14


Table 1. Average trunk diameters, ABTS antioxidant capacity and Ellenberg’s climate climate quotient of the investigated varieties (upper part of the table). Lower table includes average peak areas for each compound according to variety. The column MRM denotes the MRM transition used for the quantitative assessment of the individual compounds by HPLC-MS/MS.


Variety


Farchau (26)


Average trunk diameter (cm) ABTS (mg TE/g dw.)


7.2 120.7 Ellenberg's climate quotient (EQ) 25.59


Farchau (26)


Peak tr 1


2 3 4 5 6 7 8 9


10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44


Compound (min)


1.12 Unknown 1 1.12


1.13 Unknown 2 1.16 Unknown 3 1.22 Unknown 4


1.83 Procyanidin C trimer 1 2.89 3.76


4.26 Unknown 5 4.35 4.67 4.93 5.15 6.04 6.05 6.76 6.85 7.28 7.56 7.57 7.65 7.95 8.26 10.3


Procyanidin B dimer 2 (+)-Catechin


Procyanidin C trimer 2 Chlorogenic acid isomer 2 Procyanidin C trimer 3 Procyanidin B dimer 3 Procyanidin B dimer 4 Procyanidin C trimer 4 Chlorogenic acid isomer 3 Procyanidin C trimer 5 (‒)-Epicatechin Coniferin isomer


Feruloylthreonic acid Procyanidin B dimer 5 Procyanidin B dimer 6


10.53 Procyanidin C trimer 6 11.04 Procyanidin B dimer 7 11.51 Procyanidin C trimer 7 11.56 Naringenin-C-hexoside 1 11.88 Naringenin-C-hexoside 2 12.10 Naringenin-C-hexoside 3 12.92 Coniferin derivative 1 12.97 Procyanidin C trimer 8 13.22 Procyanidin B dimer 8 13.31 Unknown 6


13.31 Quercetin-O-hexoside 1 13.48 Quercetin-O-glucuronide 13.58 Quercetin-O-hexoside 2 14.55 Kaempferol-O-hexoside 1 14.56 Quercetin-O-pentoside 14.81 Coniferin derivative 2 15.12 Kaempferol-O-hexoside 2 15.88 Kaempferol-O-pentoside 17.34 Kaempferol-O-deoxyhexoside 17.49 Coniferin derivative 3


Chlorogenic acid isomer 1 Procyanidin B dimer 1


Caffeic acid-O-hexoside


1108903 1520138 634390 99875 119092


220996 6202 3713 4882


1042701 1583385 159716


151258


7057350 7192719 9668604 9323563 9277990 4995


4466 11780 4969 5954


2753521 2822531 2000214 2346925 2742760 8104


8411


462570 1729884 1245079 1256243 1926291 81988 43640 22372 21766 48844 102503 293761 312222 24508 30374 135649 226371 5027


31206 22494 61294


281298 20417


7041


111154 67065 25125 26716 2623


4205


88896 302113 9297


6442


287559 340465 58473 12404 213856 103320 3849 3987


4087 1534


35264 25114 16625 15103 5275


7613


507213 459191 479059 423406 485056 450656 32005 56295 10399 16633 8586


7799


92009 102055 162861 199833 171037 297618 148883 217488 32337 59616 44859 50624 79828 142732 170793 298970 25580 40539 108089 281827 28218 56044


253157 1966


18202 3319 1807


204019 1334


321264 11776 25679 355 494


13559 11948 4093


450860 428712 435646 64734 11764 7984


38175


694786 389595 619850 180108 329658 139712 847707 204306 470130 59056


29297 31983 71662


330125 13305


310972 4219


76385 27253 1419


256807 6075


272781 28797


180360 8223 1624


21691 11558 2992


398299 377377 386641 39880 8964 5106


86840


447560 404062 525273 183194 204093 126775 988934 186284 353089 35345


36538 30732 57385


397467 16825


182708 11238 76568 44473 3025


491469 6023


304503 14960


198504 6330 1806


28529 12366 4100


486457 470857 456820 55822 12014 5953


99820


574521 218821 536788 147505 213871 206644 727697 127813 457432 54539


685351 228172


Pidkamin (59)


8.3


155.8 29.58


Pidkamin (59)


Torup (23)


3.3


202.1 26.18


Variety


Torup (23)


Peak aras


B.szent- györgy (H1)


M.egregy (52)


Gråsten (21)


MRM


(Q1/Q3) 439/97


341/179


9475302 533/191 3729


481/191


2006856 191/85 16063


865/125


1498860 353/191 7996 509


137391 632065 625


272789 45088 844


141798 10308


285044 3629


512905 25977 54449 845


1100 1634 5923 1990


349447 345113 343611 55037 6514 1086 83


967900 499648 814838 200752 383701 311128


577/125 311/149 577/125 289/109 865/125 353/191 865/125 577/125 577/125 865/125 353/191 865/125 289/109 341/59 311/193 577/125 577/125 865/125 577/125 865/125 433/313 433/313 433/313 451/341 865/125 577/125 413/57


