secondary metabolites ......................................................... 32 3 Material and Methods ...................................................................................... 35 3.1 Soil and climate conditions ................................................................................ 35 3.1.1 Experimental site Gross­Gerau .......................................................................... 35 3.1.2 Experimental site Rauischholzhausen ............................................................... 36 3.2 Design and experimental procedure .................................................................. 37 3.2.1 Experimental design .......................................................................................... 37 3.2.1.1 Field experiment Gross­Gerau ........................................................................... 39 3.2.1.2 Field experiment Rauischholzhausen ................................................................ 45 3.2.2 Plant parameters ............................................................................................... 51 3.2.3 Biomass and leaf yield ....................................................................................... 51 Table of contents II 3.3 Laboratory analysis ............................................................................................ 52 3.3.1 Plant material ..................................................................................................... 52 3.3.2 Chemicals and reagents .................................................................................... 52 3.3.3 Essential oil content ........................................................................................... 52 3.3.4 Methanolic extraction ......................................................................................... 52 3.3.5 Total phenolic content ........................................................................................ 53 3.3.6 Antioxidant capacity ........................................................................................... 53 3.3.7 Rosmarinic acid content ..................................................................................... 54 3.4 Statistical analysis ............................................................................................. 55 4 Results ............................................................................................................. 56 4.1 Field experiment Gross­Gerau ........................................................................... 56 4.1.1 Plant parameters ............................................................................................... 56 4.1.1.1 Plant height ........................................................................................................ 56 4.1.1.2 Leaf area index (LAI) ......................................................................................... 60 4.1.1.3 SPAD values ..................................................................................................... 62 4.1.1.4 Number of shoots per plant ................................................................................ 65 4.1.2 Yield parameters................................................................................................ 68 4.1.2.1 Biomass yield (FM) ............................................................................................ 68 4.1.2.2 Biomass yield (DM) ............................................................................................ 71 4.1.2.3 Leaf yield (FM) ................................................................................................... 74 4.1.2.4 Leaf yield (DM) .................................................................................................. 77 4.1.2.5 DM content of the leaves ................................................................................... 80 4.1.2.6 Leaf:stem ratio ................................................................................................... 83 4.1.3 Essential oil content ........................................................................................... 86 4.1.4 Total phenolic content ........................................................................................ 90 4.1.5 Antioxidant capacity ........................................................................................... 94 4.1.6 Rosmarinic acid content ..................................................................................... 98 4.2 Field experiment Rauischholzhausen .............................................................. 102 4.2.1 Plant parameters ............................................................................................. 102 4.2.1.1 Plant height ...................................................................................................... 102 4.2.1.2 Leaf area index (LAI) ....................................................................................... 105 4.2.1.3 SPAD values ................................................................................................... 107 4.2.1.4 Number of shoots per plant .............................................................................. 110 4.2.2 Yield parameters.............................................................................................. 113 4.2.2.1 Biomass yield (FM) .......................................................................................... 113 4.2.2.2 Biomass yield (DM) .......................................................................................... 116 4.2.2.3 Leaf yield (FM) ................................................................................................. 119 Table of contents III 4.2.2.4 Leaf yield (DM) ................................................................................................ 122 4.2.2.5 DM content of the leaves ................................................................................. 125 4.2.2.6 Leaf:stem ratio ................................................................................................. 128 4.2.3 Essential oil content ......................................................................................... 131 4.2.4 Total phenolic content ...................................................................................... 134 4.2.5 Antioxidant capacity ......................................................................................... 137 4.2.6 Rosmarinic acid content ................................................................................... 140 5 Discussion ..................................................................................................... 143 5.1 Plant parameters ............................................................................................. 143 5.1.1 Plant height ...................................................................................................... 143 5.1.2 Leaf area index (LAI) ....................................................................................... 146 5.1.3 SPAD values / Chlorophyll content .................................................................. 148 5.1.4 Shoots per plant .............................................................................................. 150 5.2 Yield parameters.............................................................................................. 152 5.3 Essential oil content ......................................................................................... 159 5.4 Phenolic compounds and antioxidant properties .............................................. 168 6 Summary ........................................................................................................ 180 7 Zusammenfassung ........................................................................................ 182 Bibliography ................................................................................................................. 184 Appendix ...................................................................................................................... 213 Scatterplots of correlations............................................................................................. 213 Three-factorial analyses for Gross-Gerau ...................................................................... 216 Three-factorial analyses for Rauischholzhausen ............................................................ 230 Declaration / Erklärung ................................................................................................ 244 Abbreviations IV Abbreviations 4CL 4-coumarate-CoA ligase a.i. Active ingredient AAPH 2,2'-azobis(2-amidinopropane) dihydrochloride ABA Abscisic acid ANOVA Analysis of variance APX Ascorbate peroxidase AUC Area under the curve BHA Butylated hydroxyanisole BHT Butylated hydroxytoluene C4H t-cinnamic acid 4-hydroxylase CAT Catalase DHAR Dehydroascorbate reductase DM Dry matter DMAPP Dimethylallyl pyrophosphate DOXP 1-deoxy-D-xylulose 5-phosphate DPPH Diphenyl-1-picrylhydrazyl dt Decitonne DXP 1-deoxy-D-xylulose 5-phosphate DXR DXP reductoisomerase DXS DXP synthase EC Enzyme Commission number EO Essential oil ET Electron transfer FC Folin-Ciocalteu assay FM Fresh matter FNR Fachagentur Nachwachsende Rohstoffe (Agency for Renewable Resources) Abbreviations V FPP Farnesyl pyrophosphate FPPS Farnesyl pyrophosphate synthase FRAP Ferric ion reducing antioxidant parameter GAE Gallic acid equivalents GG Gross-Gerau GGPP Geranylgeranyl pyrophosphate GPP Geranyl pyrophosphate GPPS Geranyl pyrophosphate synthase GST Glutathione S-transferase ha Hectare HAT Hydrogen atom transfer HMGB1 High mobility group box-1 HMG-CoA 3-hydroxy-3-methylglutaryl-CoA HMGR HMG-CoA reductase HPLC High-performance liquid chromatography HPPR Hydroxyphenylpyruvate reductase HSV-1 Herpes simplex virus type 1 IPP Isopentenyl pyrophosphate ISO International Organization for Standardization K Potassium LAI Leaf area index LS Linalool synthase LS means Least-squares means MDAR Monodehydroascorbate reductase MEP 2-C-methyl-D-erythritol 4-phosphate Mg Magnesium N Nitrogen n.a. Not applicable, not available Abbreviations VI n.d. Not determined ORAC Oxygen radical absorbance capacity P Phosphorus PAL Phenylalanine ammonia lyase PAR Photosynthetically active radiation PEP Phosphoenolpyruvate Ph. Eur. European Pharmacopoeia PPFD Photosynthetic photon flux density R:FR Red:far-red ratio RA Rosmarinic acid RAS Rosmarinic acid synthase RH Rauischholzhausen RNS Reactive nitrogen species ROS Reactive oxygen species S Sulfur SAS Shade avoidance syndrome SOD Superoxide dismutase SPAD Soil & Plant Analyzer Development TAT Tyrosine aminotransferase TE Trolox equivalents TEAC Trolox equivalent antioxidant capacity TPC Total phenolic content TRAP Total radical trapping antioxidant parameter VDLUFA Verband deutscher landwirtschaftlicher Untersuchungs- und Forschungsanstalten (Association of German Agricultural Analytic and Research Institutes) List of figures VII List of figures Fig. 1: Lemon balm plant in vegetative stage. .......................................................... 6 Fig. 2: Flowering lemon balm plant. .......................................................................... 