Availability of extractives from various Norway spruce Picea abies stumps assortments

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY-NC-ND 4.0 https://creativecommons.org/licenses/by-nc-nd/4.0/ Availability of extractives from various Norway spruce Picea abies stumps assortments © 2022 The Author(s). Published with license by Taylor & Francis Group, LLC Published version Halmemies, Eelis S.; Brännström, Hanna E.; Karjalainen, Mikko; Nurmi, Juha; Alén, Raimo Halmemies, E. S., Brännström, H. E., Karjalainen, M., Nurmi, J., & Alén, R. (2023). Availability of extractives from various Norway spruce Picea abies stumps assortments. Journal of Wood Chemistry and Technology, 43(1), 13-27. https://doi.org/10.1080/02773813.2022.2152049 2023 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=lwct20 Journal of Wood Chemistry and Technology ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lwct20 Availability of extractives from various Norway spruce (Picea abies) stumps assortments Eelis S. Halmemies, Hanna E. Brännström, Mikko Karjalainen, Juha Nurmi & Raimo Alén To cite this article: Eelis S. Halmemies, Hanna E. Brännström, Mikko Karjalainen, Juha Nurmi & Raimo Alén (2022): Availability of extractives from various Norway spruce (Picea�abies) stumps assortments, Journal of Wood Chemistry and Technology, DOI: 10.1080/02773813.2022.2152049 To link to this article: https://doi.org/10.1080/02773813.2022.2152049 © 2022 The Author(s). Published with license by Taylor & Francis Group, LLC View supplementary material Published online: 09 Dec 2022. Submit your article to this journal View related articles View Crossmark data Availability of extractives from various Norway spruce (Picea abies) stumps assortments Eelis S. Halmemies a , Hanna E. Br€ annstr€ om b , Mikko Karjalainen c , Juha Nurmi b , and Raimo Al en a a Department of Chemistry, University of Jyv€ askyl€ a, Jyv€ askyl€ a, Finland; b Natural Resources Institute Finland, Kokkola, Finland; c UPM Kymmene, Lappeenranta, Finland ABSTRACT Stumps and knotwood of Norway spruce (Picea abies) are valuable sources of wood extractives. Although lignans from knotwood have already been utilized in value-added products, the behavior and valorization of stump-derived extractives are less studied. In this study, the composition of lipophilic and hydrophilic extractives, particularly lignans, from various spruce stump samples (stump bottom, stump heart, and crushed stump samples) stored outside were studied. Lipophilic and hydrophilic extracts were separated with an accelerated solvent extraction (ASE) apparatus using n-hexane and hot water, respectively. The detailed extractives content of samples was then determined by gas chromatography equipped with a flame ionization detector and a mass detector (GC-FID/MS) and high-performance liquid chromatography (HPLC). In stump bottom samples, an apparent decrease in total dissolved solids was observed in all the major extractives groups during storage: lignans, sugars, stilbene–glucosides, organic acids, resin acids, fatty acids, diterpenoids, and sterols. While a definitive decrease in extractives could not be demonstrated due to the moderately high variation of extractives among different samples, a good indication of the accessibility of important extractives in weathered stumps was obtained. Of the identified hydrophilic extractives, 79% were lignans, 53% of them being composed of 7-hydroxymatairesinol (HMR), 16% conidendric acid, and 12% todolactol. After 12 weeks of storage, the total amount of lignans was 15.3 mg/g of dry matter in stump bottom, 17.0 mg/g of dry matter in stump heart samples, and 10.2 mg/g of dry matter in crushed stump samples. KEYWORDS Lignan; 7-hydroxymatairesinol; high-performance liquid chromatography; gas chromatography-mass spectrometry; stump extractives; Norway spruce Introduction In recent years, biomaterials containing high contents of extractives, such as forest industry side-stream wood bark, are commonly used for producing energy, but their utilization for other purposes is also gradually increasing. Traditionally, the extractives from aged and pine trees (especially stumps) comprise an interesting substance group, which has been utilized, for example, to prepare tar