Č ufar , Tišler : Lipophilic Extractives in Heartwood of European Larch

The heartwood of two European larch trees was examined for the content of lipophilic extractives. Hexane was used as a solvent for extractions, while gas chromatography with fl ame ionisation detection and gas chromatography coupled to mass spectrometry were applied for analyses. Different lipophilic groups of compounds, such as fatty acids, resin acids, diterpenoids, sterols, steryl esters and triglycerides were identifi ed and quantitatively evaluated as well as individual low molecular mass components. Distribution of heartwood lipophilics in relation to the trunk heights was also determined and their most likely biological function in wood tissues discussed. The content of hexane extract increased with stem height. Various fatty and resin acids as well as diterpenoid alcohols and sterols were characterized. The predominating lipophilic compounds identifi ed were isopimaric acid and diterpenoid alcohol larixyl acetate. Their average concentrations in examined samples was between 2.0 and 2.5 mg/g. Higher molecular mass lipophilics, e.g. steryl esters and triglycerides, were also present with concentrations between 0.5 and 2.2 mg/g.


INTRODUCTION
1. UVOD Hydrophilic extractives are nonstructural wood components, which are readily soluble in polar solvents, such as acetone, methanol, ethanol and water.In contrast to lipophilic compounds in resin canals and parenchyma cells of sapwood (Zule et al., 2015), they mainly originate from the transition zone between heartwood and sapwood.Their concentrations are the highest at the boundary, after which they gradually decrease in radial direction towards the pith.Hydrophilics are mostly composed of phenolic compounds.Their synthesis is genetically regulated so each tree species contains specifi c compounds by which it differs from other species.Phenolic type compounds are comprised of aromatic components from simple phenols to complex phenolic polymers.
Lignans are an important group of plant phenols and are widespread in the plant world.Typical for them is oxidative coupling of two phenylpropane (C 6 C 3 ) units via linkage between β-positions on propane side chains.In addition to dimeric lignans, minor quantities of trimers (sesquilignans) and tetramers (dilignans) can also be found in wood.They are generally called oligolignans.Several tens of lignans have been identifi ed so far in wood tissues.However, the predominating are dimeric components, e.  Bohm, 1998;Stenius, 2000;Willför et al., 2006).
On account of their specifi c chemical structure and related antioxidative properties, phenolic compounds are toxic for numerous microorganisms, fungi and insects.They function as powerful natural biocides and enable chemical protection of wood against harmful impacts and biological degradation (Rennerfelt and Nacht, 1955;Hart, 1989;Willför, 2002).
Wood of larch (various species of the genus Larix) is generally known as durable as well as resistant against weather conditions, acids and water.So far, several authors have studied chemical composition of hydrophilic extractive fraction of the heartwood of dif-ferent larch species (Gripenberg, 1952;Brewerton, 1956;Nair and von Rudloff, 1960;Gardner and Barton, 1960;Sasaya et al., 1970;Lepteva et al., 1971;Tyukavkina et al., 1973;Giwa and Swan, 1975;Keith and Chauret, 1988;Chui and Mackinnon-Peters, 1995).They found out that the predominating hydrophilics were fl avonoids.Their concentrations, measured in radial direction at representative stem height (1.5 m), were highest at the heartwood/sapwood boundary.
Recently, many studies have also been dedicated to evaluation of larch wood as potential source of technologically important highly bioactive phenolic compounds, such as taxifolin, dihydrokaempferol and secoisolariciresinol (Babkin et al., 1999 Most authors focused on chemical characterization of larch heartwood at the representative stem height (1.5 m), while there is almost no data on the content of extractives at different positions along the stem and in branches.
The aim of the present work was to make a detailed chemical characterization of hydrophilics in the heartwood of European larch (Larix decidua Mill.) and to determine their variability in longitudinal direction within a tree and between two trees selected for analyses.This article is the continuation of the previously published work (Zule et al., 2015), where lipophilic extractive compounds of the same samples were characterized.

Uzorci
Two larch trees (Larix decidua Mill.) were felled at the end of June, 2009 in the Alpine region of Slovenia at the altitude of 1000 m.They originated from a mixed forest where beech (Fagus sylvatica L.) and spruce (Picea abies Karst.)predominated.Both larch trees were 180 years old and about 30 m high.They were healthy without visual damage.About 5 cm thick discs were cut from the trunks at the base (50 cm above ground) as well as at the heights of 8, 18 and 28 m.The discs were debarked and representative sections of heartwood (circular sectors from the pith to the heartwood/sapwood boundary) were cut into smaller pieces, which were subsequently frozen at -24°C prior to analysis.