6 Fig. 3: Structural formula of phenol ........................................................................ 13 Fig. 4: Structural formula of rosmarinic acid ........................................................... 13 Fig. 5: Biosynthetic pathway of rosmarinic acid in Melissa officinalis ...................... 17 Fig. 6: Structural formulas of major compounds in lemon balm essential oil ........... 20 Fig. 7: Schematic, simplified illustration of isopentenyl pyrophosphate (IPP) biosynthesis in plants .................................................................................. 21 Fig. 8: Biosynthesis of terpenoids, starting from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) .......................................... 22 Fig. 9: Phenoxy radical, resonance-stabilized ........................................................ 24 Fig. 10: Example curves of ORAC measurements on a 96-well microplate. ............. 28 Fig. 11: Design of the field experiment in Gross-Gerau 2013-2015. ........................ 39 Fig. 12: Plot dimensions of the field experiment Gross-Gerau. ................................. 40 Fig. 13: Photosynthetically active radiation (PAR) under full sunlight and moderate shading. Field experiment Gross-Gerau, 2013-2015. ................. 43 Fig. 14: Global radiation under full sunlight and moderate shading. Field experiment Gross Gerau, 2013-2015. ........................................................ 43 Fig. 15: Differences in temperature between light and shade treatment during the vegetation periods 2014 and 2015. Field experiment Gross-Gerau. ...... 44 Fig. 16: Differences in relative humidity between light and shade treatment during the vegetation periods 2014 and 2015. Field experiment Gross-Gerau. .............................................................................................. 44 Fig. 17: Design of the field experiment in Rauischholzhausen 2013-2014. .............. 45 Fig. 18: Design of the field experiment in Rauischholzhausen 2015. ........................ 46 Fig. 19: Plot dimensions of the field experiment Rauischholzhausen 2013-2014. ................................................................................................. 47 Fig. 20: Photosynthetically active radiation (PAR) under full sunlight and strong shading. Field experiment Rauischholzhausen, 2013-2015. ....................... 48 List of figures VIII Fig. 21: Global radiation under full sunlight and strong shading. Field experiment Rauischholzhausen, 2013-2015. .............................................. 48 Fig. 22: Differences in temperature between light and shade treatment during the vegetation periods 2014 and 2015. Field experiment Rauischholzhausen. .................................................................................... 49 Fig. 23: Differences in relative humidity between light and shade treatment during the vegetation periods 2014 and 2015. Field experiment Rauischholzhausen. .................................................................................... 49 Fig. 24: Pipetting scheme for ORAC assay on a 96-well plate. ................................. 53 Fig. 25: Plant height [cm] of lemon balm. First cut in Gross­Gerau, 2013-2015. ...... 58 Fig. 26: Plant height [cm] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ................................................................................................. 59 Fig. 27: Leaf Area Index (LAI) of lemon balm. First and second cut in Gross- Gerau, 2014 and 2015................................................................................. 61 Fig. 28: SPAD values of lemon balm (first fully developed leaf). First cut in Gross­Gerau, 2013-2015. ........................................................................... 63 Fig. 29: SPAD values of lemon balm (first fully developed leaf). Second cut in Gross­Gerau, 2013-2015. ........................................................................... 64 Fig. 30: Shoots per plant of lemon balm. First cut in Gross­Gerau, 2013-2015........ 66 Fig. 31: Shoots per plant of lemon balm. Second cut in Gross­Gerau, 2013-2015. ................................................................................................. 67 Fig. 32: Fresh matter biomass yield [dt FM/ha] of lemon balm. First cut in Gross­Gerau, 2013-2015. ........................................................................... 69 Fig. 33: Fresh matter biomass yield [dt FM/ha] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 70 Fig. 34: Dry matter biomass yield [dt DM/ha] of lemon balm. First cut in Gross­Gerau, 2013-2015. ........................................................................... 72 Fig. 35: Dry matter biomass yield [dt DM/ha] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 73 Fig. 36: Fresh matter leaf yield [dt FM/ha] of lemon balm. First cut in Gross­Gerau, 2013-2015. ........................................................................... 75 Fig. 37: Fresh matter leaf yield [dt FM/ha] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 76 List of figures IX Fig. 38: Dry matter leaf yield [dt DM/ha] of lemon balm. First cut in Gross­Gerau, 2013-2015. ........................................................................... 78 Fig. 39: Dry matter leaf yield [dt DM/ha] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 79 Fig. 40: DM content of the leaves [%] of lemon balm. First cut in Gross­Gerau, 2013-2015. ................................................................................................. 81 Fig. 41: DM content of the leaves [%] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 82 Fig. 42: Leaf:stem ratio of lemon balm. First cut in Gross­Gerau, 2013-2015. ......... 84 Fig. 43: Leaf:stem ratio of lemon balm. Second cut in Gross­Gerau, 2013-2015. ................................................................................................. 85 Fig. 44: Essential oil content [%] of lemon balm leaves. First cut in Gross­Gerau, 2013-2015. ........................................................................... 88 Fig. 45: Essential oil content [%] of lemon balm leaves. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 89 Fig. 46: Total phenolic content [mg GAE/g DM] of lemon balm. First cut in Gross­Gerau, 2013-2015. ........................................................................... 92 Fig. 47: Total phenolic content [mg GAE/g DM] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 93 Fig. 48: Antioxidant capacity [µmol TE/g DM] of lemon balm. First cut in Gross­Gerau, 2013-2015. ........................................................................... 96 Fig. 49: Antioxidant capacity [µmol TE/g DM] of lemon balm. Second cut in Gross­Gerau, 2013-2015. ........................................................................... 97 Fig. 50: Rosmarinic acid content [%] of dried lemon balm leaves. First cut in Gross­Gerau, 2013-2015. ......................................................................... 100 Fig. 51: Rosmarinic acid content [%] of dried lemon balm leaves. Second cut in Gross­Gerau, 2013-2015. ......................................................................... 101 Fig. 52: Plant height [cm] of lemon balm. First cut in Rauischholzhausen, 2013-2015. ............................................................................................... 103 Fig. 53: Plant height [cm] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. ............................................................................................... 104 Fig. 54: Leaf Area Index (LAI) of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 105 List of figures X Fig. 55: SPAD values of lemon balm (first fully developed leaf). First cut in Rauischholzhausen, 2013-2015. .............................................................. 108 Fig. 56: SPAD values of lemon balm (first fully developed leaf). Second cut in Rauischholzhausen, 2013-2015. .............................................................. 109 Fig. 57: Shoots per plant of lemon balm. First cut in Rauischholzhausen, 2013-2015. ............................................................................................... 111 Fig. 58: Shoots per plant of lemon balm. Second cut in Rauischholzhausen, 2013-2015. ............................................................................................... 112 Fig. 59: Fresh matter biomass yield [dt FM/ha] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 114 Fig. 60: Fresh matter biomass yield [dt FM/ha] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 115 Fig. 61: Dry matter biomass yield [dt DM/ha] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 117 Fig. 62: Dry matter biomass yield [dt DM/ha] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 118 Fig. 63: Fresh matter leaf yield [dt FM/ha] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 120 Fig. 64: Fresh matter leaf yield [dt FM/ha] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 121 Fig. 65: Dry matter leaf yield [dt DM/ha] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 123 Fig. 66: Dry matter leaf yield [dt DM/ha] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 124 Fig. 67: DM content of the leaves [%] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 126 Fig. 68: DM content of the leaves [%] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 127 Fig. 69: Leaf:stem ratio of lemon balm. First cut in Rauischholzhausen, 2013-2015. ............................................................................................... 129 Fig. 70: Leaf:stem ratio of lemon balm. Second cut in Rauischholzhausen, 2013-2015. ............................................................................................... 130 List of figures XI Fig. 71: Essential oil content [%] of lemon balm leaves. First cut in Rauischholzhausen, 2013-2015. .............................................................. 132 Fig. 72: Essential oil content [%] of lemon balm leaves. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 133 Fig. 73: Total phenolic content [mg GAE/g DM] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 135 Fig. 74: Total phenolic content [mg GAE/g DM] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 136 Fig. 75: Antioxidant capacity [µmol TE/g DM] of lemon balm. First cut in Rauischholzhausen, 2013-2015. .............................................................. 138 Fig. 76: Antioxidant capacity [µmol TE/g DM] of lemon balm. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 139 Fig. 77: Rosmarinic acid content [%] of dried lemon balm leaves. First cut in Rauischholzhausen, 2013-2015. .............................................................. 141 Fig. 78: Rosmarinic acid content [%] of dried lemon balm leaves. Second cut in Rauischholzhausen, 2013-2015. .............................................................. 142 List of tables XII List of tables Tab. 1: Rosmarinic acid (RA) contents of lemon balm (Melissa officinalis) from literature. ..................................................................................................... 14 Tab. 2: Air temperature and precipitation at the experimental station Gross- Gerau in 2013-2015. .................................................................................. 35 Tab. 3: Air temperature and precipitation at the experimental station Rauischholzhausen in 2013-2015. ............................................................. 