in the Nordic countries. [1] This manufacturing process was started in the forest three to four years before the actual production in the tar pit by removing bark from pine trees, thus inducing excessive oleoresin formation of the trees. [2] In the case of pine stumps, the stump hearts may become increasingly resin-hardened and thus rot-resistant after felling. [3] This has historically made them especially favorable materials for tar production. However, this kind of resin saturation over time does not seem to occur readily in spruce stumps. Hence, the critical question is if there is a good rationale for the recovery and further utilization of spruce stump extractives, and is it worth the cost. The knotwood of many wood species has a high concentration of valuable extractives. For example, the knot wood of aspen has been found to contain increased amounts of flavonoids, [4] and the knotwood of pine lumber has been found to contain ten times higher terpenoid concentration compared to sapwood. [5] Similarly, the knotwood of Norway spruce (Picea abies) is known to be saturated by the fraction of lignans, the most prominent compound being 7hydroxymatairesinol (HMR). [6] However, P. abies stumps are also known to contain high levels of lignans and stilbene–glucosides. [7] Lignans are natural CONTACT Eelis S. Halmemies [email protected] Department of Chemistry, University of Jyv€ askyl€ a, Jyv€ askyl€ a, Finland. Supplemental data for this article can be accessed online at https://doi.org/10.1080/02773813.2022.2152049 ß2022 The Author(s). Published with license by Taylor & Francis Group, LLC This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/bync-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY https://doi.org/10.1080/02773813.2022.2152049 polyphenolic antioxidants that have been attributed with various health benefits, such as anti-breast cancer and prostate cancer activity. [8,9] The prospect of utilizing leftover Norway spruce stumps as feedstock materials, similarly to how spruce knotwood is already being used, for producing value-added chemicals, offers an attractive possibility. However, the development of spruce stump extractives-based products requires a broad chemical understanding of extractable compounds, together with the influence of stump aging on the content of extractives, which are of primary importance. The degradation of spruce bark, forest residues, and stumps was studied in the EU-funded research project called BioHub. This project’s general aim was to understand better the effects of storage on the chemical composition of various forest industrial sidestreams and, consequently, to find ways to improve their procurement practices. The main goal of this study was to increase the general knowledge and provide a broad picture of extractives behavior during stump storage by investigating the effects of outside storage on the chemical composition of various stump assortments. Our primary focus was on the most prominent compound fraction of stumps, namely, lignans. Materials and methods Storage studies and sampling A mature, Norway spruce-dominated stand was clearcut in Kannus, Finland, in early May 2017. The tree stumps were extracted and split into 2–3 segments with a standard stump rake attached to an excavator. This was followed by an immediate forwarding to a landing for storage during May 15–19, 2017. Stump segments were placed in 3 102.5 m (WLH) piles. Care was used not to contaminate stump sections with the underlying soil by placing them on an older set of stumps. Three types of samples were collected: (a) crushed stump, (b) stump heart, and (c) stump bottom (Figure 1). Collection times were at the initiation of the study (zero-sample, taken on May 23–24, 2017), 4, 12, and 25weeks after the initiation. The bottom and heart samples were collected using a chain saw without bar oil at each sampling time. These samples were debarked and comminuted to a smaller particle size with a 5.6 kW Murray Mulch Maker. The crushed stump samples were stored as whole until sampling and then crushed with a 708kW Vermeer HG6800TX horizontal grinder. After comminution, all samples were closed in plastic bags and a cooler box for immediate transport to the laboratory cold