Hofmann, Tamás

Nebehaj, Esztella

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THE LEAF POLYPHENOLS OF FOREST TREES: OPTIMIZATION OF THE EXTRACTION SOLVENT, SEASONAL CHANGES, AND STRUCTURAL ELUCIDATION BY HPLC-ESI-MSn   E. Nebehaj*, T. Hofmann University of West-Hungary, Faculty of Forestry, Institute of Chemistry. Sopron, Hungary nebehaj.esztella@gmail.com HPLC 2015, 42nd International Symposium on High Performance Liquid Phase Separations and Related Techniques, June 21-25, 2015, Geneva, Switzerland INTRODUCTION Leaf extracts of some tree species can exhibit several beneficial effects (anti-oxidant, anti-inflammatory, anti-rheumatic, anti-cancerogenic) on human health. One of the most bioactive groups of extractives, responsible for these effects are the polyphenols. We have investigated the total phenol content of the leaves of some of the most important Hungarian tree species (Fagus sylvatica, Carpinus (C.) betulus, Quercus (Q.) robur, Q. petraea, Q. cerris, Q. pubescens, Robinia pseudoacacia, Pinus (P.) sylvestris, P. nigra, Acer platanoides, Castanea sativa, Populus x euramericana). We have optimized extraction solvent composition and have determined seasonal variations of the total phenol contents. The July and August leaves of Q. pubescens and C. betulus had the highest total phenol levels. Using HPLC and MS/MS measurements the structural identification of leaf polyphenols of these species have been carried out for the first time.   SAMPLING SITE, TIME Botanic Garden of the University of West-Hungary, Sopron (H), between May and September 2014, on 5 occasions. One healthy tree from each species was sampled by taking 15 shade- and 15 sun leaves per occasion. SAMPLE AND EXTRACTS PREPARATION Leaf samples were treated with microwave irradiation for enzyme inactivation and ground to a fine powder. Extraction: 0.2 leaf powder + 20 ml 4:1 methanol:water/ 4:1 ethanol:water/ water, 20 min in ultrasonic bath.  DETERMINATION OF TOTAL PHENOLICS Total phenol content was measured using the Folin-Ciocalteu assay [1] applying  quercetin as standard. Absorbance was measured at 760 nm.  RESULTS TOTAL PHENOLICS (mg*g-1 dried leaf) – Extraction optimization SPECIES 4:1 METHANOL:WATER 4:1 ETHANOL:WATER WATER Fagus sylvatica  37.82 ± 1.01B 39.71 ± 1.33B 23.68 ± 1.09A Carpinus betulus 78.81 ± 0.59A 76.84 ± 2.07A 86.75 ± 2.51B Robinia pseudoacacia 29.30 ± 0.99B 28.91 ± 0.12B 23.55 ± 0.84A Acer platanoides 46.63 ± 2.80B 47.03 ± 1.28B 7.48 ± 0.00A Castanea sativa 71.30 ± 3.53B 66.20 ± 1.33B 38.67 ± 1.34A Populus x euramericana 29.03 ± 0.29B 31.22 ± 1.40B 25.55 ± 0.68A Quercus robur 59.85 ± 2.27C 51.55 ± 2.16B 37.14 ± 0.31A Quercus petraea 56.47 ± 1.34C 47.62 ± 2.01B 32.84 ± 0.72A Quercus pubescens 37.52 ± 0.54B 29.33 ± 1.07A 30.81 ± 0.81A Quercus cerris 78.99 ± 3.35B 73.21 ± 5.50B 51.68 ± 1.81A Pinus sylvestris 27.12 ± 0.78B 32.64 ± 0.63C 21.78 ± 0.24A Pinus nigra 27.37 ± 0.97B 27.16 ± 0.91B 22.38 ± 1.01A TOTAL PHENOLICS (mg*g-1 dried leaf) – Seasonal changes SPECIES MAY JUNE JULY AUGUST SEPTEMBER Fagus sylvatica  37.82 ± 1.01AB 56.83 ± 0.99CD 48.12 ± 1.28BBC 47.38 ± 3.14BB 57.71 ± 2.59CCB Carpinus betulus 78.81 ± 0.59AD 93.08 ± 3.29BH 105.93 ± 5.57CH 94.27 ± 5.38BG 80.81 ± 3.05AE Robinia pseudoacacia 29.30 ± 0.99BA 29.73 ± 0.73BA 43.25 ± 0.21CBA 19.89 ± 2.89AF 49.63 ± 1.84DB Acer platanoides 46.63 ± 2.80AE 57.37 ± 1.98BD 80.21 ± 1.47EG 65.88 ± 2.83CDEF 73.73 ± 1.26DDE Castanea sativa 71.30 ± 3.53ABF 78.52 ± 1.13BG 62.51 ± 1.59ADE 76.32 ± 5.25BF 75.49 ± 4.67BBD Populus x euramericana 29.03 ± 0.29AA 53.63 ± 3.24BDC 73.73 ± 3.05DFG 61.96 ± 2.74CCDE 57.61 ± 0.58BCBC Quercus robur 59.85 ± 2.27BC 58.46 ± 2.50BDE 48.32 ± 4.82ABC 49.36 ± 4.21ABC 72.21 ± 0.46CD Quercus petraea 56.47 ± 1.34AC 51.23 ± 3.89ADC 59.15 ± 4.06ADE 53.70 ± 2.14ABCD 70.01 ± 2.42BD Quercus pubescens 37.52 ± 0.54AB 71.68 ± 2.32CFG 63.80 ± 3.31BCE 92.85 ± 3.67DG 60.31 ± 5.73BC Quercus cerris 78.99 ± 3.35CD 65.82 ± 1.59ABEF 65.89 ± 1.54ABEF 73.73 ± 5.81BCEF 59.69 ± 2.52AC Pinus sylvestris 27.12 ± 0.78AA  42.56 ± 1.62BB 37.47 ± 3.95BA 24.63 ± 3.98AA 36.07 ± 1.74BA Pinus nigra 27.37 ± 0.97AA 46.61 ± 4.23BBC 53.80 ± 1.56BCD 45.39 ± 5.51BB 35.78 ± 2.30AA This research was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and financed by the VKSZ_12-1-2013-0034 Agrárklíma.2 project. • In the case of most trees methanol containing mixture resulted the highest polyphenol yields, yet for C. betulus water worked best. • The total phenol content increased gradually from spring to autumn in the leaves except of Q. cerris.  • Overall the highest values were measured in C. betulus and Q. pubescens followed by Acer platanoides and Castanea sativa. • Carpinus betulus contains a wide variety of polyphenols including (+)-catechin, (-)-epicatechin, chlorogenic acid isomers, gallic acid, ellagic acid, ellagitannins and gallotannins, methyl-gallate, quercetin-, myricetin-, apigenin and kaempferol glycosides. • Quercus pubescens contains gallic acid, ellagic acid, ellagitannins, and gallotannins, gallocatechin, (+)-catechin, (-)-epicatechin, dimeric procyanidins, caffeic acid derivatives, quercetin-, isorhamnetin-, kaempferol and myricetin-glycosides.   [1] Singleton, J. A. Rossi. 1965. Colorimetry of total phenolics with phosphomolibdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture 161: 144-158. tr (min) COMPOUND NAME [M-H] - MS/MS 5.8;  16.0 unknown-hexoside 355 337, 311, 161, 133, 121, 6.4; 7.9; 16.0; 28.4 monogalloyl glucose isomers 331 169, 125, 271, 331 8.7 gallic acid 169 125 11.3 chlorogenic acid derivative 371 353, 341, 191, 173, 161 18.3 gallocatechin 305 261, 219, 179, 167, 137 18.6 vanillic acid 167 167, 152, 123, 108 19.7; 21.6 galloyl-hexoside isomers 325 281, 169, 155, 137, 125 21.5; 30.2 methyl-gallate isomers 183 168, 139, 124 25.8 syringic acid 197 182, 167, 153, 138, 123 29.7 gallic acid derivative 325 169, 137, 125, 111, 107 30.4; 51.3 unknown-C-hexoside 359 341, 299, 239, 211, 197 33.1 epigallocatechin 305 261, 219, 179, 167, 137 33.1 chlorogenic acid isomer 353 191 33.7 dimeric procyanidin 577 533, 425, 407, 289, 245 33.8 chlorogenic acid derivative 706.6 533, 462.8, 353, 191, 179 34.6 dimeric procyanidin 577 533, 425, 289, 245, 124.9 35.1 (+)-catechin 289 245, 221, 203, 125, 109 35.8 chlorogenic acid derivative 706.6 706.6, 533, 462.8, 353, 191 36.8 gallic acid