36 Tab. 4: Investigated factors and levels for the field experiments in Gross-Gerau (GG) and Rauischholzhausen (RH). ............................................................ 37 Tab. 5: Harvest dates, vegetation days, air temperature, precipitation and irrigation of the field experiment in Gross-Gerau. ......................................... 41 Tab. 6: Harvest dates, vegetation days, air temperature, precipitation and irrigation of the field experiment in Rauischholzhausen. .............................. 50 List of figures in appendix XIII List of figures in appendix Fig. A 1: Correlation of Leaf Area Index (LAI) and plant height of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 213 Fig. A 2: Correlation of SPAD values and DM leaf yield of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 213 Fig. A 3: Correlation of SPAD values and essential oil content of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 213 Fig. A 4: Correlation of FM and DM biomass yield of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 213 Fig. A 5: Correlation of FM and DM leaf yield of lemon balm in Gross­Gerau, 2013-2015. ............................................................................................... 213 Fig. A 6: Correlation of leaf:stem ratio and plant height of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 213 Fig. A 7: Correlation of essential oil content and leaf:stem ratio of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 214 Fig. A 8: Correlation of essential oil content and plant height of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 214 Fig. A 9: Correlation of essential oil content and DM leaf yield of lemon balm in Gross­Gerau, 2013-2015. ......................................................................... 214 Fig. A 10: Correlation of antioxidant capacity and total phenolic content of lemon balm in Gross­Gerau, 2013-2015. ............................................................ 214 Fig. A 11: Correlation of rosmarinic acid and antioxidant capacity of lemon balm in Gross­Gerau, 2013-2015. ..................................................................... 214 Fig. A 12: Correlation of rosmarinic acid and total phenolic content of lemon balm in Gross­Gerau, 2013-2015. ..................................................................... 214 Fig. A 13: Correlation of FM and DM biomass yield of lemon balm in Rauischholzhausen, 2013-2015. .............................................................. 215 Fig. A 14: Correlation of FM and DM leaf yield of lemon balm in Rauischholzhausen, 2013-2015. .............................................................. 215 Fig. A 15: Correlation of leaf:stem ratio and plant height of lemon balm in Rauischholzhausen, 2013-2015. .............................................................. 215 Fig. A 16: Correlation of antioxidant capacity and total phenolic content of lemon balm in Rauischholzhausen, 2013-2015. .................................................. 215 List of figures in appendix XIV Fig. A 17: Correlation of RA content and total phenolic content of lemon balm in Rauischholzhausen, 2013-2015. .............................................................. 215 Fig. A 18: Correlation of RA content and antioxidant capacity of lemon balm in Rauischholzhausen, 2013-2015. .............................................................. 215 List of tables in appendix XV List of tables in appendix Tab. A 1: Plant height [cm], Gross­Gerau 2013-2015. Results of the three- factorial analysis. ....................................................................................... 216 Tab. A 2: Leaf Area Index (LAI), Gross­Gerau 2013-2015. Results of the three- factorial analysis. ....................................................................................... 217 Tab. A 3: SPAD values, Gross­Gerau 2013-2015. Results of the three-factorial analysis. .................................................................................................... 218 Tab. A 4: Shoots per plant, Gross­Gerau 2013-2015. Results of the three- factorial analysis. ....................................................................................... 219 Tab. A 5: Biomass yield (FM) [dt FM/ha], Gross­Gerau 2013-2015. Results of the three-factorial analysis. ........................................................................ 220 Tab. A 6: Biomass yield (DM) [dt DM/ha], Gross­Gerau 2013-2015. Results of the three-factorial analysis. ........................................................................ 221 Tab. A 7: Leaf fresh matter yield [dt FM/ha], Gross­Gerau 2013-2015. Results of the three-factorial analysis. ........................................................................ 222 Tab. A 8: DM leaf yield [dt DM/ha], Gross­Gerau 2013-2015. Results of the three-factorial analysis............................................................................... 223 Tab. A 9: DM content of the leaves [%], Gross­Gerau 2013-2015. Results of the three-factorial analysis............................................................................... 224 Tab. A 10: Leaf:stem ratio, Gross­Gerau 2013-2015. Results of the three-factorial analysis. .................................................................................................... 225 Tab. A 11: Essential oil content [%], Gross­Gerau 2013-2015. Results of the three-factorial analysis............................................................................... 226 Tab. A 12: Total phenolic content [mg GAE/g DM], Gross­Gerau 2013-2015. Results of the three-factorial analysis. ....................................................... 227 Tab. A 13: ORAC [µmol TE/g DM], Gross­Gerau 2013-2015. Results of the three- factorial analysis. ....................................................................................... 228 Tab. A 14: Rosmarinic acid content [%], Gross­Gerau 2013-2015. Results of the three-factorial analysis............................................................................... 229 Tab. A 15: Plant height [cm], Rauischholzhausen 2013-2015. Results of the three-factorial analysis............................................................................... 230 Tab. A 16: Leaf Area Index (LAI), Rauischholzhausen 2013-2015. Results of the three-factorial analysis............................................................................... 231 List of tables in appendix XVI Tab. A 17: SPAD values, Rauischholzhausen 2013-2015. Results of the three- factorial analysis. ....................................................................................... 232 Tab. A 18: Shoots per plant, Rauischholzhausen 2013-2015. Results of the three- factorial analysis. ....................................................................................... 233 Tab. A 19: Biomass yield (FM) [dt FM/ha], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ....................................................... 234 Tab. A 20: Biomass yield (DM) [dt DM/ha], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ....................................................... 235 Tab. A 21: Leaf fresh matter yield [dt FM/ha], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ....................................................... 236 Tab. A 22: DM leaf yield [dt DM/ha], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ........................................................................ 237 Tab. A 23: DM content of the leaves [%], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. .................................................................... 238 Tab. A 24: Leaf:stem ratio, Rauischholzhausen 2013-2015. Results of the three- factorial analysis. ....................................................................................... 239 Tab. A 25: Essential oil content [%], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ........................................................................ 240 Tab. A 26: Total phenolic content [mg GAE/g DM], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ................................... 241 Tab. A 27: Antioxidant capacity [µmol TE/g DM], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. ....................................................... 242 Tab. A 28: Rosmarinic acid content [%], Rauischholzhausen 2013-2015. Results of the three-factorial analysis. .................................................................... 243 Introduction 1 1 Introduction Lemon balm (Melissa officinalis) is, due to its content of secondary metabolites, an important medicinal and aromatic plant in the Lamiaceae family. The typical lemon-like fragrance is caused by its content of essential oil, and makes it an appreciated flavoring ingredient for culinary uses. The dried leaves are also used for the preparation of herbal infusions, but especially important is the wide pharmaceutical use of lemon balm. It is, for instance, used internally for treating tenseness, restlessness and irritability, nervous sleeping disorders, or functional gastrointestinal complaints (Blumenthal et al., 2000, 1998). Externally it is used in the form of extracts, e.g. as the active ingredient in ointments against Herpes labialis (cold sores) (ESCOP, 2003; Koytchev et al., 1999). In Germany, it is licensed as a standard medicinal tea for sleep disorders and disorders of the gastrointestinal tract (BfArM, 2015). The pharmaceutically used parts of the plant are the dried leaves (Melissae folium), and the corresponding requirements, as well as those for the dried extract (Melissae folii extractum siccum), are described in the European Pharmacopoeia (Ph. Eur. 7, 2011). Two classes of secondary metabolites are of special interest in lemon balm: On the one hand the essential oil, a mixture of several mainly terpenoid lipophilic compounds, and on the other hand the phenolic compounds, such as phenolic acids and flavonoids. The main component among the phenolic substances in lemon balm is rosmarinic acid (Weitzel and Petersen, 2010). For meeting the requirements of the European Pharmacopoeia, dried lemon balm leaves have to contain a minimum of 1%, and lemon balm leaf dry extract a minimum of 2% rosmarinic acid (Ph. Eur. 7, 2011). Phenolic substances, mainly rosmarinic acid and flavonoids, also lead to a high antioxidant capacity of lemon balm. In the human body, oxidative stress has been related with the development of several diseases (Aksenov et al., 2001; Cai et al., 2011; Reuter et al., 2010; Sayre et al., 2008). In food products, oxidative processes, especially the oxidation of fatty acids, lead to the deterioration of the product quality, and may even produce substances that are harmful for the health of the customers (Choe and Min, 2006). Therefore, synthetic antioxidants, like butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT), are used as food additives (EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), 2012, 2011). However, many consumers prefer natural antioxidants to synthetic antioxidants. For those reasons, natural antioxidants are of great interest for the food industry, for instance for replacing synthetic antioxidants in meat products (Berasategi et al., 2011; Introduction 2 Falowo et al., 2014; Fernandes et al., 2016; Lara et al., 2011), or even already