storage at –20 C for chemical analysis. Sample pretreatment and basic characterization The moisture content of fresh stump samples was determined by a standard method (CEN/TS 147742:2004). [10] The stump samples were dried at a temperature of 105C until a constant mass was achieved. All the measurements were performed in duplicate. The stumps were lyophilized (for three days) and ground with a Retsch SM 100 cutting laboratory mill (Retsch GmbH, Haan, Germany) equipped with a bottom sieve with trapezoidal holes (perforation size <1.0 mm) for chemical analysis. Samples were stored in a frozen state (<–20C). The dry matter content of each lyophilized stump sample was determined by drying about 1 g of stump powder in a tared crucible in an oven at 105 C oven overnight. Chemicals The solvents used in the sample preparation of extractives were analytical grade acetone (BDH), n-butanol (Merck), high-performance liquid chromatography (HPLC)-grade n-hexane (VWR), methyl tert-butyl ether (MTBE, Lab-Scan), pyridine (BDH), and 95% ethanol (EtOH, >94%, ETAX A, Altia Corporation). The silylation reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS), were from Regis Technologies. HPLC-grade methanol (MeOH, Merck), HPLC-grade acetonitrile (VWR International), formic acid (98%; Sigma-Aldrich, Espoo, Finland), and trans-polydatin (99%, PhytoLab) were used in the HPLC analysis stump samples. The compounds used as internal standards in the gas chromatography (GC) analysis of extractives were heneicosanoic acid (99%, Sigma), betulinol (98%, Sigma), cholesteryl margarate (97%, TCI America), and 1,3-dipalmitoyl-2-oleylglycerol (99%, Sigma). Other chemicals used in the analyses were NaOH (>98%, VWR), HCl (37%, VWR), Na 2 CO 3 (99.8%, Sigma), H 2 SO 4 (95–97%, Sigma), and bromocresol green (>95%, Sigma). Separation of stump extractives The extractions of stump samples were made via a Dionex Accelerated Solvent Extractor (ASE 100) using n-hexane and water as solvents to extract lipophilic and hydrophilic extractives, respectively. The extraction temperature was 120 C, static extraction time 2 E. S. HALMEMIES ET AL. was 10min, extraction cell flush was 60%, nitrogen purge was 70s, and extraction pressure was 1500 psi. Approximately, 2 g of dried stump powder was loaded to a 34-mL extraction cell plugged with a cellulose filter for each extraction. Each sample was first extracted with n-hexane and then with water. The extractions were performed in duplicates for each sample. Gravimetric analysis of total dissolved solids and stock solutions The total dissolved solids (TDS) of stump extracts were determined gravimetrically. The n-hexane extracts were evaporated to near dryness in a rotary evaporator, transferred to tared Kimax test tubes in acetone, and finally evaporated to dryness under nitrogen flow. The mass of the dried extract was the TDS of n-hexane extracts. Stock solution (100 mL) of the lipophilic extract was then prepared by dissolving the dried extract in acetone. Stock solutions of the hydrophilic extract were prepared by diluting the raw extract to 100 mL with ultra-high quality (UHQ) water. Then, 10 mL of the stock solutions was lyophilized and the TDS of the hydrophilic extracts was determined based on the lyophilized sample mass. Chromatographic analysis methods Qualitative analysis by GC-MS For qualitative analysis, 3 mg of extracts (based on dry mass) were dried (either by nitrogen flow or lyophilization) and dissolved in 500 lL of pyridine and 300 lL of the silylation reagent (BSTFA/TMCS (95/5, vol/vol)). The silylation was accelerated by keeping the sample in an oven at 70 C for 1 h. The sample was then analyzed by GC-MS using an HP-5 column (30 m 0.32 mm, with 0.25 lm film), injecting the sample at 290C and detecting the compounds with a mass selective detector (EI) at 300 C. The temperature program was: at 100 C (1.5 min), to 180 C (6 C/min) to 290 C(4 C/min), at 290 C (13 min), to 300 C(4C/min), and at 300 C (20 min). Quantitative analysis by GC-FID For quantitative analysis of individual lignans, approximately 3 mg of stump extracts were dried together with 100 lg of internal standards (heneicosanoic acid and betulinol), the mixtures were dissolved in 500lL of pyridine and 300 lL of the