DOI: 10.3303/CET23107110 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Paper Received: 08 April 2023; Revised: 29 June 2023; Accepted: 20 October 2023 
Please cite this article as: Visiné-Rajczi E., Németh L., Albert L., Mátyás C., Hofmann T., 2023, Applicability of Beech (Fagus Sylvatica L.) Leaf 
Polyphenols as Indicators for Growth and Acclimation and Their Potential Contribution to Sustainable Forestry, Chemical Engineering 
Transactions, 107, 655-660  DOI:10.3303/CET23107110 
  

 CHEMICAL ENGINEERING TRANSACTIONS  
 

VOL. 107, 2023 

A publication of 

 

The Italian Association 
of Chemical Engineering 
Online at www.cetjournal.it 

Guest Editors: Petar S. Varbanov, Bohong Wang, Petro Kapustenko 

Copyright © 2023, AIDIC Servizi S.r.l. 

ISBN 979-12-81206-07-6; ISSN 2283-9216 

Applicability of Beech (Fagus Sylvatica L.) Leaf Polyphenols 

as Indicators for Growth and Acclimation and Their Potential 

Contribution to Sustainable Forestry 

Eszter Visiné-Rajczia,*, László Némethb, Levente Alberta, Csaba Mátyása, Tamás 

Hofmanna  

aInstitute of Environmental Protection and Natural Conservation, Faculty of Forestry, University of Sopron, 4 

 Bajcsy-Zsilinszky str, 9400, Sopron, Hungary. 
bInstitute of Informatics and Mathematics, Faculty of Wood Engineering and Creative Industries, University of Sopron, 4  

 Bajcsy-Zsilinszky str, 9400, Sopron, Hungary. 

 visine.rajczi.eszter@uni-sopron.hu 

Sustainable forestry is one of the most important challenges of the 21st century, which requires the cultivation 

of species adapted to the changing climate and, in the case of existing species, the selection of climate-resistant 

propagation materials. European beech (Fagus sylvatica L.) is the most important hardwood tree species in 

Europe. Its ecological and economic importance is outstanding. The species, however, is significantly affected 

by the change in climate, so it is essential to select the appropriate propagating material for future cultivation 

and afforestation. The aim of this work was to measure and evaluate the polyphenolic composition of the leaves 

of different beech provenances to find relationships between average stem diameter, climate index (EQ), ABTS 

(2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid) antioxidant capacity and polyphenolic composition to 

assess the adaptability of the tested beech provenances using factor analysis. According to the results from the 

altogether 44 identified polyphenols, flavonoid (quercetin, kaempferol) glycosides and flavan-3-ol compounds 

were the strongest antioxidants, and the two types of compounds probably participate differently in the 

antioxidant defence system, but both have a significant role. A significant relationship between the concentration 

of individual polyphenols (Procyanidin B dimer 5, Feruloylthreonic acid, Unidentified 1) and average stem 

diameter was found: these compounds and factors can be chemical indicators of growth and climatic adaptation 

and can be used in predicting the future effects of climate change and in the future selection of beech 

propagation material. 

1. Introduction 

European beech (Fagus sylvatica L.) is one of Europe's most significant hardwood species from an ecological 

and economic point of view. It is particularly sensitive to the drying of the climate and stress, so its future 

sustainability raises many questions. Climate projections predict significant warming and drought throughout 

Hungary (Bartholy et al., 2014). While the tree species accounted for 5.5 % of Hungary's forest area (103 

thousand hectares) in 2019 (URL1), according to forecasts, by 2050, only 1 % of the country's territory can 

possess optimal climatic conditions for the species (Gálos and Führer, 2018). In 1998 a provenance trial was 

initiated by the International Union of Forest Research Organizations (IUFRO) throughout Europe to study the 

climatic adaptability of the species (von Wuehlisch and Alia, 2011). Offspring of beech populations, adapted to 

the climate of purposefully chosen sites of origin were planted at assigned test sites. According to the 

hypothesis, the climatic difference between the sites of origin and the test site imitates rapid climate change and 

reveals the possibilities and limitations of inherited adaptability (Horváth and Mátyás, 2014). The Hungarian test 

location was Bucsuta (Zala county), located in an ecologically privileged location, closest to the xeric limit of the 

tree species (Horváth and Mátyás, 2016). 

