Review on Lipophilic and Hydrophilic Extractives in Tissues of Common Beech

Common beech (Fagus sylvatica L.) is one of the most widespread and economically important tree species in Europe and, therefore, represents a potential source of high value added extractives. The aim of this paper was, therefore, to review the existing data regarding the composition of lipophilic and hydrophilic extractives of beech tissues, and the extraction systems and analytical techniques used for their examination. The lipophilic extractable fraction of beech is characterized mainly by saturated and unsaturated fatty acids, fatty alcohols and sterols while the hydrophilic extractives of beech consist of soluble sugars, i.e. monosaccharides, oligosaccharides, sugar alcohols and sugar acids, as well as of simple phenols and fl avonoids. Chromatography has been recognized as the convenient and most frequently used technique for the chemical analysis of extractives. This overview showed that the information about the composition of low-molecular extractives of beech is satisfactory, but the data on oligomeric extractives are still fragmentary.


INTRODUCTION 1. UVOD
Utilization of hardwood represents one of the main industrial and research issues of the wood sector in Europe.Common beech (Fagus sylvatica L.) accounts for approximately one third of the wood stock in Slovenian forests (Report of Slovenian Forest Service for 2011) and represents an economically important tree species with a relatively high potential in wood industry.One of the main defi ciencies of this tree species is its tendency to develop discoloured wood in the central part of the tree, also known as the red heart (Bosshard, 1974;Torelli, 1984).Red heart in beech has been investigated from various aspects, ranging from physiology, gross and minute anatomy, chemical composition and physical, mechanical and technological properties (Dietrichs, 1964a;Bauch, 1984;Sachsse, 1991;Torelli et al., 1994;Baum and Schwarze, 2002;Koch et al., 2003;Hofmann et al., 2004;Wernsdörfer et al., 2005;Oven et al., 2008;Mali et al., 2009;Vek et al., 2014;Vek et al., 2015).Generally, if compared to the unaffected wood, the value of discoloured round wood, as well as wood elements on the market, is usually lower (Zell et al., 2004) and, therefore, attempts were made to commercially promote discoloured beech wood (Koch, 2002).In addition to the main structural components of the cell wall, i.e. cellulose, hemicelluloses and lignin, different tissues of a living tree also contain smaller amounts of compounds, which can be removed from the plant tissue with the process of extraction and are, therefore, known as extractives.These are natural low-molecular compounds, the non-structural components of wood, located in the lumina of cells and extraneous to a lignocellulose cell wall.From the physiological aspect of view, the extractive compounds of plant tissue are primary and secondary metabolites (Rowe and Conner, 1979;Fengel and Wegener, 1989;Kai, 1991;Holmbom, 1999).Primary metabolites are present in every plant species, whereas specifi c secondary metabolites can be found only in some tree species or related group of species.Secondary metabolites were sometimes referred to as accessory compounds (Fengel and Wegener, 1989), because their important ecological functions in the tree have not been understood for a long time.It is known that these in general contribute to protection of plants and against herbivories and microbial pathogens, serve as attractants for pollinators and seed-dispersing animals and function as agents of plant-plant competition and plant-microbe symbioses (Taiz and Zeiger, 2002).
Extractives can be classifi ed according to their chemical similarities, with respect to biochemical paths of their synthesis or regarding the solvent, in which they are soluble.Based on solubility, extractives are divided into classes of lipophilic and hydrophilic extractives (Willför et al., 2006;Jansson and Nilvebrant, 2009).
As proposed by Holmbom (1999), extractives are usually analyzed at three levels, i.e. gravimetric determination of total extractives, determination of different component groups and analysis of individual components.The fi rst level of extractive analysis includes gravimetric and other determination of total extractives.It is generally known that the xylem of tree species in the temperate climate zone contains a relatively small amount of extractives, up to 5 -10 % (Umezawa, 2000).Anyway, the concentration can be much higher in certain parts of the tree, e.g. in branch bases, bark and roots.Moreover, higher amounts of extractives are also found in some tropical and subtropical woods (Fengel and Wegener, 1989;Holmbom, 1999).As reported by Wagenführ (1996), beech wood consists of 33.7 % to 46.4 % of cellulose, 11.6 % to 22.7 % of lignin, 11.8 % to 25.5 % of hemicelluloses, 3 % to 5 % of extractives, meanwhile the share of inorganic compounds represents 0.3 % to 1.2 %.By applying standardized analytical methods used in wood chemistry, 47.66 % of cellulose, 25.53 % of lignin, 69.01 % of holocellulose, 0.93 % substances soluble in benzene-alcohol, 2 % of substances soluble in hot water, 13.15 % substances soluble in 1 % NaOH and 0.3 % of mineral substances (ash) were determined by Bodirlau et al. (2008).The pH value of beech wood ranges from 5.06 up to 5. 13 (Hillis, 1987).Fengel and Wegener (1989) stated that the average pH value is 5.4.Even more, authors reported that the pH value of cold water extract is 5.5, while the pH value of hot water extract is slightly lower (5.3).Sixta et al. (2004) found 24.2 % of lignin in freshly prepared beech wood chips.Further carbohydrate analysis revealed 41.6 % glucan, 17.9 % xylan, 1.3 % mannan, 1.2 % galactan and 0.5 % arabinan (Sixta et al., 2004).It was reported that intact sapwood cont ains less lignin (19.75 %) than biologically decayed wood (20.82 %) (Košikova et al., 2008), whereas the origin of decay was not described in this research.Generally, beech wood is characterized b y a relatively low amount of extractives (Rowe and Conner, 1979).Furthermore, the information regarding t he content and composition of extractives in beech wood is poor.In the case of fresh beech wood chips, Sixta et al.
(2004) stated that 0.2 % of the compound are extracted with dichloromethane, 1.0 % with acetone, 1.7 % of extractives are soluble in ethanol, while 2.7 % are obtained with water.According to Košikova et al. (2008), in comparison to healthy sapwood, from which 1.78 % of extractives were gained, decayed wood contains less extractives soluble in acetone, i.e. 0.98 %.Furthermore, Kubel and Weissmann (1988) report that the amount of extractives, soluble in petrol ether and diethyl ether, is relatively low and it amounts to 0.2% and 0.1%, respectively.More compounds can be extracted by the mixture of alcohols and water, 1.6 % with acetone/water (9:1, v/v) and 1.2 % with ethanol/water (8:2, v/v).A relatively low amount of water soluble extractives (0.3 %) can be attributed to the successive extraction procedure.
The basic gravimetrical estimation of the amount of total extractives is usually upgraded by the second and third level of analyses, i.e. determination of the component groups in extracts and evaluation of composition of individual extractives.For the determination of groups of extractives in wood extracts, e.g. total phenols, fl avonoids or proanthocyanidins, a spectrophoto-metric analysis (UV-Vis) represents a quick and reliable technique (Baum and Schwarze, 2002;Albert et al., 2003;Brighente et al., 2007;Vek et al., 2013a;Vek et al., 2013b).The lack of semi-qualitative evaluation is that UV-Vis analysis gives relative and not absolute results as a consequence that the amount of component group is estimated towards the component of external standard, e.g.gallic acid, quercetin of catechin.Therefore, more precise chromatographic techniques are used, i.e. thin-layer chromatography (TLC), gas chromatography (GC) or high performance liquid chromatography (HPLC).Qualitative determination is possible by the 13 C NMR and eventually with Furrier transform infrared spectroscopy (FTIR) as well.Furthermore, for the determination of different groups of extractives by means of HPLC, both the size-exclusion (HPLC-SEC) and reversed-phase (RP) modes can be applied.Precise qualitative evaluation of individual compounds separated by chromatographic techniques (GC or HPLC) is most conveniently made by mass spectrometry (MS).Therefore, GC/MS and HPLC/MS represent an adequate technique for the identifi cation of extractives in complex mixtures such as plant extract.
The aim of the present work was to review the literature on extractives, which have been qualitatively evaluated as the extracts of beech tissue so far.In the following, the results of various chemical analyses of extrac-tives occurring in different types of beech tissue are presented separately for lipophilic and hydrophilic components (Holmbom, 1999;Naczk and Shahidi, 2007).