silylation reagent, and kept in an oven at 70 C for 1 h. Long column GC-FID equipped with an HP-5 column (30 m 0.32 mm, with 0.25 lm film) with injection at Figure 1. Preparation of the different stump samples for chemical analysis. (a) Crushed stump samples were prepared directly from pre-split stumps with roots intact. (b) The stump heart samples were cut as 10 cm 7cm 7 cm pieces from the de-rooted stump, while (c) the stump bottom samples were cut at 10 cm height from the bottom of the de-rooted stump and debarked. JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY 3 290 C and detection at 300 C was used for the analysis. The temperature program was: at 100 C (1.5 min), to 180 C(6 C/min), to 290C(4 C/min), at 290C (13min), to 300C(4 C/min), and at 300 C (20 min). Analysis by HPLC For the qualitative analysis of stump extractives with HPLC, 1 mg/mL dilutions of stump hot-water extracts were prepared in MeOH/H 2 O (50/50, vol/vol) and filtrated through a 0.2-lm polytetrafluoroethylene (PTFE) filter. The HPLC analysis was performed with an Agilent 1290 LC (liquid chromatography) instrument equipped with a ZORBAX StableBond column (80 Å C18, 2.1 mm 100 mm, 1.8 lm, 1200 bar), a ZORBAX SBC18 UHPLC guard column (2.1 mm, 1.8 lm), 1290 Infinity II Diode Array Detector, and a 6460 triple quadrupole mass spectrometer (LC/DAD/ QQQ). The LC columns were maintained at 30 C. Two solvents were used for the mobile phase: (A) 0.1% formic acid in UHQ water and (B) 0.1% formic acid in acetonitrile. The mobile phase flow rate was 0.4 mL/min. The run method was as follows: 5% B (from 0.0–20.0 min), 5–30% B (from 20.0–22.0 min), 30–80% B (from 22.0–24.0 min), and 80–5% B (from 24.0–25.0 min). Mass spectrometry analyses were performed in negative mode with a range of m/z 100–1200. The drying gas used was nitrogen at 10 L/ min at 350 C with a nebulizer pressure of 40 psi. The capillary voltage was 3100V. The Bruker Data Analysis 3.2 software was used for data processing. Polydatin was used as an external standard for quantifying the hydrophilic compounds from the stump hotwater extracts. Results and discussion Due to an unfortunate random accident during sampling, four stump samples (weeks 4 and 25 from crushed stump, and zero-sample and 25-weeks sample from the stump heart) were lost, effectively making stump bottom the only whole stump series. Despite this, the authors of this article decided that presenting the remaining results from the other series as directive references would still be helpful. Change in total dissolved solids The combined TDS from n-hexane and water extracts in the studied stump samples are presented in Figure 2. Depending on assortment and storage time, TDS varied between 2.9% and 8.4% of dry weight. These are slightly higher concentrations to those reported by Hakkila, who found acetone extracts of P. abies stumps to total 2.2%–4.3% of dry matter. [11] Sj€ oholm has similarly reported central stump of P. abies to contain, on average, 2.8% and 1.5% of dry matter acetone and dichloromethane extractives. [12] It should be noted that acetone is more selective solvent than hot water and ASE-extraction (with more harsh extraction conditions) was utilized by us while Sj€ oholm used more mild Soxhlet-extraction. In stump bottom, the hot-water extract totaled 70% of the extractives, 74% in crushed stump, and 73% in stump heart. In stump bottom, hydrophilic extractives ranged between 1.5% and 6.6% of dry matter, in crushed stump, 4.1%–5.8% of dry matter, and in stump heart, 1.9%–7.0% of dry matter. The total amount of n-hexane extractives ranged, in stump bottom, between 1.0% and 2.0% of dry matter, in crushed stump, 1.5% and 1.8% of dry matter, and in stump heart, 1.2% and 1.7% of dry matter. The crushed stump samples had consistently relatively high hydrophilic and lipophilic TDS, presumably caused by the bark material included in the samples. The root bark of Norway spruce is known for containing even up to 15%–29% of extractives. [13–15] In contrast, the lowest hydrophilic and lipophilic TDS seemed to be found in the debarked stump bottom samples. These results are comparable to those by Latva-M€ aenp€ a€ a, who found that P. abies stump wood contained 1.93% of dry matter acetone-soluble