The trees at the test sites are exposed to several stressors at the same time, which can affect the functioning 

of the cell and the entire organism, from genes to plant metabolites. The vast majority of stress factors trigger 

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oxidative stress, which is a shift in the balance between oxidants and antioxidants in favour of oxidants (Sies, 

1991). In response, the plant activates its enzymatic and non-enzymatic antioxidant systems to eliminate 

reactive oxygen species and damaging free radicals. The glutathione system and other specific stress proteins, 

oxidase enzymes and their isoenzymes, and some polyphenols indirectly characterize plant stress through 

changes in their qualitative and quantitative spectra and can be considered biomarkers (Vieito et al., 2018) of 

the stress response (Tausz et al., 2004). In beech leaves, (Formato et al., 2022) and bark (Dübeler et al., 1997) 

the role of polyphenols against environmental stress has been already investigated in detail. 

During the year 2013, the general state of the non-enzymatic antioxidant system of six selected beech 

provenances (Farchau, Pidkamin, Torup, Gråsten, Bánokszentgyörgy, Magyaregregy) was assessed in the 

Bucsuta provenance trial through the quantitative evaluation of polyphenolic compounds and the determination 

of ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) antioxidant capacity of the leaves (Visi-Rajczi et 

al., 2021). By the assessment of the polyphenolic composition of the leaves of the different beech provenances 

relationship with the average trunk diameter, climate index, sun exposure and the ABTS antioxidant capacity 

was established using multivariate statistical analysis (factor analysis).  

This study is the first to investigate the effect of acclimation stress in different European beech provenances by 

the assessment of the leaf polyphenolic antioxidant system using multivariate statistical methods. To the best 

of our knowledge, such an evaluation has not been yet conducted. The present research investigated if stress 

responses in different beech provenances could be characterized by chemical analysis of selected leaf 

metabolites, and that among these metabolites, which may be stress indicators that characterise acclimation 

and adaptation at the molecular level. The results of the work aimed at answering the following questions in 

particular: 

1. Which compounds are most responsible for the measured ABTS antioxidant capacity, i.e. which leaf 

polyphenols could participate most effectively in defence reactions in case of biotic and abiotic stress in the 

case of beech? 

2. How does sunlight (exposure to light) affect the polyphenol content and ABTS antioxidant capacity of the 

leaves, thus the defence capacity (resilience)? 

3. Are there compounds that can be used as tree growth indicators or related to climate adaptation? 

2. Materials and methods 

2.1 Sample collection and processing 

Figure 1. depicts the origin of the investigated provenances and the location of the test site (Bucsuta) compared 

with the occurrence of beech. Six provenances were selected for the investigations and 8 trees of each of the 

provenances were sampled in June 2013 (Visi-Rajczi et al., 2021). The average diameter at the breast height 

of the trees (ASD) was determined for each provenance. From each tree, 30 leaves were collected from different 

parts of the canopy. Leaves were stored in dry ice until the extraction process. Before extraction leaves were 

treated to inactivate their polyphenol-oxidizing enzymes (2 min at 750 W in a household microwave oven), then 

the leaves were ground and the polyphenols were extracted as follows: 0.15 g ground leaves were mixed with 

15 ml MeOH:H2O 4:1 (v/v) mixture and stirred for 24 h in the dark, then the extract was filtered through a 0.45 

μm cellulose acetate filter, diluted twice fold and taken to analysis 

 

Figure 1: Origin of the investigated provenances and the location of the test site (Bucsuta) compared with the 

occurrence of beech. Investigated provenances: 21: Gråsten (DK), 23: Torup (S), 52: Magyaregregy (H), H1: 

Bánokszentgyörgy (H), 59: Pidkamin (UA), 26: Farchau (D) 

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The aim of this work was to measure and evaluate the polyphenolic composition of the leaves of different beech 

provenances to find relationships between average stem diameter, climate index (EQ), sun exposure factor, 

ABTS (2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid) antioxidant capacity and polyphenolic composition 

to assess the adaptability of the tested beech provenances using factor analysis. 