as a feed supplement to influence the later meat quality (Kasapidou et al., 2014; Marcinčáková et al., 2011). According to FNR (2014), there is a high demand for lemon balm in Germany. However, most of it is imported from other countries, although it can be cultivated under the given climate conditions. Therefore, increasing the cultivation area and productivity to produce high-quality lemon balm in Germany is of great interest. Yield and quality of medicinal and aromatic plants can be influenced by genetic, phenological, and environmental factors, as well as by the cultivation management (Azizi et al., 2009; Mortensen, 2014; Mrlianová et al., 2002; Novak et al., 2000; Sellami et al., 2009). Therefore, it is important for quality assurance to better understand the factors that determine yield and quality of lemon balm. A certain degree of variation between different genotypes in the content of secondary metabolites has been shown in several Lamiaceae plants. In Ocimum basilicum, for instance, the total phenolic content varied fivefold, and rosmarinic acid content even a hundredfold between the tested genotypes (Kwee and Niemeyer, 2011). A variation in the content of phenolic substances was also reported for other plants in the Lamiaceae family (Chizzola et al., 2008; Javanmardi et al., 2003; Kiferle et al., 2011; Lamien-Meda et al., 2010; Müller-Waldeck et al., 2010; Yan et al., 2016). Only few studies on the variation between different lemon balm genotypes are available, especially for plants grown under field conditions. Therefore it is of interest to compare different genotypes of lemon balm regarding their content of secondary metabolites and their antioxidant capacity in a field trial. The development stage of a plant can have an influence on the content of essential oil (Sangwan et al., 2001). However, in investigations on different Lamiaceae plants, differing results regarding the stage with the highest content of essential oil have been reported (Ben Farhat et al., 2016; Mastelić and Jerković, 2003; Mirjalili et al., 2006; Nurzyńska- Wierdak et al., 2017; Tahmasebi et al., 2016). Also for the content of phenolic substances, such differences have been observed (Kiferle et al., 2011; Ozkan et al., 2010; Raudone et al., 2017; Vassão et al., 2006). Thus, further investigations are needed to gather information regarding changes in these quality-determining secondary metabolites in lemon balm. In the cultivation of a diverse range of plants, especially in horticulture, viticulture and the cultivation of medicinal and aromatic plants, the use of different kinds of nets is getting more and more popular. Among others, they are utilized for the protection of plants from Introduction 3 insects, excessive sunlight, or adverse climatic conditions, as well as for the modification of their morphology, quality, and yield characteristics (Ben-Yakir et al., 2012, 2008; Castellano et al., 2008; Oren-Shamir et al., 2001). For the cultivation of medicinal and aromatic plants, protection nets have been proposed for the prevention of leafhopper infestations (Blum et al., 2011; Meyer et al., 2010). Besides, it has been found that the use of agro-textile coverings for the improvement of the microclimate led to higher essential oil contents in lemon balm cultivated under Swiss conditions (Carron et al., 2008). In Brazil, investigations on the influence of differently colored shading nets reported to some extent higher leaf yields of lemon balm plants cultivated under the nets (Brant et al., 2009; Oliveira et al., 2016). However, it remains an unanswered question how lemon balm plants react on shading nets under the temperate climate conditions in Germany. As any kind of covering reduces the light intensity, this might impair the photosynthetic processes and thus the energy supply of the plants, and could therefore possibly have an impact on the yield. At the same time, plants receive far more sunlight than they can use for photosynthesis, even under temperate climate conditions, and can therefore experience light stress and photoinhibition (Long et al., 1994; Wilhelm and Selmar, 2011). Plants protect themselves from these harmful conditions by certain mechanisms, like photorespiration (Peterhansel et al., 2010), or the formation of secondary metabolites, such as phenolic compounds (Grace and Logan, 2000). However, these processes also divert precursors from the primary metabolism, are therefore costly for the plants, and might as a result affect the biomass yield (Gershenzon, 1994; Logemann et al., 2000; Walker et al., 2016; Wilhelm and Selmar, 2011). On the other hand, several secondary metabolites, such as phenolic compounds and essential oil, are the valuable components of medicinal and aromatic plants like lemon balm. Too much solar radiation, however, might also increase the loss of the volatile substances that constitute the essential oil. It is therefore of interest to identify suitable cultivation conditions that lead to an improved leaf yield, without impairing the content of valuable secondary metabolites. Especially the influence of light intensity on phenolic substances, essential oil content and yield parameters in Melissa officinalis cultivated in Germany has not been reported. Therefore, it was the aim of the current study to clarify the effect of shading on yield and quality parameters (like the contents of essential oil, total phenolics, and rosmarinic acid) of lemon balm under field conditions in Germany. Investigations took place in two sites with different soil and climatic conditions, in plant stands observed over a common cultivation period of three years. It was further of interest whether different genotypes react in different ways, and if there are interactions with the development stages of the plants. Introduction 4 Thus, the following questions were addressed in this investigation:  What is the crop yield potential of different lemon balm genotypes at two locations in Germany with different soil and climate conditions?  Does the rosmarinic acid content of the tested genotypes meet the requirements of the European Pharmacopoeia under the chosen cultivation conditions?  Do the tested lemon balm genotypes differ in yield and quality?  How do yield and quality change at different harvest stages?  How are yield and quality parameters of lemon balm plants influenced when cultivated under nets with a moderate or a strong light reduction?  Are there interaction effects between the tested factors genotype, harvest stage, and light reduction? Literature review – Botanical characterization of lemon balm 5 2 Literature review 2.1 Botanical characterization of lemon balm (Melissa officinalis L.) Lemon balm (Melissa officinalis L.) is a perennial plant in the Lamiaceae family, classified in the subfamily Nepetoideae, the tribe Mentheae, and the subtribe Salviinae (Bomme et al., 2013; Moon et al., 2008). Its name is derived from the Greek word μέλι (honey) or μέλισσα (honeybee), as it is said to attract honeybees (Burgett, 1980). It is assumed to be originating from an area between the Mediterranean region and the western Tien Shan (Hanelt and IPK, 2001), but is nowadays widely cultivated not only under subtropical, but also under temperate conditions, as in Germany (Bomme et al., 2013). Wild populations of Melissa officinalis can be found in different habitats, ranging from moist temperate forest regions to dry mountain steppe habitats (Abrahamyan et al., 2015). With an Ellenberg value (Hill et al., 2004) / Ellenberg-Pignatti value (Vitasović Kosić et al., 2017) of 6, lemon balm can therefore be described as a plant of a habitat between semi-shade and well lit places. Three subspecies of Melissa officinalis have been described, namely ssp. officinalis, ssp. altissima, and ssp. inodora (Bomme et al., 2013). However, only ssp. officinalis is the pharmaceutically used lemon balm (Hänsel et al., 1993). Lemon balm has been described as a diploid species, with a chromosome number of 2n = 32 (Bomme et al., 2013). However, recent investigations found further ploidy levels among the tested accessions, namely triploid (2n = 3× = 48 chromosomes) as well as tetraploid (2n = 4× = 64 chromosomes) types (Kittler et al., 2015). Lemon balm is a perennial plant, which is normally cultivated for two to three years, and can typically reach plant heights of about 50-90 cm (Bomme et al., 2013). The stem of the plants is quadrangular, carrying the leaves in decussate position (Fig. 1). The leaves can reach a length of about 5-9 cm, and a width of about 3-6 cm (Bomme et al., 2013). They are petiolate, broadly ovate to almost cordate in the vegetative stage, with crenate or serrate margins, and have a distinct venation on the lower surface, giving them an embossed appearance (Wichtl, 2002). During the further development of the plant, the leaf form changes. Flowering shoots show leaves with pointed ends, and a reduced leaf size (Bomme et al., 2013). The surface of the leaves is more or less pubescent, with hairs being visible mainly on the adaxial side of the leaves, and only to a lesser extent on the abaxial side (Wichtl, 2002). Both glandular (essential oil producing) as well as non- glandular trichomes have been described for lemon balm (Chwil et al., 2016; Moon et al., 2009). Literature review – Botanical characterization of lemon balm 6 The flowers, which are of pale color, have a two-lobed calyx, and are grouped in the axils of the leaves (Wichtl, 2002) (Fig. 2). The bicarpellate, superior ovary with a false septum and two ovules per carpel develops into four nutlets of around 1.5-2 mm, with an average thousand seed weight of around 0.6 g (Bomme et al., 2013). For germination and a fast development of the seedling, a minimum temperature of 18 °C is required, and optimal growth temperature for the plant is 20-30 °C (Bomme et al., 2013). The aerial parts of the plants die back in winter, and new shoots will regrow from the roots in spring (Bomme et al., 2013). While the root system is quite frost tolerant, late spring frost can severely damage the plants once the regrowth of the new shoots has started (Bomme et al., 2013). Among the different genotypes, two main growth types can be distinguished: An upright growth type, with erect plants already in the first year, and a procumbent growth type, with the plants growing along the ground in the first year. However, also intermediate types exist. All types will have an upright growth in the second year or after vernalization (Bomme et al., 2013). Fig. 1: Lemon balm plant in vegetative stage. The typical quadrangular stem carries the leaves in decussate position (own photo). Fig. 2: Flowering lemon balm plant. The pale-colored flowers are grouped in the axils of the leaves (own photo). Literature review – Cultivation of lemon balm 7 2.2 Cultivation of lemon balm Lemon balm needs adequate soil conditions, and an appropriate supply with water and nutrients, especially nitrogen, because of the high biomass production (Bomme et al., 2013). An appropriate soil should consist of sandy and loamy fractions, be rich in humus, and warm up easily. It is important that no waterlogging occurs. A pH value of the soil between 5 and 7 is recommended (Bomme et al., 2013). Because of the origin in the Mediterranean region, lemon balm prefers warmer temperatures, but