extractives. [16] Overall, there appeared to be no significant change in the total amount of lipophilic compounds in stump bottom or crushed stump samples during storage. Of the studied lipophilic and hydrophilic compounds, about 40% and 50% were identifiable by GCFID/MS, respectively. Unidentified compounds by GC-FID, in general, represent group of compounds that are poorly volatile and of higher molecular weight. Interestingly, unidentified hydrophilic compounds exhibited the most variation between individual stump samples. Variation between trees is not surprising by itself. The disparity in extractives content even between two trees of similar age can be high. Analogous results have also been reported regarding the extractives content between individual spruce logs. [17] Hydrophilic compounds in samples stored outside are generally lost more easily and prone to external influences, such as photodegradation via UV-light or leaching via rain. [18–21] In stump bottom, based on the identifiable (smaller molecular weight) compounds by GC, and despite the relatively low hydrophilic content of the zero-sample, an apparent 4 E. S. HALMEMIES ET AL. loss of hydrophilic extractives could be seen, aligning well with our expectations –namely, gradual loss of hydrophilic extractives. However, due to the relatively high variation between individual samples, especially in the amount of unidentifiable compounds, a definite conclusion on the decrease of stump extractives cannot be drawn based on these results. Qualitative and quantitative results by GC-FID/ GC-MS The hydrophilic extractives groups of the stump samples are presented in Figure 3. Among the studied samples, approximately 43% of the extractives were identified, of which 79% were lignans, 8% sugars, 8% stilbene–glucosides and other aromatics, and 2% organic acids. Hydrophilic extractives, in general, seem to be most abundant in the crushed stump samples, explained by the inclusion of rootbark known for its high extractives content. Picea abies bark is also known to contain oligosaccharides and distilbenes unidentifiable with GC-FID. [17,22] This could also explain the abundance of unidentified hydrophilic compounds in the crushed stump samples. Lignan concentrations, on the other hand, appeared to be higher in the stump bottom and heart samples, in particular. This result would agree with the known data that lignan concentration in stumps is high, especially in the heart of the stump. [7,23] In stump bottom, despite the exceptionally high concentration of unidentified compounds in the stump bottom samples at week 12, the general trend appeared to be that the hydrophilic extractives content is decreasing during the storage. The 25-week samples from the stump bottom had, on average, 32% less hydrophilic compounds compared to the zero-sample. The high variation in unidentified compounds may reflect the fact that the excavator-cut stump pieces used for sampling were unevenly shielded in the storage pile from weathering. As has been previously demonstrated regarding bark in pile storage, it could be assumed that the stump samples in the middle of the pile have higher hydrophilic content than those in the outer layers. However, to confirm this, more studies should be conducted. For this study, assuming that all of the stumps in a given pile would be utilized for valorization, choosing between individual stump pieces regarding the location in a pile (whether in the middle or on the top) was not done. Only the stumps at the bottom of the pile were Figure 2. The total dissolved solids of the stump samples. JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY 5 disregarded due to a risk of contamination from the soil. The concentration of lignans in the stump water extracts is presented in Figure 4. The greatest lignan concentration was found in spruce heart samples (on average 22 mg/g of dry matter) and the lowest in the crushed stump samples (on average 8 mg/g of dry matter). The average concentration of lignans in all the stump samples was 13.6 mg/g of dry matter. Much higher lignan concentrations have been found in the knotwood of spruce. Willf€ or et al. [24] found as high as 6%–24% and Mansikkala et al. [25] 16% of lignans in P. abies