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Article

Role of Extractable and Non-Extractable Polyphenols in the Formation of Beech (Fagus sylvatica L.) Red Heartwood Chromophores

by
Tamás Hofmann
*,
Eszter Visi-Rajczi
and
Levente Albert
Institute of Environmental Protection and Nature Conservation, Faculty of Forestry, University of Sopron, 9400 Sopron, Hungary
*
Author to whom correspondence should be addressed.
Forests 2025, 16(10), 1557; https://doi.org/10.3390/f16101557
Submission received: 13 September 2025 / Revised: 3 October 2025 / Accepted: 7 October 2025 / Published: 9 October 2025
(This article belongs to the Section Wood Science and Forest Products)
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Abstract

Despite the long history of beech (Fagus sylvatica L.) red heartwood research, there has been no experimental proof on the structure of the chromophores yet. For the first time, using high-performance liquid chromatography/diode array detection/multistage electrospray ionization mass spectrometry, it was evidenced that red heartwood chromophores are water/methanol solvent extractable high molecular weight (400–2200 Da) compounds, which are polymerized, transformed, and oxidized products of (epi)catechin and taxifolin. Acid soluble non-extractable polyphenols (flavonoids, tannins) were not evidenced in the cell wall structure, while alkaline soluble compounds (ferulic acid, dehydrodiferulic acid, p-coumaric acid) have been identified for the first time from the sapwood/red heartwood boundary tissues: these supposedly play a role in the structural reinforcement of the cell wall structure and in the antioxidant protection and have a lesser role in color formation. Results on the structure of chromophores and on cell wall composition may enhance color homogenization technologies and contribute to a better utilization of red-heartwooded timber in the future.