2.2 Ellenberg climate quotient (EQ) 

For the characterization of the climate of the origin of the provenances the Ellenberg climate quotient (EQ) was 

used, which is calculated from the ratio of the mean temperature of the warmest month (July, T07 [oC]) and 

annual precipitation (Pann, [mm]) as shown in Eq(1) (Ellenberg, 1988):  

 𝐸𝑄 = 1000 ∙ 𝑇07 ∙ 𝑃𝑎𝑛𝑛
      −1 (1) 

2.3 Sun exposure factor (Sun) 

For all samples, the average of the light intensity was measured which the leaves were exposed to using a 

Sunche HS1010A type handheld light meter. From the measured light intensity, a sun factor was calculated by 

normalizing light intensity values to the 0-100 scale, where 0 corresponded to the leaves exposed to the lowest 

light intensity and 100 to the samples exposed to the highest intensity. 

2.4 ABTS antioxidant capacity 

The ABTS assay was run as described by Stratil et al. (2007) at 734 nm, using the ABTS•+ radical ion and trolox 

standard. Reaction time was 10 min. ABTS antioxidant capacity was evaluated as mg trolox g-1 dry leaf units. 

Measurements and evaluations were run in triplicates.  

2.5 Chromatographic analysis 

For the identification and quantitative assessment of polyphenolic compounds high-performance liquid 

chromatography/photodiode array detection/tandem electrospray mass spectrometry (HPLC-PDA-ESI-MSn) 

has been applied. Detailed methods for analysis and evaluation are included in the work of Hofmann et al. 

(2017). In this study, the authors have identified 44 polyphenolic compounds from beech leaves. The results 

and list of compounds are consistent with later non-targeted studies on the polyphenol profiling of beech leaves 

(Formato et al., 2021). 

2.6 Statistical evaluation  

Factor analysis was run using the Statistica 12 software (StatSoft Inc, Tulsa, USA). For data analysis and 

interpretation, the most commonly-used varimax normalized method was applied.  

The scheme of the sample collection, chemical analysis and evaluation is summarized in Figure 2. 

 

Figure 2: Graphical scheme of the study approach 

3. Results and discussion 

Factor analysis was applied for the evaluation of the data. Factor analysis helps to find connections between 

independent variables (vectors). Factors are calculated from the transformation of individual vectors, grouped 

by variance. In this manner, compounds are grouped in a way that also respects their relationship with 

antioxidant capacity (ABTS), environmental parameters (Sun, EQ) and growth (ASD). 

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In the factor analysis, first, the scree-plot was determined (Figure 3), in which the sum of variance of the 

eigenvalues for the analysis is plotted in descending order. In the scree-plot, it can be seen that the sum of 

variance for the first four eigenvalues is higher than 95 % of the sum of all the eigenvalues, which indicates that 

the 48 variables (44 polyphenol concentrations, ABTS, Sun, ASD, EQ) can be investigated and well displayed 

in a four-dimensional factor space, where the four factors correspond to > 95 % of the total variance of the 

eigenvalues. 

 

Figure 3: The scree plot 

By plotting the 4 factors against one another the factor coordinates of the individual vectors can be displayed, 

and the vectors closely related to each other can be revealed. The results are presented in Figure 4 (Factor 1 

vs. Factor 2) and Figure 5 (Factor 1 vs. Factor 4). The vectors that best define each factor (loading > |0.7|) were 

marked in both figures. The + or – signs indicate whether the factor weights (loadings) are positive or negative. 