knotwood by dry weight; 65%–85% of which was HMR. Latva-M€ aenp€ a€ a et al. [7] also found HMR lignan in some of the Norway spruce root neck samples at a concentration of 10% of total dry matter. Although several different variables could explain the difference, such as the soil of the growth area or the age of the trees, it appears that one of the significant reasons for the higher concentration of lignans reported by Latva-M€ aenp€ a€ a et al. [7] is due to their more precise way of cutting and separating the heartwood from the stump samples. Poller and Storkan [3] have demonstrated already in 1978 that the stump center is the most concentrated with extractives. Latva-M€ aenp€ a€ a has also demonstrated that the lowermost part of Norway spruce root neck has the highest saturation of lignans. [16] Thus, given that lignans are also localized predominately in and around the heartwood, it could be assumed that the more actual heartwood in the sample is included, the higher the lignan concentration would be. Of the lignans discovered by us, in stump bottom, on average, 53% was HMR, 17% conidendric acid, 12% todolactol, 4% lignan A, 3% conidendrin, and 2% iso-HMR, isolariciresinol, lariciresinol, and other lignans, and 1% todolactol guiaiacyl ether, lignan A guaiacylglyceryl ether, nortrachelogenin, and oxo-matairesinol. In crushed stump, on average, 45% was HMR, 19% conidendric acid, 16% todolactol, 4% lignan A, 3% isolariciresinol and other lignans, 2% iso-HMR, lariciresinol, conidendrin, and 1% todolactol guiaiacyl ether, lignan A guaiacylglyceryl ether, nortrachelogenin, and oxo-matairesinol. In stump heart, on average, 56% was HMR, 15% conidendric acid, 10% todolactol, 4% conidendrin, 3% lignan A, 2% isoHMR, lariciresinol, and 1% isolariciresinol, other lignans, todolactol guiaiacyl ether, lignan A Figure 3. The hydrophilic extractives groups from the stump hot-water extracts as analyzed by GC-FID/MS. 6 E. S. HALMEMIES ET AL. guaiacylglyceryl ether, nortrachelogenin, and oxo-matairesinol. It was also likely that among the unidentified hydrophilic compounds determined by GC-FID/ MS, there was also a small amount of larger sesquilignans or dilignans, such as those discovered from P. abies knotwood by Mansikkala et al. [25] They found approximately 30% of the acetone extract of knotwood to consist of these larger lignan species. Although the amount of lignans in the studied stump samples exhibited significant variation, indications of losses due to storage time could also be seen. Even despite the seemingly unusually low concentration at the stump bottom zero-sample, the apparent degradation of lignans after 25 weeks of storage was still 59%. The amount of sugars in the stump hot-water extracts is presented in Figure 5. The average amount of sugars was approximately 1 mg/g of dry matter in the stump bottom and stump heart and 3 mg/g of dry matter in the crushed stump –a relatively insignificant amount compared to the amount of sugars in bark and sapwood. In P. abies bark, the hot-water extractable monosaccharides at 120 C can reach up to 65 mg/g of dry matter. [17,26] The higher sugar concentration in the crushed stump can be explained by its carbohydrate-rich bark material. Of the identified sugars and their derivatives, in stump bottom, 37% was glucose, 23% galactose, 19% sucrose, 8% pinitol, 5% palatinose, and 2% inositol and other sugars, and 1% mannitol, arabitol. In crushed stump, 45% was glucose, 24% sucrose, 11% pinitol, 10% galactose, 2% inositol, mannitol and arabitol, and 1% palatinose and other sugars. In stump heart, 30% was glucose, 25% sucrose, 24% galactose, 8% pinitol, 4% palatinose, 3% other sugars, 2% arabitol, and 1% inositol and mannitol. The amount of sugars, mainly glucose and sucrose, appeared to decrease systematically during storage among all samples, while the relative proportion of galactose increased, following the pattern of degradation observed in previous studies regarding the degradation of P. abies bark extractives. [17,26] Interestingly, a relatively high amount of galactose and pinitol was also found in the stump samples. Pinitol is a well-researched methoxy derivative of chiro-inositol –a cyclitol with noted self-defensive and medicinal capabilities. For example, plants with high pinitol concentrations have traditionally been utilized to treat diabetes and cancer. [27] The amount of stilbene–glucosides and other aromatic compounds from the stump samples is presented in Supplementary Figure S1. It should be noted that all of the stilbenoid species detected in the stump Figure 4. The lignans quantified by GC-FID/MS from stump hot-water extracts. JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY 7 References [1] Hennius, A. Viking Age Tar Production and Outland Exploitation. Antiquity 2018,92, 1349–1361. DOI: 10. 