Drying and grinding 2.2. Sušenje i mljevenje
Frozen samples were fi rst conditioned at room temperature, after which they were cut into short splin-ters, freeze-dried for 24 hours (Martin Christ Gefriertrocknungsanlagen GmbH) and ground by means of a Wiley laboratory mill (0.150 mm) to wood meal.The latter was freeze-dryed again for 24 hours in order for volatile compounds to be removed.

Extraction 2.3. Ekstrakcija
Extractions were conducted by means of an accelerated solvent extraction system (ASE) using the instrument Dionex ASE 200.About 5 g of each freezedried powdered sample was weighed into a metal extraction cell and sequentially extracted fi rst with hexane (V-50 ml) to remove lipophilic fraction (Zule et al., 2015) and afterwards with 95 % ethanol (V-50 ml) to isolate hydrophilic extractive portion.The temperature of the fi rst extraction step was 90 ºC, pressure 13.8 MPa and extraction time 10 minutes (2 static cycles with static time of 5 minutes).Ethanol extraction was carried out at 100 ºC under the same experimental conditions.Both extractions were performed under the stream of nitrogen.

Derivatization of extractives 2.4. Derivatizacija ekstraktiva
All ethanol extracts were derivatized prior to chromatographic analyses (GC-FID, GC-MS) by which components with hydroxyl groups, such as fl avonoids and lignans, were converted to the corresponding trimethylsilyl (TMS) derivatives, which where less polar and so more convenient for subsequent chromatographic analyses.2 ml of internal standard solution were added to each extract, containing about 0.5 mg of extractive compounds.Internal standards were heneicosanoic acid (S1), betulinol (S2), cholesteryl heptadecanoate (S3) and 1.3-dipalmitoyl-2-oleyl glycerol (S4), all having concentration of 0.02 mg/ml.The mixture of a sample and standards was dried under the stream of N 2 and in vacuum desiccator at 40 ºC, after which sylilation reagents were added: 80μl BSTFA (bis-trimethylsilyl-trifl uoroacetamide) and 20 μl TMCS (trimethyl-chlorosilane) in 20 μl of pyridine.The reaction mixture was heated for 1 hour at 70 ºC, than it was cooled and injected into gas chromatograph (Willfor, 2007).

Identifi cation of extractive compounds by
GC-MS 2.5.1.Identifi kacija ekstraktivnih spojeva metodom GC-MS Characteristic components of the representative ethanol extracts were identifi ed by means of gas chromatography coupled to mass spectroscopy (GC-MS).The analyses were performed on the HP 6890-5973 GC-MSD instrument.The separation was carried out on the HP-1 (30 m x 0.25 mm x 0.25 μm) capillary column under the following experimental conditions: temperature program of column heating 80 ºC, 8 ºC/ min, 290 ºC; carrier gas He (0.9 ml/min); split injector (1:15) -260 ºC; MS-EI detector (source temp.280 ºC, 70 eV, quadrupole temp.180 ºC).Mass range (m/z) was between 10 and 1050.For positive identifi cation of individual compounds, the mass spectra of their chromatographic peaks were compared with spectra of pure compounds from the Wiley Registry NIST 2008 Mass Spectral Library.On both GC-FID and GC-MS chromatograms there was practically the same sequence of chromatographic peaks of individual compounds as similar long capillary columns were used, by which reliability of identifi cation was ensured and quantitative work facilitated (Willfor, 2007).

GC-FID analysis on long capillary column 2.5.2. GC-FID analiza na dugoj kapilarnoj koloni
The determination of individual fl avonoids, lignans and other hydrophilic compounds was accomplished by means of gas chromatography on the Perkin Elmer AutoSystem XL instrument.HP-1 (25 m x 0.20 mm x 0.11 μm) capillary column was used for separation of compounds under the following experimental conditions: temperature program of column heating: 120 ºC, 6 ºC/min, 300 ºC (10 min); carrier gas H 2 (0.8 ml/min); split injector (1:20) -160 ºC, 8 ºC/min, 260 ºC (15 min); FID detector: 320 ºC; injection volume 1 μl.Betulinol (S2) served as standard in determination of phenolic compounds.The latter were calculated by comparison of the corresponding peak areas using correction factor 1.2.All results are expressed as milligram per gram of dry sample weight, where the limit of quantifi cation was about 0.01 mg/g (Willfor, 2007).