1. Introduction

Red heartwood formation in beech (Fagus sylvatica L.) is a complex physiological and biochemical process that has significant implications for both the wood industry and forest management. Its research dates back already more than 120 years [1,2,3]. Most studies indicate that red heartwood formation is rare in young trees and becomes increasingly common as trees age, particularly after 80–100 years, with a marked increase in incidence and extent in stands older than 110 years. The process is gradual, with the transition from white to red heartwood occurring over a period of years rather than days or months [4]. To the best of our current knowledge, the most important predisposing factors of occurrence are trunk diameter and tree age [5,6,7]; however, other factors, like stem geometry, canopy structure, various environmental factors (soil quality, drought, extreme weather conditions, pollution), and forest management methods (thinning, stand composition) have been reported also to have an effect on red heartwood formation [3,4,8,9]. Presumably, the climatic extremes of recent years, the general physiological decline of beech forests [10], and the decrease in their vitality [11] also indirectly increase the frequency of red heartwood [12,13] due to the weakened health status of beech forests [14,15].
In the context of wood technology and industry, the phenomenon is characterized by a distinct reddish-brown discoloration in the central part of the stem, which is often perceived as a defect, reducing the commercial value of beech timber for high-grade applications [4,5,7,16,17]. The underlying mechanisms responsible for this color anomaly have been the subject of extensive research, with particular attention given to the role of polyphenolic compounds—secondary metabolites that are abundant in plant tissues and known for their diverse chemical structures and biological functions [18,19,20,21,22]. The formation of colored heartwood is primarily associated with the aging of trees, physiological changes in the xylem, and the accumulation of secondary metabolites, particularly polyphenolic compounds [18,23,24,25].
Polyphenols are secondary metabolites in plants. They are mostly categorized according to their different properties and traits (e.g., structure, biosynthesis, function, etc.). Regarding their location in the plant cell and extractability, they are classified as extractable polyphenols (EPs) and non-extractable polyphenols (NEPs). EPs are typically low- to medium-molecular-weight compounds (flavonoids, phenolic acids, and tannins) that are readily solubilized by aqueous-organic solvents and are often the focus of nutritional and functional studies [26,27,28]. In contrast, NEPs, also known as bound polyphenols, remain in the plant matrix after conventional extraction and are composed of high-molecular-weight polymers or small phenolics covalently or non-covalently bound to macromolecules such as cell wall polysaccharides, lignin, and proteins [29,30,31]. Both EPs and NEPs contribute significantly to the antioxidant capacity, color, and health-promoting properties of plant tissues and foods, but NEPs have historically been underappreciated due to analytical challenges [29,31,32,33]. Recent advances in extraction and analytical techniques have improved our understanding of the composition and function of both EPs and NEPs, highlighting their complementary roles in plant defense, human health, and potential industrial applications [29,32,34].
The distinction between these two classes is crucial for understanding the chemical processes that lead to the development of chromophores—molecular structures responsible for the absorption of visible light and, consequently, the coloration of red heartwood.
Recent advances in analytical techniques, such as high-performance liquid chromatography coupled with mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), have enabled the detailed characterization of polyphenolic profiles in beech wood. These studies have identified a wide array of compounds, including procyanidins, flavonoids (e.g., catechins, taxifolin, naringenin), and phenolic acids, many of which show increased concentrations at the boundary between normal wood and red heartwood [19,25]. Notably, the transformation of specific flavan-3-ols, such as (+)-catechin and (−)-epicatechin, has been linked to the enzymatic and oxidative processes that generate the red chromophores characteristic of heartwood [18,19]. The activity of phenol-oxidizing enzymes, such as peroxidases and polyphenol oxidases, is also elevated at the color boundary, further supporting the role of polyphenol metabolism in chromophore formation [18,19].
The interplay between extractable and non-extractable polyphenols is further complicated by the dynamic nature of heartwood formation, which involves both the synthesis of new polyphenolic compounds and the transformation of existing ones. Studies have shown that the concentration and composition of polyphenols vary radially within the stem, with notable changes occurring in the transition zone between sapwood and heartwood [22,25]. In addition, the presence of other extractives, such as lipophilic compounds and sugars, may influence the overall chemical environment and affect the stability and reactivity of polyphenols [17,35].
Comparative research on other hardwood species, such as walnut (Juglans regia L.), cherry (Prunus avium L.), and Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) has provided valuable insights into the general principles governing heartwood coloration and the role of polyphenols in chromophore formation [24,36,37]. Across tree species, chromophoric polyphenols in colored heartwood have a molecular weight range of 150–1500 Da. The specific types and concentrations of these compounds vary by species, but their weight range is consistent and seems to be central to heartwood coloration [38,39,40,41,42,43,44]. These studies highlight the importance of both the type and distribution of polyphenolic compounds, as well as the enzymatic and non-enzymatic reactions that lead to the development of colored pigments in heartwood tissues.
Despite significant progress, several questions remain regarding the precise chemical pathways and the relative contributions of extractable versus non-extractable polyphenols in the formation of chromophores in beech red heartwood, as well as the structure of chromophores. According to previous results, it was assumed that the chromophoric substances in the red heartwood of beech are either low molecular weight EPs or NEP compounds, or both types contribute to color [25]. Yet, these theories have not yet been investigated in detail.
The present research focuses on the comparative analysis of EP and NEP content of red heartwood in timber for the first time, for the elucidation of their contribution to red heartwood color. The EP and NEP content of selected tissues (sapwood, red heartwood transition zone, and inner red heartwood) of red heartwood in beech was investigated. First, the EPs were extracted from the wood tissues by conventional solvent extraction. Extracted (EP-free) samples were then subjected to acid/alkaline hydrolysis to release NEPs. Extracts were then subjected to high-performance liquid chromatography/diode array detection/multistage electrospray ionization mass spectrometry (HPLC-PDA-ESI-MS/MS) analysis to study their composition and to analyze and identify the structure of red heartwood chromophores.
Results of the present article contribute significantly to the understanding of the role and structure of polyphenols and chromophores in beech red heartwood formation. Despite the growing scientific interest in NEPs, only a limited number of studies are available that focus on the NEPs of woody tissues; a research gap that this also attempted to fill. Apart from academic significance, results on the structure of extractable chromophores and on the composition of cell wall NEPs may enhance color homogenization technologies and contribute to a better utilization of red-heartwooded timber for the wood industry.