15184/aqy.2018.22. [2] Holmbom, B. Extraction and Utilisation of NonStructural Wood and Bark Components. In Biorefining of Forest Resources, 1st ed.; Al en, R., Ed.; Paper Engineers’Association: Helsinki, Finland, 2011; Vol. 20, pp 176–224. [3] Poller, S.; Storkan, O. Decay of Pine Wood by Trametes Pini.Holztechnologie 1978,19, 107–111. [4] Pietarinen, S. P.; Willf€ or, S. M.; Vikstr€ om, F. A.; Holmbom, B. R. Aspen Knots, a Rich Source of Flavonoids. J. Wood Chem. Technol. 2006,26, 245–258. DOI: 10.1080/02773810601023487. [5] Ingram, L. L., Jr., Templeton, M. C.; McGraw, G. W.; Hemingway, R. W. Knot, Heartwood, and Sapwood Extractives Related to VOCs from Drying Southern Pine Lumber. J. Wood Chem. Technol. 2000,20, 415–439. DOI: 10.1080/02773810009351892. [6] Cosentino, M.; Marino, F.; Ferrari, M.; Rasini, E.; Bombelli, R.; Luini, A.; Legnaro, M.; Delle Canne, M. G.; Luzzani, M.; Crema, F.; et al. Estrogenic Activity of 7-Hydroxymatairesinol Potassium Acetate (HMR/Lignan TM ) from Norway Spruce (Picea abies) Knots and of Its Active Metabolite Enterolactone in MCF-7 Cells. Pharmacol. Res. 2007,56, 140–147. DOI: 10.1016/j.phrs.2007.05.001. [7] Latva-M€ aenp€ a€ a, H.; Laakso, T.; Sarjala, T.; W€ ah€ al€ a, K.; Saranp€ a€ a, P. Root Neck of Norway Spruce as a Source of Bioactive Lignans and Stilbenes. Holzforschung 2014,68,1–7. DOI: 10.1515/hf-2013-0020. [8] Bylund, A.; Saarinen, N.; Zhang, J.-X.; Bergh, A.; Widmark, A.; Johansson, A.; Lundin, E.; Adlercreutz, H.; Hallmans, G.; Stattin, P.; M€ akela, S. Anticancer Effects of a Plant Lignan 7-Hydroxymatairesinol on a Prostate Cancer Model In Vivo.Exp. Biol. Med. (Maywood) 2005,230, 217–223. DOI: 10.1177/ 153537020523000308. [9] Miura, D.; Saarinen, N. M.; Miura, Y.; Santti, R.; Yagasaki, K. Hydroxymatairesinol and Its Mammalian Metabolite Enterolactone Reduce the Growth and Metastasis of Subcutaneous AH109A Hepatomas in Rats. Nutr. Cancer 2007,58,49–59. DOI: 10.1080/ 01635580701308133. [10] CEN/TS 14774-2: 2004. Solid Biofuels—Methods for the Determination of Moisture Content-Oven Dry Method-Part 2: Total Moisture-Simplified Method; British Standards Institute: London, UK, 2004. [11] Hakkila, P. Bark Percentage, Basic Density, and Amount of Acetone Extractives in Stump and Root Wood. Folia For. 1975,224,1–14. [12] Sj€ oholm, R. T. Acetone–Water Extractives from the Stump and Root Softwood and the Influence of the Removal of These through Pre-Extraction on the Pulping. Acta Acad. Aboensis 1977,37, 130. [13] Matthews, S.; Mila, I.; Scalbert, A.; Donnelly, D. M. Extractable and Non-Extractable Proanthocyanidins in Barks. Phytochemistry 1997,45, 405–410. DOI: 10. 1016/S0031-9422(96)00873-4. [14] Mart ınez-I~ nigo, M. J.; Immerzeel, P.; Gutierrez, A.; Rio, J. d.; Sierra-Alvarez, R. Biodegradability of Extractives in Sapwood and Heartwood from Scots Pine by Sapstain and White-Rot Fungi. Holzforschung 1999,53, 247–252. DOI: 10.1515/HF.1999.042. [15] Richter, A. K.; Frossard, E.; Brunner, I. Polyphenols in the Woody Roots of Norway Spruce and European Beech Reduce TTC. Tree Physiol. 2007,27, 155–160. DOI: 10.1093/treephys/27.1.155. [16] Latva-M€ aenp€ a€ a, H. Bioactive and Protective Polyphenolics from Roots and Stumps of Conifer Trees (Norway Spruce and Scots Pine). Doctoral Dissertation, University of Helsinki, Faculty of Science, Chemistry, Helsinki, Finland, 2017. [17] Halmemies, E. S.; Br€ annstr€ om, H. E.; Nurmi, J.; L€ asp€ a, O.; Al en, R. Effect of Seasonal Storage on Single-Stem Bark Extractives of Norway Spruce (Picea abies). Forests 2021,12, 736. DOI: 10.3390/f12060736. [18] Zahri, S.; Belloncle, C.; Charrier, F.; Pardon, P.; Quideau, S.; Charrier, B. UV Light Impact on Ellagitannins and Wood Surface Colour of European Oak (Quercus petraea and Quercus robur). Appl. Surf. Sci. 2007,253, 4985–4989. DOI: 10.1016/j.apsusc.2006. 