463/300 477/301 463/300 447/227 433/300 451/341


1211637 447/227 208348 46


15766


417/255 431/285 451/341


According to correlation analysis, the most efficient antioxidants in beech leaf were Quercetin-O-hexoside 1 and 2, Coniferin derivative 2, (+)-Catechin,(-)-Epicatechin, Quercetin-O-pentoside, Caffeic acid-O-hexoside, Kaempferol-O-hexoside 2, Procyanidin B dimer 3 and Procyanidin C trimer 3 and 4 respecting the p<0.05 significance level. Interestingly, for some compounds significant negative correlations were indicated (R < -0.812), which could possibly be explained that these compounds have prooxidant effects in beech leaf extracts assessed by the ABTS method. Additionally, the antioxidant behaviour of isomers (especially those of Procyanidin B and C isomers) seems to be markedly different, which may be attributed to structural differences of these isomers.


By comparing the compounds’ concentrations to trunk diameter and to the EQ parameter it was shown that the varieties with higher EQ (originating from warmer and more arid regions of Europe) had lower levels of some of the most efficient antioxidant compounds by showing a significant negative correlation ((+)-catechin, Procyanidin C trimer 3 and Procyanidin B dimer 4) at the p<0.05 level. These results indicate that the varieties which were originally adapted to dryer and warmer climate do not tend to produce efficient antioxidant polyphenols in excess, as they are not ‘stressed’ in the Bucsuta region, hence their adaptability is good. These varieties also showed better growth parameters (trunk diameters) compared to low EQ varieties (see Table 1). Interestingly, some of the compounds (Procyanidin C trimer 2 and 8, Unknown 6) showed elevated levels in these varieties (significant positive correlations with the EQ value) which requires further explanation.


According to Table 1 there are apparent differences between varieties respecting trunk diameter, polyphenolic composition and antioxidant capacities. As a general tendency it was observed that varieties with the poorest growth parameters (Gråsten, Torup) had the highest ABTS levels and in these varieties the concentrations of some of the compounds were also the highest (Caffeic acid-O-hexoside, Unknown 2; Quercetin-O-hexoside 1 and 2; Quercetin-O-pentoside; Kaempferol-O-pentoside) or surprisingly the lowest (Unknown 1, 3 and 6; Procyanidin B dimer 5 and 6; Procyanidin C trimer 6). From these results the following questions arise: which compounds are the most powerful antioxidants? Which compounds can take part most efficiently as antioxidants in the defence reactions of the leaves? Can certain compounds act as markers of climatic adaptation and vitality, and is there a direct and statistically provable relationship between polyphenol levels and trunk


B.szent- györgy (H1)


7.5


163.5 26.77


M.egregy (52)


11.0


178.2 26.87


Gråsten (21)


5.2


296.2 20.26


Compound


Quercetin-O-hexoside 1 Coniferin derivative 2 (‒)-Epicatechin


Quercetin-O-hexoside 2 Quercetin-O-pentoside (+)-Catechin


Caffeic acid-O-hexoside Procyanidin C trimer 3 Procyanidin B dimer 4 Procyanidin C trimer 4 Kaempferol-O-hexoside 2 Quercetin-O-glucuronide Procyanidin B dimer 2 Kaempferol-O-hexoside 1 Kaempferol-O-pentoside Unknown 2


Procyanidin C trimer 1 Coniferin derivative 1


Chlorogenic acid isomer 2 Chlorogenic acid isomer 1 Chlorogenic acid isomer 3


Coniferin derivative 3 Procyanidin B dimer 5 Procyanidin C trimer 8 Unknown 1 Unknown 3


Procyanidin B dimer 6 Procyanidin C trimer 7 Feruloylthreonic acid Procyanidin C trimer 5 Naringenin-C-hexoside 2 Unknown 4 Unknown 5


Naringenin-C-hexoside 1 Naringenin-C-hexoside 3 Procyanidin C trimer 2 Procyanidin B dimer 1 Procyanidin B dimer 8 Unknown 6


Procyanidin B dimer 3 Procyanidin C trimer 6 Procyanidin B dimer 7


ABTS 0.937


0.919 0.903 0.889 0.876 0.873 0.872 0.870 0.825 0.817 0.815 0.811 0.761 0.740 0.732 0.650 0.605 0.534 0.519 0.382 0.251


Kaempferol-O-deoxyhexoside -0.343 Coniferin isomer


-0.359 -0.392 -0.558 -0.561 -0.572 -0.594 -0.639 -0.665 -0.683 -0.694 -0.733 -0.759 -0.785 -0.807 -0.824 -0.829 -0.849 -0.849 -0.900 -0.908 -0.944 -0.954


EQ


-0.756 -0.706 -0.781 -0.678 -0.693 -0.834 -0.632 -0.851 -0.838 -0.757 -0.627 -0.555 -0.513 -0.505 -0.531 -0.435 -0.525 0.007 -0.200 0.163 0.135 0.660 -0.265 0.791 0.493 0.872 0.677 0.707 0.097 0.784 0.314 0.304 0.539 0.656 0.793 0.638 0.672 0.869 0.441 0.778 0.848 0.574 0.688 0.782