It was supposed that the polyphenol vectors closely located to each other are also behaving similarly and the 

vectors “clustered” closely together have a significant relationship with either of the ABTS, Sun, ASD or EQ 

vectors that they are located closely to. 

 

Figure 4: Results of the factor analysis; factor loadings Factor 1 vs. Factor 2. F1 denotes the group of vectors 

corresponding to Factor 1, and F2 and F4 are used similarly. For interpretation of tags of compounds, please 

refer to Figure 2 

Based on Figure 4, it can be stated that ABTS antioxidant capacity and Sun are most closely related to factors 

F1 and F2 and they are also close to each other in the F1 vs F2 factor space, indicating their close relation, 

658



which can be explained that strong sunlight (UV radiation) increases the accumulation of polyphenols in leaves 

(Holub et al., 2019). Nevertheless, the factor coordinates of both vectors are at the limit of the significance for 

both of the factors, which can be interpreted as both influencing F1 and F2 equally, but none of F1 and F2 are 

the exclusive antioxidant factors.  

 

Figure 5: Results of the factor analysis; factor loadings Factor 1 vs. Factor 4. F1 denotes the group of vectors 

corresponding to Factor 1, and F4 is used similarly. For interpretation of tags of compounds, please refer to 

Figure 2 

Factor 1 has a strong significant relationship with flavonoid (quercetin, kaempferol) glycosides, while Factor 2 

has a strong and significant relationship with catechins and procyandins. According to this, both Factors 1 and 

2 are "antioxidant factors", since ABTS has a relationship with both of these factors. Based on the results, it can 

be assumed that there are two types of antioxidant mechanisms in beech leaves with regard to polyphenols, 

i.e., the compounds connected with Factors 1 and 2 are all strong antioxidants, but they exert their effects in 

different ways. As polyphenols play key roles in the growth, regulation and structure of plants (Watson, 2014), 

it seems feasible that there must be compounds of those concentrations that are directly correlated with the 

growth performance of plants. According to Figure 5, the average trunk diameter (ASD) showed the strongest 

correlation with Factor 4 (loading > 0.9) and the vectors close to ASD (PBd5 (Procyanidin B dimer 5): 0.915, Fta 

(Feruloylthreonic acid): 0.798, Ui1 (Unidentified 1): 0.729) can be indicators of tree growth, thereby also 

indicators of the future adaptability of the investigated beech provenances. Future tests need, however, be 

carried out to study the effects and precise functions of these compounds. 

4. Conclusion 

Sustainable management of beech in Europe requires the proper choice of propagation material for growing 

healthy beech forests that can withstand changing climatic conditions. The assessment of plant stress via 

antioxidant compounds as markers can help to elaborate methods for this purpose. In the present work leaf 

polyphenols and ABTS antioxidant capacity, as well as Ellenberg’s climate index, average stem diameter, and 

sun exposure factor have been assessed and evaluated using factor analysis based on an international 

provenance trial at the Bucsuta test site. According to the results in the polyphenolic antioxidant system of beech 

leaves, flavonoid (quercetin, kaempferol) glycosides and flavan-3-ol compounds were shown to be the strongest 

antioxidants. The two types of compounds probably participate differently in the antioxidant defence system, but 

both have a significant and determining role. Strong relationships were found between the concentration of 

Procyanidin B dimer 5, Feruloylthreonic acid, Unidentified 1 and the average stem diameter, which suggest that 

these compounds are indicators of growth in the beech provenances. Certain polyphenol concentrations may 

serve as chemical indicators of the acclimation potential of populations and may contribute to the forecasting of 

the effects of climate change. The applicability of the results for forestry practice requires further research and 

evaluation. Future measurements will focus on the analysis of other provenances and a larger number of trees 

to investigate the applicability of the results. 

659



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