11.005. [19] Mallory, F. B.; Mallory, C. W. Photocyclization of Stilbenes and Related Molecules. Org. React. 2004,30, 1–456. DOI: 10.1002/0471264180.or030.01. [20] Olsson, V. Wet Storage of Timber: Problems and Solutions. Master’s Thesis, KTH Royal Institute of Technology, Stockholm, Sweden, 2005. [21] Krigstin, S.; Wetzel, S. A Review of Mechanisms Responsible for Changes to Stored Woody Biomass Fuels. Fuel 2016,175,75–86. DOI: 10.1016/j.fuel.2016. 02.014. [22] Bianchi, S.; Koch, G.; Janzon, R.; Mayer, I.; Saake, B.; Pichelin, F. Hot Water Extraction of Norway Spruce (Picea abies [Karst.]) Bark: Analyses of the Influence of Bark Aging and Process Parameters on the Extract Composition. Holzforschung 2016,70, 619–631. DOI: 10.1515/hf-2015-0160. [23] Hakkila, P. Utilization of Residual Forest Biomass. In: Utilization of Residual Forest Biomass; Springer Series in Wood Science; Springer: Berlin, Heidelberg, 1989; pp 352–477. DOI: 10.1007/978-3-642-74072-5_8.. [24] Willf€ or, S.; Hemming, J.; Reunanen, M.; Eckerman, C.; Holmbom, B. Lignans and Lipophilic Extractives in Norway Spruce Knots and Stemwood. Holzforschung 2003,57,27–36. DOI: 10.1515/HF.2003.005. [25] Mansikkala, T.; Patanen, M.; K€ ark€ onen, A.; Korpinen, R.; Pranovich, A.; Ohigashi, T.; Swaraj, S.; Seitsonen, J.; Ruokolainen, J.; Huttula, M.; et al. Lignans in Knotwood of Norway Spruce: Localisation with Soft X-Ray Microscopy and Scanning Transmission Electron Microscopy with Energy Dispersive X-Ray Spectroscopy. Molecules 2020,25, 2997. DOI: 10.3390/ molecules25132997. [26] Halmemies, E. S.; Al en, R.; Hellstr€ om, J.; L€ asp€ a, O.; Nurmi, J.; Hujala, M.; Br€ annstr€ om, H. E. Behaviour of Extractives in Norway Spruce (Picea abies) Bark during Pile Storage. Molecules 2022,27, 1186. DOI: 10. 3390/molecules27041186. 14 E. S. HALMEMIES ET AL. [27] S anchez-Hidalgo, M.; Le on-Gonz alez, A. J.; G alvezPeralta, M.; Gonz alez-Mauraza, N. H.; MartinCordero, C.; Pinitol, D. A Cyclitol with Versatile Biological and Pharmacological Activities. Phytochem. Rev. 2021,20, 211–224. DOI: 10.1007/s11101-02009732-2. [28] Kebbi-Benkeder, Z.; Colin, F.; Dumarc¸ay, S.; G erardin, P. Quantification and Characterization of Knotwood Extractives of 12 European Softwood and Hardwood Species. Ann. For. Sci. 2015,72, 277–284. DOI: 10. 1007/s13595-014-0428-7. [29] Reena, R.; Sindhu, R.; Balakumaran, P. A.; Pandey, A.; Awasthi, M. K.; Binod, P. Insight into Citric Acid: A Versatile Organic Acid. Fuel 2022,327, 125181. DOI: 10.1016/j.fuel.2022.125181. [30] Ramachandran, S.; Fontanille, P.; Pandey, A.; Larroche, C. Gluconic Acid: Properties, Applications and Microbial Production. Food Technol. Biotechnol. 2006,44, 185–195. http://ir.niist.res.in:8080/jspui/handle/123456789/2172. [31] Alakoski, E.; J€ ams en, M.; Agar, D.; Tampio, E.; Wihersaari, M. From Wood Pellets to Wood Chips, Risks of Degradation and Emissions from the Storage of Woody Biomass–a Short Review. Renew. Sust. Energ. Rev. 2016,54, 376–383. DOI: 10.1016/j.rser. 2015.10.021. [32] Krigstin, S.; Helmeste, C.; Jia, H.; Johnson, K. E.; Wetzel, S.; Volpe, S.; Faizal, W.; Ferrero, F. Comparative Analysis of Bark and Woodchip Biomass Piles for Enhancing Predictability of Self-Heating. Fuel 2019,242, 699–709. DOI: 10.1016/j.fuel.2019.01.056. [33] Li, X.; Yuan, J.; Xu, S.; Wang, J.; Liu, X. Separation and Determination of Secoisolariciresinol Diglucoside Oligomers and Their Hydrolysates in the Flaxseed Extract by High-Performance Liquid Chromatography. J. Chromatogr. A 2008,1185, 223–232. DOI: 10.1016/j. chroma.2008.01.066. [34] Davin, L. B.; Wang, H.; Crowell, A. L.; Bedgar, D. L.; Martin, D. M.; Sarkanen, S.; Lewis, N. G. Stereoselective Bimolecular Phenoxy Radical Coupling by an Auxiliary (Dirigent) Protein without an Active Center. Science 1997,275, 362–366. DOI: 10.1126/science.275.5298.362. [35] Swanson Health Products. Ultra-7-HMRlignans from Norwegian Spruce Tree. https://www.swansonvitamins. com/swanson-ultra-7-hmrlignans-from-norwegianspruce-tree-40-mg-60-caps (accessed Nov 11, 2022). JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY 15