2. Materials and Methods

2.1. Sample Collection and Extraction of EPs

For the present study, two mature and healthy trees were selected, which contained a typical and most frequent type (cloudy-shaped) red heartwood. Both trees have been processed and investigated fully; however, because of the identical results obtained for both trees, only one is presented in the article as a representative example. The presented tree originated from the forests of the TAEG Forestry Company (Tanulmányi Erdőgazdaság, Sopron, Hungary) and was felled in December. The age of the trees was between 100 and 110 years according to forest management records. After felling, the sample disk was cut from a height of 1.3 m. The disk diameter was 36 cm, and the red heartwood diameter was 18 cm. The sample disk was stored at −20 °C until processing.
Sampling followed the earlier published method of the authors [25]. In short, half of the quarter was cut out of the disk, and 8 sections (a–h) were assigned between the bark and the pith as depicted in Figure 1. The letters f and g represent the two sides (0.5 cm) of the color boundary (transition zone).
In the present study, only the sapwood (sample b), red heartwood boundary tissue (f and g combined, hereafter only referred to as sample f), and inner red heartwood tissue (h) were investigated to track changes in polyphenolic compositions during red heartwood formation. Wood samples (20 g) were separated using a wood rasp. The rasped wood particles were mixed to prepare representative and homogenized wood samples, then extracted (1.6 g wood sample + 30 mL methanol/water 50:50 (v/v) solution for 2 h in a 70 °C water bath). One extraction was carried out for each sample. Extracts were filtered and separated from the wood particles using filter paper. Extracts (10 mL) were evaporated to dryness under reduced pressure at 40 °C using a Büchi Rotavapor device (Büchi, Flawil, Switzerland) and redissolved in a 0.5 mL methanol/water 50:50 (v/v) solution for the analysis of EPs. Extracted wood particles were collected and dried for 4 h at 70 °C, then stored in a desiccator to reach and maintain complete dryness of the EP-free wood samples. Each sample yielded around 1 g of EP-free wood particles.

2.2. Hydrolysis of Extracted Wood Samples—Preparation of NEPs

Dried extracted wood samples were subjected to acid and base hydrolysis using three different methods: acid hydrolysis was conducted using 1 M HCl and 1 M H2SO4, while alkaline hydrolysis was carried out using 2 M NaOH. In all experiments same liquid/solid ratios were applied (0.25 g wood + 10 mL reagent), and reaction was carried out at 70 °C in a water bath for 4 h. After hydrolysis reaction solutions were cooled down to room temperature, neutralized (1 mL HCl hydrolysate + 0.050 g Na2CO3; 1 mL H2SO4 hydrolysate + 0.10 g Na2CO3; 1 mL NaOH hydrolysate + 100 µL formic acid) and centrifuged (at 12,000 rpm for 2 × 10 min in an Eppendorf MiniSpin G centrifuge (Eppendorf AG, Hamburg, Germany) then analyzed for NEPs. One hydrolysis was carried out for each sample.

2.3. HPLC-PDA-ESI-MS/MS Analysis of Extracts

The chromatographic separation and compound identification were conducted based on the previously described methodology of the authors [25]. For compound separation, a Shimadzu LC-20 type high-performance liquid chromatograph was used (Shimadzu Corporation, Kyoto, Japan) coupled with a diode array detector (Shimadzu SPD 20M, Shimadzu Corporation, Kyoto, Japan) and with an AB Sciex 3200 QTRAP triple quadrupole/linear ion trap mass spectrometer (Sciex, Framingham, MA, USA). Stationary phase: Phenomenex, Synergy Fusion C18, 2.6 µm, 150 mm × 4.6 mm column and Phenomenex SecurityGuard ULTRA LC guard column. Separation was performed at 40 °C, using solvent gradient of A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) as follows: 3% B (0–2 min), 15% B (36 min), 44% B (54 min), 100% B (66–72 min), 3% B (73–80 min) with a total flow rate of 1.2 mL/min. Sample injection: 15 µL. Diode array detection was performed in the visible (VIS) absorbance range characteristic of red/brown chromophoric substances (400–500 nm) and in the UV range (250–380 nm). The mass spectrometric detection was used for the profiling (identification) and structural analysis of polyphenolic compounds. Ion source settings: negative electrospray ionization (ESI), spray voltage: −4500 V, temperature: 500 °C, curtain gas (N2) pressure: 40 psi, spray gas (N2) pressure: 30 psi, drying gas (N2) pressure: 30 psi. Identification of polyphenols was accomplished by the IDA (Information Dependent Analysis) evaluation in the mass range of 150–1500 m/z using the RIKEN [45], MassBank [46], and MoNA [47] mass spectrometric databases. Data was acquired using Analyst 1.6.3. software (Sciex, Framingham, MA, USA).