can also be grown in a temperate climate. However, severe winter losses may occur, especially in the case of a lacking snow cover. Lemon balm plants seem to be especially prone to winter killing in the first winter, and different genotypes seem to exhibit differing degrees of frost tolerance. Thus, especially in regions with low winter temperatures, the choice of proper genotypes is important (Bomme, 2001). Before the establishment of the crop, the soil needs to be loosened up appropriately. Especially ploughing may also help to reduce weed pressure. Cultivators and harrows may be employed to reach a finer soil texture. A fine-crumbled soil is especially important for direct sowing of lemon balm. The usage of harrows is recommended as a means of preventive weed control (Bomme, 2001). An appropriate weed control is of great importance to reach a good quality of the harvested plant material. Plant stands of lemon balm are normally cultivated for two to three years (Bomme et al., 2013). Included in a crop rotation, lemon balm can therefore prevent soil erosion and leaching of nutrients. Suitable pre-crops are legumes, potatoes, or cereals. As a subsequent crop, cereals are recommended, which facilitate the control of possible volunteers by herbicides. No plants from the Lamiaceae family should be cultivated before or after lemon balm for four to five years to prevent the spreading of diseases or pests (Bomme, 2001). A lemon balm plant stand may be established either by direct sowing or by planting of young plantlets. If direct sowing is chosen, a good seedbed preparation is essential. The seeds are quite fine, with a thousand grain weight of around 0.6 g. They must therefore not be placed deeper than 0.5 cm in the ground. To ensure a good germination of the seeds, direct sowing has to be performed when air and especially soil temperature reach at least 18 °C, which means not earlier than May or June in a temperate climate. However, drying out of the germinating seeds has to be prevented. Heavy rainfall in this period can negatively affect the success of direct sowing (Bomme et al., 2013). Because of the difficulties with direct sowing, lemon balm is normally sown under greenhouse conditions. Alternatively to sowing, the use of cuttings may be performed as a means of Literature review – Cultivation of lemon balm 8 plant propagation in the greenhouse. The pre-grown plantlets will later be transferred to the field, with a density of 64.000-80.000 plants/ha (Bomme, 2001). Although lemon balm plants can withstand a certain degree of drought stress, an appropriate water supply is important to reach good yields (Bomme et al., 2013). Irrigation is of special importance after planting the young plantlets to the field to ensure taking root of the plants, after each cut to help the plants to regrow quickly, as well as generally in the case of longer dry periods (Bomme, 2001). Due to the high biomass production, an appropriate nutrient supply is essential. The amount of fertilizer should be based on the harvested biomass as well as an analysis of the soil. The nutrient removal for 100 dt fresh matter biomass has been estimated to be 49 kg N, 14 kg P2O5, 76 kg K2O, 9 kg MgO, and 19 kg CaO (Bomme, 2001). Especially the application of N fertilizer should be staggered over the growing season. The first dose should be applied as a basal dressing at the start of the growth phase in spring. After each harvest, an additional dose of N fertilizer is needed. Although an application of organic fertilizer during the cultivation of the pre-crop is regarded as positive for lemon balm, no farmyard manure or liquid manure should be applied on lemon balm due to its bioburden (Bomme, 2001). During the cultivation of lemon balm, an appropriate crop protection has to be kept in mind. If chemical plant protection agents are used, however, this needs to be cleared with the purchaser of the produced plant material. Weed control should already be started in the pre-crop. Also before sowing or planting, as well as during the growing period, weed should be controlled mechanically or by the use of appropriate, approved herbicides. However, lemon balm may react quite sensitively on the use of herbicides. Among fungal diseases, Septoria melissae is quite meaningful, furthermore Puccinia menthae and Neoerysiphe galeopsidis might occur. A typical insect pest are leafhoppers, mainly Eupteryx sp., that damage the leaves by sucking the plant sap and therefore lead to a deterioration of the quality of the harvested plant material (Bomme et al., 2013). Lemon balm can be harvested several times a year. In the establishing year, up to two harvest cuts may be possible, whereas in the following years two to four cuts might be achieved. Typically, harvest takes place before flowering. If the cutting of the plants is performed too late, the lower leaves start yellowing, therefore decreasing the quality of the product, or might even fall off completely. Additionally, the leaf:stem ratio changes disadvantageously. Cutting height should be around 10 cm above ground, as regrowth might be impaired if cutting is performed too low. If the harvest takes place in a dry period, adequate watering is necessary for a successful regrowth. The last cut must not be Literature review – Cultivation of lemon balm 9 performed too late in the year, as winter hardiness would be reduced. The time frame for harvest is around mid July to end of August as well as September for the two cuts in the first year, and in the following years end of May to beginning of June, mid July, as well as August. Harvesting can be performed with combine harvesters or mowers. Plants are then chopped as fresh plant material after harvest and then undergo a winnowing process to separate the leaves from the stems, and only the leaves are dried. An alternative is to dry the whole harvested plants, and to cut and winnow them after drying. Whichever of the two methods is chosen, a fast but gentle drying (at not more than 40 °C) of the plant material to a target moisture content of 8 to 10% is important to reach a good quality of the final product (Bomme, 2001; Bomme et al., 2013). Literature review – Yield characteristics of lemon balm 10 2.3 Yield characteristics of lemon balm For field trials under German climate conditions, biomass yields of 160-350 dt FM (fresh matter)/ha in the first year and 180-450 dt FM/ha for the following years have been described, and leaf yields reached 90-230 dt FM/ha (19-40 dt/ha dried leaves) in the first year and 100-250 dt FM/ha (20-45 dt/ha dried leaves) in the following years (Bomme et al., 2013). In investigations under Polish climate conditions, biomass yields of 93 dt FM/ha in the first year and 73 dt FM/ha in the second year were described, with a yield of air-dried leaves of 16 dt/ha and 14 dt/ha, respectively (Dzida et al., 2015). In another Polish study, biomass yields at four organic and two conventional farms were investigated over three consecutive years. Averaged annual biomass yields were stated as 44.9 to 241.6 dt FM/ha (9.3 to 55.1 dt/ha dried biomass) at the organic farms, and 81.9 to 149.6 dt FM/ha (29.1 to 13.8 dt/ha dried biomass) at the conventional farms (Seidler- Łożykowska et al., 2015). In a Romanian study with six different lemon balm accessions, biomass yields of 40.4 to 107.7 dt FM/ha in the first year, 216.2 to 288.6 dt FM/ha in the second year, as well as 527.9 to 611.4 dt FM/ha in the third year have been obtained (Marian, 2012). In an investigation under Slovak climate conditions, a biomass yield of 55.6 dt/ha dried plant material as well as a leaf yield of 21.1 dt/ha dried plant material was found in a three-year-old plant stand (Mrlianová et al., 2002). Under the climate conditions of Northern India, biomass yields of 149.0 dt FM/ha (32.8 dt/ha dried plant material) were obtained 180 days after planting (Singh et al., 2014). In a Turkish investigation, yields of 48.2 and 75.8 dt/ha dried biomass as well as 30.0 and 46.0 dt/ha dried leaves were obtained in the first and second year, respectively (Saglam et al., 2004). Biomass and dry matter (DM) leaf yield of the first cut have been described to be higher than for the second cut (Bomme et al., 2013; Özgüven et al., 1999). In investigations of Özgüven et al. (1999) on the yield parameters of different lemon balm accessions with two to three harvests per year, the highest biomass and leaf yields were almost always found for the first harvest of the year. Under German conditions, up to three cuts within a year have been described, with a tendency of decreasing leaf yields from harvest to harvest (Bomme, 2001). The percentage of leaves from the harvested biomass has been described as 44-52% (equalling a leaf:stem ratio of 0.79-1.08) for the first cut, and up to 68% (equalling a leaf:stem ratio of 2.13) for the second cut (Bomme et al., 2013). Literature review – Requirements of the European Pharmacopoeia 11 2.4 Requirements of the European Pharmacopoeia In the European Pharmacopoeia (Ph. Eur. 7, 2011), the drug Melissae folium (lemon balm leaves) is described as the dried leaves of Melissa officinalis. A minimal rosmarinic acid content of 1.0% in the dried drug is requested. The drug needs to have a lemon-like odor. However, no requirements for a minimal content of essential oil are described. Testing for identity has to take place macroscopically, microscopically, and with thin layer chromatography, with citronellal and citral (consisting of neral and geranial) as reference substances. The requirements regarding drug purity are a maximum of 10% stalks with a diameter above 1 mm, and a maximum of 2% impurities. Loss on drying must not exceed 10%, and ash content may reach at most 12%. The determination of rosmarinic acid content needs to be performed with an HPLC (high-performance liquid chromatography) method. In the past, a photometric had been used, with a minimal requirement of 4% hydroxycinnamic acids, calculated as rosmarinic acid (Krüger et al., 2010), which has to be kept in mind when comparing rosmarinic acid contents obtained with these different methods. Literature review – Secondary metabolites of lemon balm 12 2.5 Secondary metabolites of lemon balm The term "secondary metabolites" covers a wide variety of different substances, among others phenolics, terpenes, glucosinolates, and alkaloids (Bennett and Wallsgrove, 1994). More than 200,000 of these structures have been found (Bresinsky et al., 2013). They play important roles in plants, as they enable them to adapt to adverse environmental conditions (Behnke et al., 2007; Delfine et al., 2000; Loreto et al., 1998; Oh et al., 2009; Ramakrishna and Ravishankar, 2011; Sharkey et al., 2001), protect them from herbivores or pathogens (Bennett and Wallsgrove, 1994; Bleeker et al., 2009; De Moraes et al., 2001; Kang et al., 2010; Kessler and Baldwin, 2001; Ramakrishna and Ravishankar, 2011) as well as from ozone (Jud et al., 2016; S. Li et al., 2018), and attract pollinators (Harborne, 2001) as well as the predators of herbivores attacking the plants (Dicke et al., 1990; Turlings et al., 1995, 1990), thus even exerting an indirect herbivore defense. The secondary metabolism by which they are synthesized is connected with the primary metabolism, from which it diverts its respective precursors (Logemann et