Characterization of phenolic compoudns by
HPLC-SEC 2.5.3.Karakterizacija fenolnih spojeva metodom HPLC-SEC High performance size exclusion chromatography (HPLC-SEC) was used for the determination of molecular mass distribution of phenolic compounds.The presence of dimeric, trimeric and tetrameric phenols in representative ethanol extracts was thus confi rmed as higher phenols can not be detected by gas chromatography due to their low volatility.The selected nonderivatized ethanol extracts of heartwood samples were dried under the stream of N 2 , after which they were redissolved in THF, so that the concentration was 1 mg/ml.The analyses were carried out on the chromatographic system, composed of the following units: autosampler Spark Holland Marathon-XT, pump Shimadzu LC -10ATVP, chromatographic columns 2x Jordi Gel DVB 500A (300 mm x 7.8 mm) and detector Sedere SEDEX 85 ELSD, which is a low temperature evaporative light scattering detector.Tetrahydrofuran (THF) was used as eluent with the fl ow of 0.8 ml/min.The injection volume was 50 μl.

Gravimetric determination of the content of
hydrophilic extractives 2.6.Gravimetrijsko određivanje sadržaja hidrofi lnih ekstraktiva 10 ml aliquots of ethanol extracts were dried under the stream of N 2 and in vacuum desiccator at 40 ºC until a constant weight was reached.The gravimetric amount of extractives was calculated as milligram per gram of dry sample weight.All quantitative determinations of hydrophilic extractives in the samples of heartwood were performed in two parallels.The presented results are average values of individual determinations.

Gravimetrijsko određivanje etanolnih ekstrakata
Hexane extracted heartwood samples, from which lipophilics (free fatty and resin acids, diterpenoids, triglycerides, steryl esters and sterols) had been removed, were consecutively extracted in the same way by a polar solvent ethanol (95 %) to obtain hydrophilic extractives.Gravimetrically determined content of ethanol extract in the heartwood of both trees is presented in Figure 1.Evidently, the concentrations were slightly higher in the base of the trees (0.5 m), however they remained more or less constant further up the stems.The average values were 28.0 ± 2.8 mg/g for Tree 1 and 30.5 ± 2.7 mg/g for Tree 2, calculated on dry mass of heartwood.Obviously, there was no signifi cant difference between the two trees.
Ethanol proved to be a suitable polar solvent.It is less toxic than methanol and less fl ammable than acetone.It can be easily recycled.The ASE extraction method has many advantages over commonly used Soxhlet method.It is automated and computer controlled.The whole process is very quick due to the application of elevated temperature and pressure, while solvent consumption is signifi cantly reduced.It is indispensable method for sequential analyses of large numbers of samples.The results are comparable to Soxhlet.

Identifi cation of individual phenolic compounds (GC-MS) 3.2. Identifi kacija pojedinačnih fenolnih spojeva (GC-MS)
Identifi cation was performed by GC-MS analyses of two typical ethanol extracts of the heartwood of tree 1 at 0.5 and 28 m of height.The following fl avonoids and lignans were identifi ed: naringenin, taxifolin (2 isomers), dihydrokaempferol, secoisolariciresinol, isoliovil, lariciresinol, todolactol A and nortrachelogenin.In addition, some monomeric and dimeric sugar units were also detected in the extracts.