3. Results

3.1. Evaluation of EPs of Red Heartwood

In earlier studies by the authors, the EP composition, content, and radial distribution of red heartwood in beech stems have already been assessed in detail [25]. However, it was found that inner red heartwood tissues had very low levels of polyphenolic compounds; thus, only a low number of compounds have been identified. For more efficient identification of low-concentration compounds and red heartwood chromophores, the concentrated extract of the inner red heartwood tissue was investigated. As the identification process has already been successful in the earlier study for the other tissues, it was not repeated in the present study; thus, we only discuss the EP composition of the inner red heartwood in this chapter.
As the color of red heartwood is reddish-brown, the main absorbance range corresponding to this color is between 400 and 500 nm (violet/blue) of the visible range. To selectively detect red heartwood chromophores, the detection wavelength during HPLC separation was set to this range. For comparison, a chromatogram was also recorded simultaneously for the UV range (250–380 nm) to detect all extractives (e.g., fatty acids, sterols, monomeric and non-oxidized polyphenols, etc.) in the red heartwood extract, which in turn do not necessarily have a color. Chromatograms of the EPs of the concentrated extract of the inner heartwood tissue are depicted in Figure 2.
According to Figure 2A, red heartwood extract contains a large number of extractives, justified by the high number of peaks in the UV chromatograms and corresponding large peak heights. However, these compounds come in various types and structures, and not all of them may contribute to the color of the red heartwood. In order to identify compounds with a red/brown color, the VIS (400–500 nm) chromatogram was recorded and analyzed. By the comparison of the UV and VIS chromatograms, it was concluded that only a minority of the extractives are actually chromophores, which is proven by the much lower number and intensities of peaks in the VIS chromatogram compared to the UV chromatogram. According to the results, it can be assumed that chromophoric substances comprise only a minority of the total amount of extractives of red heartwood, which explains their difficult detection due to their low concentrations.
Figure 2B depicts the total ion chromatogram (TIC), which corresponds to the total flow of ions of a given mass range (m/z) entering the detector of the mass spectrometer. TIC enables the estimation of the mass ranges of molecules detected during the separation process. The two TIC chromatograms were recorded independently in two separate chromatographic runs (one detecting the m/z 150–700 range and one detecting the m/z 700–1500 range), which is technically very important in order to avoid the suppression of the ionization of low-concentration compounds by more abundant/ionizable compounds in the detector, and enabling the direct comparison of the two TICs. It was established that the red heartwood extract is very abundant in low molecular weight compounds (m/z 150–700) and relatively poor in high molecular weight molecules (m/z 700–1500).
According to the results in Figure 2, it was concluded that, despite the low amounts of high-molecular-weight compounds, these are the chromophoric substances in the red heartwood. The majority of these compounds are eluted between 40 and 65 min. The detailed mass spectrometric identification of individual molecules of the major peaks of the VIS chromatogram is included in Table 1. Chromophoric compounds (peaks) were highlighted by numbers with brown color in Figure 2A, while the already-known extractives of the red heartwood [25] that have previously proven not to have a distinct reddish-brown color (e.g., taxifolin, (+)-catechin, (−)-epicatechin) were marked with black numbers.
For the mass spectrometric analysis of the structure of the compounds, relevant mass spectrometric databases and studies were used. Chromophores usually showed multiple ionization in the ion source, and most frequently their mass spectra contained the characteristic fragments of catechins (m/z 289, 271, 245, 203) and/or taxifolin (m/z 285, 241, 217, 175) units, thus chromophoric compounds have been proven to be products of these basic molecule units.
Since the fragments characteristic of monomers are present in the mass spectra of the colored compounds, it can be assumed that the chromophores are formed by the polymerization and partial transformation of phenol base units. If this were not the case, and the basic structure were also significantly modified, then the fragments characteristic of monomers would not appear in the mass spectra; however, this cannot be ruled out completely, because of the apparently large number and variety of chromophores and transformation products.
Figure 3 depicts the mass spectra of compounds 10, 12, 16, 17 (m/z 575, (epi)catechin + taxifolin), and compound 13 (m/z 753.5, (epi)catechin derivative), demonstrating the presence of the basic polyphenolic units in the structure of the polymers, while Figure 4 includes the structure of the basic compounds.
To the best of our knowledge, this is the first experimental proof and analysis of the molecular structure of the chromophores of beech red heartwood. According to our results, red heartwood chromophores are water/methanol-soluble, high-molecular-weight (400–2200 Da) compounds, which are polymerized and transformed (presumably oxidized) products of (epi)catechin and taxifolin. Due to the limitations of the mass spectrometric equipment, compounds with molecular weights higher than 2200 Da were also not evidenced, but their presence cannot be excluded.
The results apparently contradict our earlier results using the MALDI-TOF technique, where no high molecular weight compounds have been evidenced in the inner red heartwood tissues [25]. However, this can be explained by the fact that chromophores are present in small quantities in the red heartwood extract, and other non-chromophoric compounds present in relatively large quantities presumably suppressed the ionization of the chromophores in the MALDI ion source, rendering them undetectable. Due to the complexity of reactions, the further structural elucidation of higher molecular weight products requires further isolation, purification of the extracts, and the involvement of high-resolution mass spectrometry as well as nuclear magnetic resonance spectroscopy techniques.