Average trunk diameter (cm)


-0.436 -0.068 -0.372 -0.386 -0.534 -0.090 -0.652 -0.181 -0.098 -0.199 -0.348 -0.617 -0.239 -0.352 -0.493 -0.290 0.023 -0.249 -0.397 0.405 0.633 0.215 -0.013 0.211 0.763 0.353 0.934 0.867 0.384 0.314 0.762 0.642 0.438 0.821 0.520 0.424 0.382 0.249 0.261 0.106 0.767 0.655 0.644 0.357


Conclusion


The present study reported on the application possibilities of the HPLC-MS/MS technique for the research on polyphenolic compounds of beech leaf as potential biomarkers of the growth and climatic adaptation of beech varieties. Results indicated that there are direct relationships between concentrations of individual compounds and growth parameters of the different varieties. Although results look promising, they need to be justified by involving other varieties and investigating more sample trees per variety. Future results of the present research could be used for the selection of beech varieties for the future afforestations in Europe.


Acknowledgments


Research was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences and by the VKSZ_12-1-2013-0034 Agrárklíma.2 project.


References


Respecting average stem diameter, as a direct measure of the growth and performance of the varieties it was indicated that it correlates positively with the levels of the compounds Unknown 1, 3 and 4. According to the results, these compounds can be regarded as direct leaf biomarkers of the growth and performance of beech varieties and climatic adaptation and can be used for future research of selecting varieties for beech afforestation in the investigated region of Europe. Further research will be done on the structural elucidation of the compounds labelled as ‘unknown’, which only have recorded MSn structure has not yet been identified.


spectra, but their


1. Hickler, T., Vohland, K., Feehan, J., Miller, P.A., Smith, B., Costa, L., Giesecke, T., Fronzek, S., Carter, T.R., Cramer, W., Kühn, I., Sykes, M.T. Global Ecol. Biogeogr. 2012, 21 (1), 50–63. 2. Czúcz, B., Gálhidy, L., Mátyás, Cs. Ann. For. Sci. 2011, 68 (1), 99–108. 3. Stojanoviç, D.B., Kržiç, A., Matoviç, B., Orloviç, S., Duputie, A., Djurdjeviç, V., Galiç, Z., Stojniç, S. Agric. For. Meteorol. 2013, 176, 94– 103.


4. Berki, I., Rasztovits, E., Móricz, N., Mátyás, Cs. Cereal Res. Commun. 2009, 37, 613–616. 5. Lakatos, F., Molnár, M. Acta Silv. Lign. Hung. 2009, 5, 75–82. 6. Mátyás, Cs., Berki, I., Czúcz, B., Gálos, B., Móricz, N., Rasztovits, E. Acta Silv. Lign. Hung. 2010, 6, 91–110. 7. Gálos, B., Jacob, D., Mátyás, Cs. Acta Silv. Lign. Hung. 2011, 7, 49–62.


8. Cadahía, E., Fernández de Simón, B., Aranda, I., Sanz, M., Sánchez-Gómez, D., Pinto, E. Phytochem. Anal. 2015, 26, 171–182. 9. Stratil, P., Klejdus, B., Kubáç, V. Talanta 2007, 71, 17411751. 10. Ellenberg, H. Vegetation Ecology of Central Europe, fourth ed. Cambridge University Press, Cambridge, 1988.


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According to correlation analysis, the most efficient antioxidan Quercetin-O-hexoside 1 and 2, Coniferin derivative 2, (+)-Cate Quercetin-O-pentoside, Caffeic acid-O-hexoside, Kaempferol- dimer 3 and Procyanidin C trimer 3 and 4 respecting the p<0.0 Interestingly, for some compounds significant negative correla 0.812), which could possibly be explained that these compoun in beech leaf extracts assessed by the ABTS method. Addition behaviour of isomers (especially those of Procyanidin B and C markedly different, which may be attributed to structural differe By comparing the compoundsʼ concentrations to trunk diamete was shown that the varieties with higher EQ (originating from regions of Europe) had lower levels of some of the most efficie by showing a significant negative correlation ((+)-catechin, Pro Procyanidin B trimer 4) at the p<0.05 level. These results indic were originally adapted to dryer and warmer climate do not ten antioxidant polyphenols in excess, as they are not ʻstressedʼ in their adaptability is good. These varieties also showed better g diameters) compared to low EQ varieties (see Table 1). Intere compounds (Procyanidin C trimer 2 and 8, Unknown 6) showe varieties (significant positive correlations with the EQ value) w explanation.


Table 2. Correlation analysis between polyphenol levels and ABTS levels, Ellenberg’s climate quotient (EQ) as well as average trunk diameter. Table includes correlation coefficient (R) values. Marked correlations (red) are significant at p < 0.05 (n=6, |R| ≥ 0.812).


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