al., 2000). As a result, their production leads to a certain cost for the plants (Gershenzon, 1994; Sharkey and Yeh, 2001). While vascular plants share similarities in their primary metabolism, they differ in the profile of secondary metabolites. Therefore, secondary metabolites are also used as chemotaxonomic markers (Bennett and Wallsgrove, 1994; Bourgaud et al., 2001). Three groups of secondary metabolites have been described in lemon balm (Awad et al., 2009; Hänsel et al., 1993): 1. Phenolic compounds, like flavonoids and phenolic acids 2. Essential oil, mainly consisting of mono- and sesquiterpenes 3. Pentacyclic triterpenoids (ursolic acid, oleanolic acid) The first two groups will be further described in the following, whereas the pentacyclic triterpenoids, which have not been analyzed in this project, are mentioned only for the sake of completeness. Literature review – Secondary metabolites of lemon balm 13 2.5.1 Phenolic compounds Phenolic compounds are different substances that derive their name from phenol (Fig. 3), a molecule with an aromatic ring and a hydroxy group, which they share as a strucural element within their molecule. The term "polyphenol" is commonly used for substances with more than one phenolic ring as part of their molecule (Cheynier et al., 2013). Fig. 3: Structural formula of phenol (according to Schirmeister et al., 2016). Phenolic substances share this structure as a part of their molecules. Among the phenolic compounds in lemon balm, rosmarinic acid (RA) is accumulated in the highest quantities (Weitzel and Petersen, 2010). The RA content of lemon balm typically reaches several percent in the dried plant material (Tab. 1). Besides RA, further phenolic acids have been found in lemon balm, like caffeic, protocatechuic, chlorogenic, m-coumaric, p-coumaric, gallic, gentisic, ferulic, and p-hydroxybenzoic acid (Dastmalchi et al., 2008; Lin et al., 2012; Proestos et al., 2005; Žiaková et al., 2003). Several flavonoids are contained in lemon balm, especially luteolin glycosides (Patora and Klimek, 2002), among which luteolin 3'-O-β-D-glucuronide has been described as the main flavonoid in this plant (Heitz et al., 2000). RA (Fig. 4) is a phenolic substance that is typically found in members of the subfamily Nepetoideae within the Lamiaceae family (Janicsák et al., 1999), and therefore also in lemon balm. It is an ester consisting of caffeic acid and 3,4-dihydroxyphenyllactic acid (Petersen and Simmonds, 2003). It is stored in the vacuoles of the cells (Häusler et al., 1993). RA is regarded an important active constituent of lemon balm. The European Pharmacopoeia therefore requires a minimal RA content of 1.0% in the dried drug (Ph. Eur. 7, 2011). Fig. 4: Structural formula of rosmarinic acid (according to Petersen and Simmonds, 2003). Chemically, it is regarded as an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. Literature review – Secondary metabolites of lemon balm 14 Tab. 1: Rosmarinic acid (RA) contents of lemon balm (Melissa officinalis) from literature. RA [%] ( * ) Treatment Genotype Extraction Experiment type Literature 4.1% – Unknown Hot water (herbal tea) Polyphenols in lemon balm tea (Carnat et al., 1998) 3.24% – Unknown Hot water Analysis of lemon balm (Ieri et al., 2017) 0.24 – 2.31% – Unknown Hot water (herbal tea) Bulgarian commercial samples (Petkova et al., 2017) 2.74% – Unknown Aqueous ethanol HPLC method development (Wang et al., 2004) 3.7% a Convective drying 'Citronella' Aqueous ethanol Different drying methods (plant material from organic farm) (Argyropoulos and Müller, 2014) 5.6% b Vacuum drying 5.7% b Freeze drying 6.63% a With arbuscular mycorrhiza 'Relax' Acidified aqueous methanol Effect of arbuscular mycorrhiza (pot experiment) (Engel et al., 2016) 5.97% b Without arbuscular mycorrhiza 3.65% – Unknown Acidified aqueous methanol/ 2-propanol RA contents in several Iranian Lamiaceae species (Shekarchi et al., 2012) 3.50% a Before flowering 'Citra' Methanol Development stage (field experiment) (Tóth, et al., 2003) 3.91% a Full flowering 3.12% a Control 'Soroksár' Methanol Water deficit (pot experiment; growth chamber) (Radácsi et al., 2016) 3.59% a Stepwise water deficit 3.83% a Sharply increased water deficit 3.17% a Permanent water deficit 2.87% ab 'Gold Leaf' Methanol Genotype comparison (pot experiment) (Szabó et al., 2016) 2.43% a 'Lemona' 3.01% ab 'Lorelei' 2.72% b 'Quedlinburger Niederliegende' 2.75% b 'Soroksár' (*) Values partly converted into % from differently stated units in the original articles. Different letters indicate significant differences between the treatments within the original experiment. Literature review – Secondary metabolites of lemon balm 15 RA is known for its antioxidant properties (Adomako-Bonsu et al., 2017; Fadel et al., 2011; Nakamura et al., 1998; Soobrattee et al., 2005). The ortho-dihydroxy structure has been suggested to be especially important for the antioxidant activity of RA (Adomako-Bonsu et al., 2017; Woo and Piao, 2004). The antioxidant properties make plant extracts rich in rosmarinic acid interesting for the food industry, as they can inhibit lipid oxidation and therefore extend the shelf life of food products (de Ciriano et al., 2010; Şahin et al., 2017; Sánchez-Escalante et al., 2003). RA is able to be incorporated into lipid membranes, where it can exert its antioxidant potential (Fadel et al., 2011). Several of the health-related properties of lemon balm can be ascribed to its content of RA. Several effects of RA have been demonstrated, for instance, its antiviral activity. In an in vitro assay, RA showed a virucidal activity on herpes simplex virus type 1 (HSV-1) (Astani et al., 2012). In a cell model, lemon balm extract and RA inhibited the attachment of HSV-1 to host cells as well as their penetration (Astani et al., 2014, 2012). Human immunodeficiency virus type 1 (HIV­1) integrase, an enzyme that is essential for the replication of the virus, was inhibited in vitro by RA (Tewtrakul et al., 2003). Antiviral and anti-inflammatory effects of RA were also shown in an experimental murine model of Japanese encephalitis, where a reduced viral replication was observed (Swarup et al., 2007). The antiviral effects of a methanolic lemon balm extract on enterovirus 71 in vitro and in a mouse model were also attributed to rosmarinic acid (Chen et al., 2017). As a practical application, the antiviral activity of lemon balm extracts is used in the form of a cream against Herpes labialis (Koytchev et al., 1999). Neuroprotective effects of RA were shown in in vitro models of neuronal death (Fallarini et al., 2009). Human dopaminergic neuronal cells were protected by RA under oxidative stress conditions (H. J. Lee et al., 2008). In an in vitro study, RA protected cells from amyloid-β peptide-induced neurotoxicity (Iuvone et al., 2006). In a transgenic mouse model for Alzheimer's disease, amyloid-β deposition in the brain was significantly decreased by RA (Hamaguchi et al., 2009). Antiinflammatory activities of RA were shown in several studies. In an in vitro investigation with rat platelets and polymorphonuclear leukocytes, RA inhibited the activities of 12-lipoxygenase and 5-lipoxygenase (Yamamoto et al., 1998). The release of HMGB1 was inhibited in primary human umbilical vein endothelial cells, and the inflammatory responses dependent on HMGB1 were down-regulated (Yang et al., 2013). In the mouse ear edema model, antiinflammatory activities of RA, such as inhibition of adhesion molecule, chemokine and eicosanoid synthesis, were observed (Osakabe et al., 2004). Antiinflammatory effects were also shown in rat models of local and systemic inflammation (Rocha et al., 2015), as well as in a mouse model infected with Japanese Literature review – Secondary metabolites of lemon balm 16 encephalitis virus (Swarup et al., 2007). In a mouse cecal ligation and puncture model, the release of HMGB1 was markedly decreased, as was the sepsis-related mortality (Yang et al., 2013). An antiinflammatory effect was also observed in lipopolysaccharide-induced mastitis in mice (Jiang et al., 2017). Also in a human study, antiinflammatory effects of RA were observed. In persons suffering from seasonal allergic rhinoconjunctivitis, several symptoms were improved after oral supplementation with RA, and the numbers of neutrophils and eosinophils in nasal lavage fluid were significantly decreased (Osakabe et al., 2004). Because of its antiinflammatory properties, RA has also been proposed for the treatment of atopic dermatitis (J. Lee et al., 2008). Antiangiogenic properties of RA were shown in an in vitro model with human umbilical vein endothelial cells, where RA inhibited several steps that are important for angiogenesis (Huang and Zheng, 2006), as well as in a human retinal endothelial cell model, and in a mouse model of retinopathy (Kim et al., 2009). In studies with mouse models, antimutagenic effects of RA were shown (De Oliveira et al., 2012; Furtado et al., 2008). Nephroprotective properties have been attributed to RA according to investigations in cell and mouse models (Domitrović et al., 2014; Makino et al., 2000). Studies on cell, mouse, and rat models showed hepatoprotective properties of RA (Domitrović et al., 2013; Li et al., 2010; Osakabe et al., 2002). RA may also exert a photoprotective activity, as it increased the melanin content and tyrosinase expression in a murine cell model (Lee et al., 2007), and protected human keratinocytes from the harmful effects of UV-A radiation (Psotova et al., 2006). The biosynthesis of phenolic compounds is based on precursors from the primary metabolism. In the shikimate pathway, the primary metabolites phosphoenolpyruvate (PEP) and erythrose-4-phosphate are used for the synthesis of chorismate (Herrmann, 1995), which is further metabolized to arogenate, the precursor for the formation of the aromatic amino acids L-phenylalanine and L-tyrosine (Schmid and Amrhein, 1995). These two aromatic amino acids are needed for the formation of rosmarinic acid (Fig. 5), as well as for other phenolic substances (Weitzel and Petersen, 2010). The first step of the phenylpropanoid pathway starts with the transformation of L-phenylalanine by the enzyme phenylalanine ammonia lyase (PAL; EC 4.3.1.24) into t-cinnamic acid, which is then further hydroxylated to 4-coumaric acid by the enzyme t-cinnamic acid 4-hydroxylase (C4H; EC 1.14.13.11). In the following step, an activation is accomplished by the enzyme 4-coumarate-CoA ligase (4CL; EC 6.2.1.12) to form 4-coumaroyl-CoA, an intermediate that is a precursor not only for rosmarinic acid, but also for other phenolic substances, like flavonoids. For the biosynthesis of rosmarinic acid, in a parallel biosynthetic route L-tyrosine is converted to 4-hydroxyphenylpyruvic acid by Literature review – Secondary metabolites of lemon balm 17 tyrosine aminotransferase (TAT; EC 2.6.1.5), and then to 4-hydroxyphenyllactic acid by hydroxyphenylpyruvate reductase (HPPR; EC 1.1.1.237). By the enzyme rosmarinic acid synthase (RAS; EC 2.3.1.140), an ester is formed between the two precursors 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid, resulting in 4-coumaroyl- 4'-hydroxyphenyllactic acid. In the following enzymatically catalyzed steps, hydroxylation at positions 3 and 3' occurs to ultimately form rosmarinic acid (Fig. 