3.2. The NEP Content of Beech Wood Tissues

3.2.1. Acid Hydrolysis

The HCl hydrolysis of the extracted wood particles yielded solutions with red color (Figure 5 inset). According to the literature, the color of beech wood shifts towards red when treated with acids, especially at higher temperatures. This is explained by the acid-catalyzed cleavage of hemicelluloses and the modification of lignin, which generate new colored compounds and chromophores within the wood [48]. The coloration apparently affected all investigated samples similarly.
The UV chromatograms of the three different tissues are completely similar, even when magnified for certain regions; no higher peaks can be detected for sapwood/heartwood and inner red heartwood samples compared to sapwood (Figure 5). These findings prove that HCl hydrolysis does not cleave any additional bound polyphenolic compounds from the cell wall structure in red heartwood tissues; thus, from these results, it cannot be justified that chromophores are bound to or incorporated in the cell wall structure as NEPs in the tissues of beech red heartwood, as suggested in the earlier study [25].
Acid hydrolysis was also conducted using H2SO4, which is not consistently better than HCl for hydrolysis and analysis of plant NEPs, but is specific to other types of NEPs: HCl hydrolysis often yields higher total phenolic and flavonoid content, while H2SO4 hydrolysis can yield higher levels of condensed tannins [49]; thus, the application of the different acids enables the study of several types of NEPs.
Figure 6 depicts the UV chromatograms of the H2SO4 hydrolysates of the samples and the reaction solution. Similarly to the HCl reaction, the solutions have the same reddish color, and the chromatograms are identical; no additional peaks were detected in the sapwood/red heartwood or in the red heartwood sample compared to the sapwood sample. Results of the chromatographic analysis of the acid hydrolysis of the extracted wood samples prove that red heartwood chromophores are not built up by additional flavonoid and tannin-type compounds bound to or incorporated into the cell wall structure during red heartwood formation in beech.

3.2.2. Alkaline Hydrolysis

Alkaline (NaOH) hydrolysis offers significant benefits over acid hydrolysis for analyzing plant NEPs, primarily by more effectively releasing bound polyphenols and increasing extraction yields [50]. NaOH breaks ester and ether bonds between polyphenols and the plant cell wall matrix more efficiently than acids, leading to greater liberation of bound phenolics [51]. Furthermore, alkaline hydrolysis can release a wider range of polyphenolic compounds, including those tightly bound to cell wall components, which may not be accessible through acid hydrolysis [52].
Figure 7 depicts the chromatograms of the NaOH hydrolysates of the samples and the reaction solution. There is an apparent difference between the color of the hydrolysate solutions, with sample b having a light brown color, while samples f and h have a dark brown color. The differences between the composition of the reaction solutions can also be tracked in the chromatograms. The most significant changes in the compositions were found in the increase in the peaks of ferulic acid isomers (peaks at 42.3 min and 51.2 min), p-coumaric acid (peak at 37.96 min), and dehydrodiferulic acid (peak at 46.0 min) at the sapwood/red heartwood boundary. The structure of the compounds was evidenced and confirmed by mass spectrometric analysis of the peaks. Structures of the identified compounds are depicted in Figure 8.
Ferulic acid and its polymers are critical for cross-linking and strengthening plant cell walls, thereby impacting rigidity, integrity, and resistance to degradation. Ferulic acid is esterified primarily to hemicelluloses (arabinoxylans) in the cell walls of plants. Through oxidative coupling, ferulic acid forms dehydrodimers, trimers, and tetramers, which cross-link arabinoxylan chains to each other and to other wall components [53,54]. Ferulic acid can also form ether bonds with lignin, creating covalent bridges between polysaccharides and lignin. This cross-linking integrates the carbohydrate and lignin networks, further reinforcing the wall structure [54,55]. These cross-links hinder enzymatic degradation, making plant biomass more resistant to microbial attack and reducing digestibility [54,56]. Ferulic acid cross-linking also plays a role in plant defense, limiting pathogen invasion and contributing to resistance against environmental stresses [53,54,57].
However, it must be emphasized that most studies on the role and occurrence of ferulic acid in plant cell walls focus predominantly on grasses and cereals, while results on woody plants are limited, not typically reporting ferulic acid as a significant NEP constituent [58].
To the best of our knowledge, this is the first research to report on the occurrence of ferulic acid in beech wood. Ferulic acid and its dimer, and possibly also other oligomers, are found in bound form as NEPs in the cell wall structure of beech wood. Moreover, its increase at the sapwood/heartwood boundary indicates that substantial wood chemical and structural changes are taking place in the cell wall structure during red heartwood formation, possibly aiming at the reinforcement of the cell wall structure due to the crosslinking with ferulic acid, which not only fortifies the structure but also provides additional resistance against biotic and abiotic stress. However, the role of ferulic acids in the formation of chromophores was not evidenced from the present study and requires further research.
Unlike ferulic acid, p-coumaric acid has a much lower propensity to form cross-links between polysaccharides or between polysaccharides and lignin. P-coumaric acid is a key modifier of lignin in plant cell walls, especially in grasses [55,59,60] and some woody plants [61]. Its main structural role is to modify lignin rather than cross-link cell wall carbohydrates [55,62]. The incorporation of p-coumaric acid into lignin can influence the spatial organization, linearity, and polymerization degree of lignin, affecting cell wall rigidity, digestibility, and resistance to degradation [59,60]. P-coumaric acid-modified lignin may contribute to plant defense, mechanical strength, and adaptation to environmental stresses, although its precise physiological roles are still being elucidated [63,64].
In earlier studies by the authors, elevated concentrations of extractable p-coumaric acid have been observed in the sapwood/red heartwood transition zone [25]. However, this is the first study to prove that p-coumaric acid is also present in the cell wall matrix as a NEP at the color boundary of red heartwood with elevated concentrations compared to other tissues. We assume that its presence is more likely to be associated with structural changes, and its role and contribution to the color formation of red heartwood need to be elucidated in the future.