derivative 495 343, 337, 325, 191, 169, 125 35.9; 43.5 digalloyl-HHDP glucose isomers 785 633, 419, 301, 275, 169 37.2; 38.4;  digalloyl-glucose isomers 483 439, 331, 313, 301, 169 37.4 chlorogenic acid isomer 353 352.8, 191, 178.9, 135 37.8 chlorogenic acid derivative 707 533, 515, 353, 191, 179 39.7; 43.0 trigalloyl glucose isomers 635 483, 465, 271, 169, 125 42.9 (-)-epikatechin 289 245, 221, 203, 125, 109 43.1 unknown ellagic acid derivative 655 463, 331, 323, 301, 297 Table 1. Total phenol content of leaves measured with different extraction solvents. Different letters in one table indicate significant difference at p=0.05 level.   Table 2. Total phenol content of leaves in the case of different species. Different letters in one table ( superscript: between species, subscript: between months)indicate  significant difference at p=0.05 level.  From the extracts showing the highest total phenol levels (C. betulus, Q. pubescens) the HPLC-ESI-MSn characterization of polyphenols has been carried out for the first time. Carpinus betulus Quercus pubescens tr (min) COMPOUND NAME [M-H] - MS/MS 43.5 ellagic acid derivative 807 505, 445, 331, 301, 275 44.3; 46.0 trigalloyl glucose isomers 635 483, 465, 271, 169, 125 46.6; 47.7 trigalloyl glucose isomers 635 483, 465, 271, 169, 125 45.3; 46.4; 47.2 unknown galloyl glucose 953 909, 783, 615, 445, 301 46.4; 48.8; 50.1 unknown ellagic acid derivative 1108 1048, 765, 463, 301, 249 47.4; 54.1; 55.6 quercetin-O-hexoside 463 301, 300, 272, 215, 191 48.1 digalloyl-HHDP glucose 785 633, 615, 419, 301, 205 49.6 trigalloyl glucose isomer 635 483, 465, 271, 169, 125 50.1 gallic acid derivative 477 433, 313, 169, 125, 119 50.7 myricetin-O-hexoside 479 316, 271, 179 50.8 myricetin-O-galactoside 479 479, 316, 271, 287, 179 51.0 quercetin-O-hexoside 463 301, 300, 272, 257, 191 51.2 myricetin-O-galactoside 479 479, 316, 271, 287, 179 51.8 chlorogenic acid derivative 505 353, 191 52.1; 54.2 trigalloyl-HHDP glucose isomers 937 785, 465, 301, 275, 169 52.3 quercetin-O-pentoside 433 300, 301 52.4; 53.0; 53.6 tetragalloyl glucose isomers 787 635, 617, 465, 313, 169 53.5 myricetin-O-xyloside 449 316, 287, 271, 259, 243 53.8 myricitrin 463 359, 316, 287, 271, 259 53.9 ellagic acid 301 284, 257, 229, 185 54.5 luteolin-O-glucoside 447 447, 327, 285, 284, 256 56.6 quercetin-arabinoside/xyloside 433 301, 300, 255, 243, 179 57.2 quercetin-O-rhamnoside 447 301, 300, 272, 255, 243 57.3 apigenin-O-hexoside 431 431, 341, 311, 240, 225 58.9 ellagic acid hexoside  463 301, 245 59.9 kaempferol-3-rhamnoside 431 431, 285, 255, 227, 211 tr (min) COMPOUND NAME [M-H] - MS/MS 2.9; 3.0; 4.5 quinic acid isomers 191 173, 149, 127, 111, 93 6.4; 7.9; 16.0; 20.2 monogalloyl glucose isomers 331 169, 125, 271, 331 8.6 gallic acid 169 125 8.7; 11.5; 15.2 ellagtannin 1100 568, 493, 467, 301, 276 13.6; 21.1 ellagtannin, mw: 1866 Da [M-2H]2- : 932 914, 783, 631, 451, 301 18.1 gallocatechin 305 261, 219, 179, 167, 137 19.5; 21.6 galloyl-hexoside 325 281, 169, 155, 137, 125 23.9 gallocatechin-(4,8)-(epi)catechin 593 575, 425, 407, 289, 125 29.5; 30.4 methyl-gallate  183 168, 139, 124 31.9; 34.4 procyanidin B dimer 577 425, 407, 338, 