5) (Weitzel and Petersen, 2010). Fig. 5: Biosynthetic pathway of rosmarinic acid in Melissa officinalis (Weitzel and Petersen, 2010). PAL, phenylalanine ammonia-lyase; C4H, cinnamic acid 4-hydroxylase; 4CL, 4-coumarate:coenzyme A ligase; TAT, tyrosine aminotransferase; HPPR, hydroxyphenylpyruvate reductase; RAS, rosmarinic acid synthase, hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase; 4C-pHPL 3H, 4C-pHPL 3'H, 4-coumaroyl-4'-hydroxyphenyllactate 3/3'-hydroxylases; Caf-pHPL 3'H, caffeoyl-4'-hydroxyphenyllactate 3'-hydroxylase; 4C-DHPL 3H, 4-coumaroyl-3', 4'-dihydroxyphenyllactate 3-hydroxylase. Literature review – Secondary metabolites of lemon balm 18 2.5.2 Essential oil The International Organization for Standardization (ISO) describes essential oils (EO) as a "product obtained from a natural raw material (...) of plant origin, by steam distillation, by mechanical processes from the epicarp of citrus fruits, or by dry distillation, after separation of the aqueous phase - if any - by physical processes" (ISO, 2013). Essential oils are therefore not single substances, but rather complex mixtures of volatile lipophilic compounds, consisting mainly of terpenoid substances (especially mono- and sesquiterpenes), but may also contain phenylpropanoids, alkanes, alcohols, ketones, or even sulfur- or nitrogen-containing substances (Grassmann and Elstner, 2003). Because of their volatility, they exert a characteristic odor. Several plants are appreciated by humans because of their typical aroma which is caused by their content of essential oil. Some of them are used mainly as herbs and spices, like rosemary, oregano, or thyme, and some are also consumed in the form of herbal teas, like peppermint or lemon balm. Despite its distinct lemon-like fragrance, lemon balm contains quite low levels of essential oil. In Polish investigations, EO contents of 0.12 and 0.19% (Kowalska et al., 2014), 0.20% and 0.21% (Dzida et al., 2015), 0.22-0.28% (Politycka and Seidler-Łożykowska, 2009), 0.08-0.22% EO (Patora et al., 2003), 0.115-0.15% (Seidler-Łożykowska et al., 2015) as well as 0.3% (Nurzyńska-Wierdak et al., 2014) were presented. The screening of 22 accessions cultivated under Polish climate conditions resulted in EO contents of 0.05% to 0.44% (Seidler-Łożykowska et al., 2013). An investigation in Slovakia presented EO contents of 0.14% in lemon balm leaves, being higher in the upper leaves of the plant, with 0.39% (Mrlianová et al., 2002). For lemon balm cultivated in Iran, 0.10% to 0.26% EO, depending on the nitrogen fertilization (Abbaszadeh et al., 2009b), 0.2% (Mohamadpoor et al., 2018), as well as 0.13% to 0.35% in a collection of different accessions (Pouyanfar et al., 2018) were found. In Algerian lemon balm leaves, EO contents of 0.17% (Feknous et al., 2014) and 0.34% (Abdellatif et al., 2014) were determined. 0.3-0.4% EO have been found in Tajikistan (Sharopov et al., 2013). EO contents of lemon balm in Spain were investigated depending on the leaf position, with 0.21% for middle-basal leaves and 0.32% for terminal leaves for a harvest in August, and 0.23% as well as 0.33% for a harvest in November, respectively (Adzet et al., 1992a). In a breeding program under the climatic conditions of the Ebro Delta in Spain, EO contents of 0.28-0.31% were found in the beginning, which were then increased to 0.68-0.80% in the fifth year (Adzet et al., 1992b). The EO content of lemon balm cultivated under Turkish climate conditions varied from 0.23 to 0.45%, depending on development stage and year (Avci and Giachino, 2016), and different accessions gave EO yields varying in a range Literature review – Secondary metabolites of lemon balm 19 from 0.03% to 0.47% in another Turkish investigation (Özgüven et al., 1999). In northwestern Turkey, EO contents of 0.20-0.28% were determined at the beginning of flowering (Saglam et al., 2004). In a field experiment in Brazil, an EO content of 0.11% was found for lemon balm cultivated under monocropping conditions, which was increased to 0.22% by intercropping with yarrow (Silva et al., 2018). In a commercial lemon balm sample from France, harvested in the stage just before flowering, an EO content of 0.32% was found in the dried leaves (Carnat et al., 1998). In Egypt, EO contents of 0.18%-0.42% were found, depending on row spacing, which was further increased by the application of active dry yeast to up to 0.64% (Rashed, 2012). A number of essential oil containing plants are used as medicinal plants, and at least parts of their medical properties are connected to their content of essential oil. Also in the case of lemon balm, several health related effects have been ascribed to the essential oil or its components. For instance, the essential oil of lemon balm has been shown to exert an antiviral action in vitro studies, where it inhibited the replication and infectivity of Herpes simplex virus type 1 and type 2 (Allahverdiyev et al., 2004; Schnitzler et al., 2008), or the replication of avian influenza virus subtype H9N2 (Pourghanbari et al., 2016). In in vitro investigations, the essential oil of lemon balm showed an acetylcholinesterase inhibitory activitiy (Ferreira et al., 2006; Perry et al., 1996). A spasmolytic activity of lemon balm essential oil as well as its main component citral was observed in an investigation on rat ileum (Sadraei et al., 2003). Antiparasitic (Mikus et al., 2000), or anti-diabetic effects (Chung et al., 2010) have been described as well. In a study with patients suffering from severe dementia, positive effects of aromatherapy with lemon balm essential oil could be seen regarding agitation of the patients (Ballard et al., 2002). The essential oil of lemon balm has been described to contain as the major compounds (cf. Fig. 6) the monoterpene aldehydes geranial (= citral a) and neral (= citral b), often summed up and stated together as "citral", as well as citronellal, furthermore the sesquiterpene β-caryophyllene and its oxidized product caryophyllen oxide, and finally several components mostly found in minor concentrations, like citronellol, linalool, geraniol, germacrene D, geranyl acetate, β-ocimene, and others (Carnat et al., 1998; Hollá et al., 1997; Kitzler, 2008; Sari and Ceylan, 2002; Seidler-Łożykowska et al., 2013; Tittel et al., 1982; Weitzel, 2009). Literature review – Secondary metabolites of lemon balm 20 geranial (citral a) neral (citral b) citronellal β­caryophyllene Fig. 6: Structural formulas of major compounds in lemon balm essential oil (according to Adams, 2007). Geranial, neral, and citronellal are monoterpenes, β­caryophyllene is a sesquiterpene. Structurally, terpenes consist of isoprene units, a molecule with five carbon atoms (C5). Biochemically, however, they are biosynthesized from the C5 units isopentenyl pyrophosphate (IPP), sometimes called "activated isoprene", and its isomer dimethylallyl pyrophosphate (DMAPP). Two biosynthetic pathways for IPP are known in plants (Bresinsky et al., 2013; Vranová et al., 2013): 1. The classical, cytoplasmatic mevalonate pathway (also called the acetate-mevalonate pathway) 2. The relatively recently discovered non-mevalonate pathway, also called MEP pathway (MEP = 2-C-methyl-D-erythritol 4-phosphate), or 1-deoxy-D-xylulose 5-phosphate pathway (= DOXP pathway), taking place in the plastids The biosynthesis of IPP in either of the two pathways is closely related to the primary metabolism, from which its precursors are taken. The mevalonate pathway (Fig. 7, left- hand side) starts with three molecules of acetyl-CoA, a key metabolite of the primary metabolism. Two molecules of acetyl-CoA are joined to form acetoacetyl-CoA, out of which 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) is produced by addition of another acetyl-CoA molecule. In the next step, HMG-CoA is enzymatically reduced to form mevalonate, the molecule that gave this pathway its name. Mevalonate is then phosphorylated in two steps to mevalonate-5-phosphate. After an ATP-dependent decarboxylation, IPP is formed. Literature review – Secondary metabolites of lemon balm 21 Fig. 7: Schematic, simplified illustration of isopentenyl pyrophosphate (IPP) biosynthesis in plants (modified from Vranová et al., 2013). Left-hand side: Cytoplasmic mevalonate pathway, starting with three Acetyl-CoA molecules from primary metabolism. Right-hand side: Plastidial non mevalonate pathway, starting with D-glyceraldehyde 3-phosphate and pyruvate from primary metabolism. Also the non-mevalonate pathway (Fig. 7, right-hand side) starts with precursors from the primary metabolism, namely the C3 units D-glyceraldehyde 3-phosphate and pyruvate. In a condensation reaction and after the release of CO2, the C5 unit 1-deoxy-D-xylulose 5-phosphate (DOXP) is produced, which is then reduced and rearranged to form 2-C-Methyl-D-erythritol 4-phosphate (MEP). After several additional enzymatically catalyzed steps, IPP is formed (Vranová et al., 2013). Mevalonate pathway 3 x Acetyl-CoA 3-hydroxy-3-methyl-glutaryl-CoA Mevalonate Mevalonate-5-phosphate Mevalonate-5-diphosphate Isopentenyl pyrophosphate (IPP) ATP ADP + Pi + CO2 HS-CoA 2 NADPH + 2 H + 2 NADPH ATP ADP ATP ADP Non-mevalonate pathway D-Glyceraldehyde 3-phosphate 1-Deoxy-D-xylulose 5-phosphate 2-C-Methyl-D-erythritol 4-phosphate (MEP) Isopentenyl pyrophosphate (IPP) Pyruvate CO2 NADPH + H + NADPH Literature review – Secondary metabolites of lemon balm 22 Fig. 8: Biosynthesis of terpenoids, starting from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) (modified from Bresinsky et al., 2013). The precursors for mono-, sesqui- and diterpenes are formed stepwise. IPP can enzymatically be converted into its isomer DMAPP by isopentenyl diphosphate isomerase, also called IPP isomerase (EC 5.3.3.2) (Berthelot et al., 2012). Both IPP and DMAPP are needed as precursors for the formation of mono-, sesqui-, and diterpenes (Fig. 8), as well as for terpenes of higher order, like tri- or tetraterpenes (not shown). In a first biosynthetic step, DMAPP and IPP undergo a condensation reaction, to form geranyl pyrophosphate (GPP), a C10 unit. GPP is the precursor for the formation of monoterpenes (C10). After the addition of another IPP unit, farnesyl pyrophosphate (FPP) is produced, the precursor for the formation of sesquiterpenes (C15). By the addition of another C5 unit in the form of IPP, geranylgeranyl pyrophosphate (GGPP) is synthesized, the precursor for the formation of several C20 molecules, the diterpenes (Bresinsky et al., 2013). Literature review – Antioxidants 23 2.6 Antioxidants Reactive oxygen species (ROS) are oxygen containing chemical species that are more reactive than O2 (Halliwell, 2015). Among others, these include superoxide anions (O2 •-), hydrogen peroxide (H2O2), peroxyl radicals (ROO•), hydroxyl radicals (HO•), singlet oxygen (1O2), and peroxynitrite (ONOO-) (Huang et al., 2005). Some ROS, like O2 •-, ROO•, and HO•, are free radicals, meaning that they contain one or more unpaired electrons, contributing to their reactive behaviour (Halliwell, 2015). Similar to ROS, also reactive nitrogen species (RNS) exist, like the free radical nitric oxide (NO•) (Valko et al., 2007). Antioxidants act as antagonists of ROS. Plants use enzymatic and non-enzymatic ways to protect themselves from oxidative stress, and enzymatic and non-enzymatic antioxidants partially work