4. Conclusions

The present article provided a comprehensive study on the extractable and non-extractable polyphenol content of red heartwood in beech tissues for the first time to assess their role in the processes of the formation of red heartwood chromophores. Major findings are summarized as follows:
  • The study was the first to provide experimental proof of the molecular structure of the chromophores of beech red heartwood. Red heartwood chromophores are water/methanol soluble high molecular weight (400–2200 Da) compounds, which are mostly polymerized, transformed, and oxidized products of (epi)catechin and taxifolin.
  • The presence and participation of other identified monomers (quercetin, isorhamnetin, gallic acid, etc.) in the structure of the chromophores was not evidenced, yet it cannot be excluded.
  • Results of the chromatographic analysis of the acid hydrolysis of the extracted wood samples proved that red heartwood chromophores are not built up by additional flavonoid and tannin-type non-extractable polyphenols bound to or incorporated into the cell wall structure.
  • Alkaline hydrolysis of beech wood tissues revealed that the sapwood/red heartwood transition zone contained significant amounts of ferulic acid, dehydrodiferulic acid, and p-coumaric acid as non-extractable polyphenols. They supposedly play a role in the structural reinforcement of the cell wall structure and in the enhancement of antioxidant protection against environmental stress during red heartwood formation, and may have a lesser role in the formation of the chromophores, yet their function needs further elucidation.
  • Results provide data for a better understanding of the biochemical reactions of red heartwood formation in beech. Novel information on the structure of chromophores and on the composition of the cell wall may enhance color homogenization technologies and contribute to a better utilization of red heartwood in timber.

Author Contributions

Conceptualization, T.H. and L.A.; methodology, T.H.; software, T.H.; formal analysis, E.V.-R.; investigation, T.H.; resources, L.A.; writing—original draft preparation, T.H.; writing—review and editing, T.H. and E.V.-R.; visualization, T.H.; funding acquisition, L.A. All authors have read and agreed to the published version of the manuscript.

Funding

This article was made in the framework of the project TKP2021-NKTA-43, which has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development, and Innovation Fund, financed under the TKP2021-NKTA funding scheme.

Data Availability Statement

Data will be made available on request.