Determination of the composition of ethanol extracts related to stem height 3.3. Određivanje sustava etanolnih ekstrakata u odnosu prema visini stabla
The composition of ethanol extracts was established from GC-FID chromatograms, recorded on a 25 m long capillary column.Qualitative composition of phenols was almost the same for all heartwood samples of both trees up to the stem height of 18 m, while it considerably changed at the height of 28 m near the top of the trees.The fl avonoids dihydrokaempferol and taxifolin predominated in all samples at lower and middle stem heights, where lignans were present only in trace amounts.On the other hand, the concentrations of lignans signifi cantly increased at the top of both trees at 28 m.Secoisolariciresinol was far the most abundant lignan in the heartwood of both trees.GC-FID chromatograms of ethanol extracts of the heartwood of tree 1 at 8 and 28 m are presented in Figures 2 and 3.
Total fl avonoid and lignan concentrations, which were calculated from the corresponding GC/FID chromatograms, varied between 10.6 and 12.6 mg/g in tree 1, while the corresponding values in tree 2 were slightly higher -between 12.4 and 15.7 mg/g.Concentrations of dihydrokaemferol were in the range between 1.8 and 7.1 mg/g and those of taxifolin between 3.3 and 8.4 mg/g.
Secoisolariciresinol was the most abundant lignan and it was even the predominant phenolic compound at the top of tree 1 (Figure 4).The concentration of secoisolariciresionol in the heartwood of tree 1 at 28 m was 4.2 mg/g and in the heartwood of tree 2 at the same height it was 3.4 mg/g.It is very interesting that its concentration never exceeded 0.2 mg/g in the lower sections of heartwood.
The content and composition of phenols in the heartwood of trees 1 and 2 in relation to stem height is shown in Figures 4 and 5.
From the comparison of gravimetric and chromatographic results, it was evident that the contents of ethanol extracts were at least twice as high as the chromatographically determined concentrations of fl avonoids and lignans in those extracts.This could be partly ascribed to the presence of different sugar units, such as mono-and disaccharides in ethanol extracts, as they are readily soluble in polar solvents.However, some substances, which were simultaneously extracted by ethanol, could not be detected by GC-FID due to their ( 3 ) ( 4 ) higher molecular mass and thus lower volatility.The presence of higher phenolic structures was unambiguously confi rmed by HPLC-SEC (Figure 6). Figure 6 shows the molecular mass distribution of a THF redissolved typical ethanol extract.The most intensive peak (L2) with the retention time of 21 min represents dimeric phenols, e.g.fl avonoids and lignans, while weaker peaks with retention times between 18 and 20 min (L3 and L4) simbolize higher phenols having three and four phenolic groups in their molecular structure.According to the peak area ratio, it could be estimated that typical ethanol extracts of larch heartwood contained averagely about 20 % of higher phenols.Sugar units and other non phenolic components were not detected by HPLC-SEC under specifi ed experimental conditions.
The most abundant phenolic compounds of European larch, e.g.dihydrokaempferol, taxifolin and secoisolariciresinol exhibit, according to available literature data, very strong antioxidative properties, which can be ascribed to their specifi c molecular structure.The structural formulas of the most typical larch fl avonoids and lignans are presented in Figure 7.The biological activity of taxifolin could be attributed to the relatively high content of phenolic hydroxyl groups in the right ring of its molecular structure, while on the other hand secoisolariciresinol is very effi cient antioxidant on account of its butanediol structure (Figure 7).Presence of higher trimeric and tetrameric phenols also positively affects antioxidative properties of extractives and thus biological resistance of wood tissues against rotting (Pietarinen et al. 2006;Willför et al. 2003;Scalbert, 1991;De Bruyne et al. 1999).
The most signifi cant fi nding in the case of larch heartwood was that chemical structure of hydrophilic extractive fraction changed towards the top of both examined trees, which was not the case with lipophilic fraction of the same tissues (Zule et al., 2015).While the fl avonoids taxifolin and dihydrokaempferol predominated in the majority of heartwood, lignans appeared at the top in abundant concentrations with secoisolariciresinol as the main phenolic compound.Such distribution points to the fact that lignans are synthesized during early growth period, while later on, during wood aging, the synthesis proceeds more and more in the direction of fl avonoid formation (Willför, 2002).
Larch forest residues, such as tree tops, damaged wood, cuttings, sawdust, knots and branches from wood processing could serve as relevant source for large scale isolation of valuable bioactive compounds.The latter may be applied as "green chemicals" or natural preservatives in farmaceutic, food, chemical and other industries.The remaining extracted wood may be further chemically converted to different platform chemicals and biofuel, which is the main idea of wood biorefi neries.

ZAKLJUČCI
Nine different phenolic compounds, such as fl avonoids and lignans were determined in ethanol ex- tracts of the heartwood of two European larch trees.All substances identifi ed, e.g.naringenin, taxifolin (2 isomers), dihydrokaempferol, secoisolariciresinol, lariciresinol, isoliovil, northrachelogenin and todolactol A, are typical for larch species (European, Siberian, Japanese, Western, Tamarack and others) and are commonly not found in other conifers in any signifi cant amounts.Their average concentrations in the heartwood of the two examined trees were 12 ± 2 mg/g and 15 ± 2 mg/g, and did not change essentially in vertical direction along the stems.Flavonoids predominated at lower and middle positions while lignans were more abundant at the top of both trees.The most important fl avonoid taxifolin and lignan secoisolariciresinol are classifi ed as very powerful antioxidants which, in combination with other phenolic substances, most likely provides effi cient chemical protection of larch heartwood against rotting and harsh environmental conditions.Our study may contribute to better understanding of the chemistry of wood tissues.