together synergistically (Gill and Tuteja, 2010). Antioxidant enzymes include, for instance, superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), glutathione S-transferase (GST, EC 2.5.1.18), as well as glutathione reductase (GR, EC 1.8.1.7), whereas glutathione (GSH), ascorbic acid (vitamin C), carotenoids, tocopherols (vitamin E), flavonoids and other phenolic substances are examples for non-enzymatic antioxidants (Gill and Tuteja, 2010; Verma and Dubey, 2003). Phenolic substances can act as antioxidants in different ways. One mechanism is the chelation of prooxidant ions of transition metals like iron and copper, and thus the inhibition of the Fenton reaction in which the aggressive radical HO• is generated from O2 -• and H2O2 (Quideau et al., 2011). The catechol or galloyl groups (two hydroxy-groups in ortho-position or three hydroxy groups in neighbouring positions, respectively) are responsible for the chelating activity of phenolic substances (Andjelković et al., 2006). Other mechanisms involve the inhibition of superoxide radical producing enzymes, like xanthine oxidase (Cos et al., 1998), or the regeneration of other important antioxidants, like α-tocopherol (Pazos et al., 2007). The most investigated effect, however, is the direct scavenging of ROS or free radicals (Leopoldini et al., 2011; Quideau et al., 2011; Wright et al., 2001). Generally, it has been stated that signs for a good radical scavenging activity of phenolic substances are multiple hydroxy groups at the aromatic ring, an ortho- dihydroxy position of these hydroxy groups, the planar structure allowing conjugation, electron delocalization, and resonance effects, as well as further functional groups, like carbonyl groups or carbon-carbon double bonds (Leopoldini et al., 2011). Literature review – Antioxidants 24 Two mechanisms by which phenolic substances exert their antioxidant activity are hydrogen atom transfer (HAT) and single electron transfer (ET) (Leopoldini et al., 2011; Wright et al., 2001). HAT involves the donation of a hydrogen atom from the phenolic group (ArOH) to a free radical R• (Leopoldini et al., 2011): ArOH + R• → ArOH• + RH The phenolic substance thus becomes itself a radical (ArOH•, phenoxy radical; Fig. 9). However, depending on the number and positions of hydroxy groups, these phenoxy radicals can be quite stable due to a delocalization of electrons within their molecular structure (Leopoldini et al., 2011, 2004; Rice-Evans et al., 1996). Fig. 9: Phenoxy radical, resonance-stabilized (modified from Quideau et al., 2011). The HAT mechanism plays an important role for the inhibition of lipid peroxidation. The phenolic function is able to donate a hydrogen atom to a lipid peroxy radical LOO•, and thus to inhibit the chain reaction of lipid peroxidation (chain-breaking antioxidant) (Quideau et al., 2011). It has been suggested that RA exerts its antioxidant activity through a H-abstraction reaction, and the forming free radical to be stabilized by a semiquinone or quinone structure (Cao et al., 2005; Fujimoto and Masuda, 2012). The ET mechanism involves the donation of an electron to a radical (Leopoldini et al., 2011): ArOH + R• → ArOH+• + R- The anion R- is energetically more stable than the former free radical, as it now contains an even number of electrons. Also the radical cation formed from the antioxidant is relatively stable (Leopoldini et al., 2011; Quideau et al., 2011). In the human organism, several metabolic pathways lead to the production of free radicals, like ROS and RNS. These processes are not per se detrimental, as ROS and Literature review – Antioxidants 25 RNS play significant physiological roles, for instance in signal transduction or pathogen defense (Valko et al., 2007). Because of possibly harmful effects, however, a balance between oxidants and antioxidants in the human body is important. In an unbalanced state, with too little antioxidant capacity, oxidative stress occurs (Reuter et al., 2010). This oxidative stress can lead to the oxidation of macromolecules (like proteins, lipids, or DNA) (Gill and Tuteja, 2010), damage of cell structures, inflammatory processes, and has been linked to the development of several diseases, like cardiovascular diseases, cancer, diabetes, or even Alzheimer's disease (Aksenov et al., 2001; Cai et al., 2011; Reuter et al., 2010; Sayre et al., 2008). Additionally, oxidative processes can lead to the oxidation of fats or other components in foods. In the case of a free radical reacting with another, non-radical molecule (e.g. a lipid), another free radical is formed (Halliwell, 2015). This can lead to a chain reaction, like the one occurring in the process of lipid peroxidation (Gill and Tuteja, 2010; Halliwell and Chirico, 1993). This does not only negatively influence the organoleptic properties, but can also reduce the content of nutrients, or even lead to the formation of oxidized substances that are harmful to the human health (Choe and Min, 2006; Marnett, 1999). Therefore, antioxidants, like the synthetic substances butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT), are used as food additives (EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), 2012, 2011). However, some concern regarding the use of these synthetic antioxidants exists, and many consumers prefer products without them. For those reasons, natural antioxidants, with a high acceptance by customers, are of great interest for the food industry for the replacement of synthetic antioxidants (Berasategi et al., 2011; Fernandes et al., 2016; Lara et al., 2011). Literature review – Antioxidants 26 Antioxidant capacity assays The antioxidant capacity of samples can be investigated by several assays. These assays can roughly be divided into two groups: Assays with a reaction based on a single electron transfer (ET), and those based on a hydrogen atom transfer (HAT) reaction (Huang et al., 2005). In an ET-based assay, the reducing capacity of an antioxidant is measured, while the HAT-based assays measure the hydrogen donating capacity of the antioxidant (Huang et al., 2005). HAT-based assays include ORAC (oxygen radical absorbance capacity), TRAP (total radical trapping antioxidant parameter), and crocin bleaching assays. The group of ET- based assays includes TEAC (trolox equivalent antioxidant capacity), FRAP (ferric ion reducing antioxidant parameter), and DPPH (diphenyl-1-picrylhydrazyl) assays, as well as the common determination of the total phenolic content (TPC) by the Folin-Ciocalteu (FC) assay (Huang et al., 2005). Total phenolic content - Folin-Ciocalteu assay The reagent used in the Folin-Ciocalteu assay was originally developed for the determination of the aromatic amino acid tyrosine in proteins (Folin and Ciocalteu, 1927). Later it was employed on wine, food and plant extracts for the determination of phenolic substances (Singleton and Rossi, 1965), which share the phenolic ring as a common structural element with the amino acid tyrosine. The Folin-Ciocalteu reagent contains, among others, sodium tungstate, sodium molybdate, as well as lithium sulfate, and is characterized by an intense yellow color. Under basic conditions, phenolic compounds can reduce this reagent via dissociation of a phenolic proton and formation of a phenolate anion. The reduction of the Folin-Ciocalteu reagent leads to a color change from yellow to blue, probably by the formation of (PMoW11O40) 4-, which can be measured photometrically to determine what is commonly called the total phenolic content (TPC) (Huang et al., 2005). Strictly speaking, the Folin- Ciocalteu assay measures the reducing capacity of the sample, including ascorbic acid as the most prominent contributor, and not only the content of phenolic substances (Everette et al., 2010; Huang et al., 2005). For plant extracts with a much higher content of phenolic compounds compared to e.g. ascorbic acid, however, the assay gives a rough approximation of the content of phenolic substances (Everette et al., 2010). Literature review – Antioxidants 27 ORAC The oxyen radical absorbance capacity (ORAC) assay was originally developed by Cao et al. (1993). The original protocol used the fluorescent protein β-phycoerythrin as an indicator, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) as the peroxyl radical generator, and the water soluble vitamin E analogue trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) as a reference standard substance. Because of some drawbacks, like high cost, a lot-to-lot inconsistency, lacking photostability over time (photobleaching), as well as nonspecific protein binding to polyphenols, β-phycoerythrin was later replaced by fluorescein as a fluorescent probe (Naguib, 2000; Ou et al., 2001). For a higher sample throughput, the ORAC protocol has later been modified for the use of 96-well microplates in combination with a microplate fluorescence reader (Huang et al., 2002). The procedure of performing the ORAC assay begins with the samples, blank controls, and trolox standards to be mixed with fluorescein. After an incubation period at 37 °C, a solution of the radical generator AAPH is added, and fluorescence intensity is measured in short time intervals for a certain time period with two different wavelengths for excitation and emission. By the reaction with AAPH, fluorescence intensity of fluorescein decreases, a process that is delayed by antioxidants. The measurements result in kinetic curves (cf. Fig. 10), of which the area under the curve (AUC) is calculated, followed by the calculation of the net AUC as the difference between the AUC of the sample and the AUC of the blank. With the net AUC of the trolox standard, a calibration curve is obtained, which is then used to calculate the antioxidant capacity of the samples, normally presented as trolox equivalents (TE). Because of the AUC approach, this method can be equally applied to antioxidants showing a distinct lag phase, and those that do not (Huang et al., 2005). Literature review – Antioxidants 28 Fig. 10: Example curves of ORAC measurements on a 96-well microplate. The left column shows the blank, where the kinetic curve of fluorescence intensity drops very quickly due to the lack of antioxidants. The following columns show the curves for trolox solutions in four concentrations in the upper four rows (increasing concentrations from the first to the fourth row). In the lower four rows, the kinetic curves of sample extracts are plotted (four replicates of the same sample within the same column). Own measurements. Literature review – Light and plant growth 29 2.7 Light and its influence on plant growth and secondary metabolites Light is a part of the electromagnetic spectrum. Typically, it is defined as the radiation that is visible to the human eye, covering the range between about 380 and 720 nm. However, sometimes the neighbouring regions of far-red (about 700 to 800 nm), infrared (800 to 3000 nm) as well as ultraviolet (about 200 to 400 nm) radiation are partly included, especially because of their effects on plants (Bresinsky et al., 2013; Sager and McFarlane, 1997; Schopfer and Brennicke, 2010). For photosynthesis, plants use radiation in the spectral region between 400 and 700 nm, which is termed as photosynthetically active radiation (PAR) (McCree, 1971). Measurements of light intensity (or radiatio