Acknowledgments

During the preparation of this manuscript, the author(s) used the DeepL AI tool (free version) for the translation of texts from the Hungarian language. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The graphic description of the assignment of wood sections from sample disks; a–e: outer and inner sapwood tissues; f/g: sapwood/red heartwood transition zone; h: inner red heartwood [25].
Figure 1. The graphic description of the assignment of wood sections from sample disks; a–e: outer and inner sapwood tissues; f/g: sapwood/red heartwood transition zone; h: inner red heartwood [25].
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Forests 16 01557 g002
Figure 2. The HPLC-PDA-ESI-MS/MS separation of the red heartwood extract. (A) UV (250–380 nm) and VIS (400–500 nm) chromatograms. Brown numbers: chromophoric substances; black numbers: non-chromophoric substances. For compound numbers, please refer to Table 1. The UV chromatogram was clipped intentionally for a better visual comparison of UV and VIS chromatograms. (B) Total ion (TIC) chromatograms for m/z 150–700 and m/z 700–1500 mass ranges.
Figure 2. The HPLC-PDA-ESI-MS/MS separation of the red heartwood extract. (A) UV (250–380 nm) and VIS (400–500 nm) chromatograms. Brown numbers: chromophoric substances; black numbers: non-chromophoric substances. For compound numbers, please refer to Table 1. The UV chromatogram was clipped intentionally for a better visual comparison of UV and VIS chromatograms. (B) Total ion (TIC) chromatograms for m/z 150–700 and m/z 700–1500 mass ranges.
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Forests 16 01557 g003
Figure 3. The mass spectrum of the red heartwood chromophores is designated as peaks 10, 12, 16, 17 ((A): m/z 575, (epi)catechin + taxifolin) and as peak 13 ((B): m/z 753.5, (epi)catechin + derivative). For peak numbers, please refer to Table 1.
Figure 3. The mass spectrum of the red heartwood chromophores is designated as peaks 10, 12, 16, 17 ((A): m/z 575, (epi)catechin + taxifolin) and as peak 13 ((B): m/z 753.5, (epi)catechin + derivative). For peak numbers, please refer to Table 1.
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Forests 16 01557 g004
Figure 4. The structure of the basic compounds of red heartwood chromophores: (+)-catechin, (−)-epicatechin, and taxifolin.
Figure 4. The structure of the basic compounds of red heartwood chromophores: (+)-catechin, (−)-epicatechin, and taxifolin.
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Forests 16 01557 g005
Figure 5. The chromatographic separation (UV 250–380 nm chromatogram) of the hydrolysates produced in the acid (HCl) hydrolysis of beech wood tissues (b: sapwood (blue); f: sapwood/red heartwood boundary (magenta); h: inner red heartwood (black)) and the reaction solutions (inset).
Figure 5. The chromatographic separation (UV 250–380 nm chromatogram) of the hydrolysates produced in the acid (HCl) hydrolysis of beech wood tissues (b: sapwood (blue); f: sapwood/red heartwood boundary (magenta); h: inner red heartwood (black)) and the reaction solutions (inset).
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Forests 16 01557 g006
Figure 6. The chromatographic separation (UV 250–380 nm chromatogram) of the hydrolysates produced in the acid (H2SO4) hydrolysis of beech wood tissues (b: sapwood (blue); f: sapwood/red heartwood boundary (magenta); h: inner red heartwood (black)) and the reaction solutions (inset).
Figure 6. The chromatographic separation (UV 250–380 nm chromatogram) of the hydrolysates produced in the acid (H2SO4) hydrolysis of beech wood tissues (b: sapwood (blue); f: sapwood/red heartwood boundary (magenta); h: inner red heartwood (black)) and the reaction solutions (inset).
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Forests 16 01557 g007
Figure 7. The chromatographic separation (UV 250–380 nm chromatogram) of the hydrolysates produced in the alkaline (NaOH) hydrolysis of beech wood tissues (b: sapwood (blue); f: sapwood/red heartwood boundary (magenta); h: inner red heartwood (black)) and the reaction solutions (inset, left). Magnified region of the chromatogram (inset, right) enables better visual comparison of peak heights of compounds A: ferulic acid isomers (m/z 193), B: p-coumaric acid (m/z 163), and C: dehydrodiferulic acid (m/z 385).