289, 125 32.1 3-p-Coumaroylquinic acid 337 191, 173, 163, 155, 119 33.3 4-p-Coumaroylquinic acid 337 191, 173, 163, 155, 111 35.0 (+)-catechin 289 245, 221, 203, 125, 109 35.7; 43.5 digalloyl-HHDP glucose isomers 785 633, 419, 301, 275, 169 37.1; 38.1 digalloyl-glucose isomers 483 439, 331, 313, 301, 169 40.3 ellagtannin mw: 1116 Da [M-2H]2- : 557 932,5 465, 301, 181, 275 40.5; 42.2 ellagtannin, mw: 1866 Da [M-2H]2- : 932 914, 783, 631, 451, 301 42.9 (-)-epikatechin 289 245, 221, 203, 125, 109 45.9 trigalloyl glucose  635 483, 465, 313, 169, 125 47.4 quercetin-O-hexoside 463 301, 300, 243, 227 48.3; 49.4; 51.5 ellagtannin 1082 631, 586, 489, 301, 273 48.3 ellagtannin 499 499, 439, 341, 323, 301 48.9; 51.7; 52.5 Reginin A/Reginin D isomer mw: 1718 Da [M-2H]2- : 858 764, 613, 451, 781, 301, 275 49.4; 52.2 ellagtannin mw: 1106 Da [M-2H]2- : 552 631, 473, 467, 457, 451, 301, tr (min) COMPOUND NAME [M-H] - MS/MS 50.7; 51.1 myricetin-O-hexoside 479 316, 287, 271, 242, 214 51.9 Quercetin-O-pentoside-O-hexoside 595 301, 300, 271, 255, 253 52.2 quercetin-O-pentoside  433 301, 300, 271, 255, 243 52.3; 53.0; 53.6; 54.0 tetragalloyl glucose isomers 787 635, 617, 465, 313, 169 52.7; 53.2 quercetin galloyl-hexoside 615 463, 301, 300, 255 52.9; 53.4; 54.7 galloyl-hexoside isomers 938.4 768.5, 616.5, 465, 429, 125 55.5; 55.8; 56.9 galloyl-hexoside isomers 938.4 768.5, 616.5, 465, 429, 125 53.7 myricitrin 463 317, 316, 288, 271, 259 53.9 ellagic acid 301 284, 257, 229, 185 54.0; 54.5 quercetin-O-hexoside 463 301, 300, 271, 255, 179 54.6; 54.8 kaempferol galloyl-hexoside 599 462, 447, 327, 313, 284 55.0 ellagitannin 499 343, 323, 301, 271, 215 55.5 trigalloyl-HHDP glucose 938 785, 615, 465, 419, 301 56.1; 56.8 kaempferol-O-hexoside 447 284, 285, 255, 227, 179 56.1 quercetin-O-pentoside  433 301, 300, 271, 255, 243 56.3 kaempferol galloyl-hexoside 599 462, 447, 327, 313, 284 57.0 isorhamnetin-O-rutinoside 623 315, 314, 299, 271, 255 57.2 quercetin-O-rhamnoside 447 301, 300, 271, 179, 151 57.3 isorhamnetin-O-hexoside_derivative 545 477, 357, 314, 257, 243 57.4 isorhamnetin-O-glucoside 477 357, 329, 285, 271,243 59.0 unknown-glycoside 447 314, 300, 285, 271, 243, 257 64.3; 64.6; 66.1 isorhamnetin-O-rutinoside 623 315, 314, 299, 271, 255 64.6; 65.1; 65.5; 66.5 kaempferol-O-hexoside-O-rhamnoside 593 447, 285, 284, 255, 227, 145, 119 Table 3/A. The identified compounds in the case of C. betulus Table 3/B. The identified compounds in the case of C. betulus • Column: Phenomenex Luna C18, 5 μm, 250 x 4.6 mm; 40°C • Mobile phase: A (H2O + 0.1% HCOOH), B (ACN + 0.1% HCOOH). • Gradient elution (4 min 3%B, 4-22 min 5%B, 22-45min 15%B, 45-84 min 57%B, 84-90 min 100%B, 90-98 min 100%B) • Sample injection: 8 μl  • UV-detection: 250-380 nm • MS settings: -ESI ionization • Identification of the compounds: IDA analysis by MS/MS spectra Table 4/A. The identified compounds in the case of Q. pubescens Table 4/B. The identified compounds in the case of Q. pubescens 

THE LEAF POLYPHENOLS OF FOREST TREES: OPTIMIZATION OF THE EXTRACTION SOLVENT, SEASONAL CHANGES, AND STRUCTURAL ELUCIDATION BY HPLC-ESI-MSn

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