Figure 7. The chromatographic separation (UV 250–380 nm chromatogram) of the hydrolysates produced in the alkaline (NaOH) hydrolysis of beech wood tissues (b: sapwood (blue); f: sapwood/red heartwood boundary (magenta); h: inner red heartwood (black)) and the reaction solutions (inset, left). Magnified region of the chromatogram (inset, right) enables better visual comparison of peak heights of compounds A: ferulic acid isomers (m/z 193), B: p-coumaric acid (m/z 163), and C: dehydrodiferulic acid (m/z 385).
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Forests 16 01557 g008
Figure 8. The structure of ferulic acid, p-coumaric acid, and dehydrodiferulic acid.
Figure 8. The structure of ferulic acid, p-coumaric acid, and dehydrodiferulic acid.
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Table 1. Chromatographic, mass spectrometric, and structural data of the identified EPs in the extract of the red heartwood tissue. Color indicates whether a certain compound has absorbance in the 400–500 nm wavelength range (Y) or not (N).
Table 1. Chromatographic, mass spectrometric, and structural data of the identified EPs in the extract of the red heartwood tissue. Color indicates whether a certain compound has absorbance in the 400–500 nm wavelength range (Y) or not (N).
Peaktr
(min)		CompoundColorParent ion [M-H]−
(m/z)		Charge (n)(Estimated) Molecular Weight
(Da)		Fragments (MS/MS)
124.7(+)-CatechinN2891290289, 271, 245, 203, 179, 125
229.55(Epi)catechin derivativeN4851486437, 361, 331, 289, 271, 245, 203
330.84(Epi)catechin derivativeN4851486437, 361, 331, 289, 271, 245, 203
432.5(−)-EpicatechinN2891290289, 271, 245, 203, 179, 125
533.8(Epi)catechin derivativeY798.221596691, 643, 289, 245, 203
635.3(Epi)catechin derivativeN4851486437, 361, 331, 289, 271, 245, 203
736.9(Epi)catechin derivativeN4851486437, 361, 331, 289, 271, 245, 203
841.35(Epi)catechin derivativeY713.41714543, 331, 289, 271, 245, 203
941.37(Epi)catechin + taxifolin derivativeY8631864693, 449, 289, 285, 245, 241, 217, 203, 175
1041.75(Epi)catechin + taxifolinY5751576539, 449, 423, 289, 285, 271, 245, 241, 217, 203
1141.8(Epi)catechin derivativeY811.521623631, 507, 315, 289, 245, 203
1242.4(Epi)catechin + taxifolinY5751576539, 449, 423, 289, 285, 271, 245, 241, 217, 203
1342.5(Epi)catechin derivativeY753.521507601, 449, 301, 289, 271, 245, 203
1443.25(Epi)catechin derivativeY771.621544441, 331, 303, 289, 271, 245, 203
1543.4TaxifolinN3031304285, 241, 217, 175
1643.8(Epi)catechin + taxifolinY5751576539, 449, 423, 289, 285, 271, 245, 241, 217, 203
1744.2(Epi)catechin + taxifolinY5751576539, 449, 423, 289, 285, 271, 245, 241, 217, 203
1845.8UnidentifiedY731.61732419, 389, 373, 359, 311
1946.1UnidentifiedY709.532130489, 471, 455, 441, 243
2046.3UnidentifiedY703.521408519, 419, 315, 169, 183
2146.7(Epi)catechin derivativeY4551456455, 301, 289, 271, 245, 203
2247.35UnidentifiedY731.732196577, 314, 285
2352.5UnidentifiedY711.821424545, 504
2454.8UnidentifiedY878.11878498, 227, 209, 183
2554.8UnidentifiedY867.211734
2660.75UnidentifiedY729.821460504, 357, 342, 193, 175
2760.83UnidentifiedY716.521434670, 557, 408, 369, 317
2861.1UnidentifiedY716.521434670, 557, 408, 369, 317
2961.2UnidentifiedY729.821460504, 357, 342, 193, 175
3061.3UnidentifiedY713.821426506, 343, 207, 207, 193, 175
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Hofmann, T.; Visi-Rajczi, E.; Albert, L. Role of Extractable and Non-Extractable Polyphenols in the Formation of Beech (Fagus sylvatica L.) Red Heartwood Chromophores. Forests 2025, 16, 1557. https://doi.org/10.3390/f16101557

AMA Style

Hofmann T, Visi-Rajczi E, Albert L. Role of Extractable and Non-Extractable Polyphenols in the Formation of Beech (Fagus sylvatica L.) Red Heartwood Chromophores. Forests. 2025; 16(10):1557. https://doi.org/10.3390/f16101557

Chicago/Turabian Style

Hofmann, Tamás, Eszter Visi-Rajczi, and Levente Albert. 2025. "Role of Extractable and Non-Extractable Polyphenols in the Formation of Beech (Fagus sylvatica L.) Red Heartwood Chromophores" Forests 16, no. 10: 1557. https://doi.org/10.3390/f16101557

APA Style

Hofmann, T., Visi-Rajczi, E., & Albert, L. (2025). Role of Extractable and Non-Extractable Polyphenols in the Formation of Beech (Fagus sylvatica L.) Red Heartwood Chromophores. Forests, 16(10), 1557. https://doi.org/10.3390/f16101557

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