Lipophilic extractives 2.1. Lipofi lne ekstraktivne tvari
Lipophilic extractives are referred to as the compounds which are soluble in nonpolar organic solvents, e.g.pentane, hexane, petroleum ether, dichloromethane, chloroform or toluene.It is well known that lipophilic extractives can have a negative infl uence and may disturb the analysis of more polar compounds.Moreover, they may have a deleterious effects on chromatographic instrumentation due to column clogging (Slanina and Glatz, 2004).Therefore, it is recommended fi rst to remove the lipophilic extractives from the sample before further extraction with polar solvents (Naczk and Shahidi, 2004; Willför et al., 2006).Waxes, fats, fatty alcohols, fatty acids, terpenoids, sterols and steryl esters are known as the characteristic representatives of lipophilic extractives.Some of them were also identifi ed in the nonpolar extractible fraction of beech.
In the case of beech wood dust, extracts were characterized by a relatively low amount of lipophilic  In addition to fatty acids and alcohols, numerous glycerides, sterols and steryl esters were identifi ed as the lipophilic extractives of beech wood.An acyclic triterpenoid squalene (Figure 3) and cyclic cycloartenyl acetat, β-amyrin acetate, acetyl methyl betulinate and dihydrositosterol were extracted from dried wood by Pišova and Souček (1973).Furthermore, relatively low levels of β-carotene and lutein were found in the

Hydrophilic extractives 2.2. Hidrofi lne ekstraktivne tvari
After removing lipophilic extractives, the hydrophilic compounds are usually further extracted by means of more polar solvents, such as methanol, ace-tone, ethanol, water, etc. Soluble sugars and various phenolic extractives, e.g.simple phenolics, stilbenes, lignans, fl avonoids and tannins are known as the characteristic representatives of hydrophilic extractives.In a broader sense, inorganic compounds are also known as hydrophilic extractives and they can be extracted by water (Fengel and Wegener, 1989).The yield of hydrophilic extractives from beech wood is usually much larger than that of lipophilic extractives (Kubel and Weissmann, 1988).
Various monosaccharides and oligosaccharides, sugar alcohols and acids, simple phenolics, fl avonoids, both oligomeric units of tannins have been determined as the hydrophilic substances of beech wood.Among the monosaccharides, glucose, galactose, arabinose, fructose, xylose, mannose and rhamnose were identifi ed in the wood extracts and wood condensate of beech (Dietrichs, 1964b; Kubel and Weissmann, 1988;Irmouli et al., 2002).Furthermore, disaccharides saccharose and trehalose as well as trisaccharide raffi nose were identifi ed in the extracts of beech wood (Dietrichs, 1964b; Vek et al., 2014).The presence of both tetrasaccharide stachyose and polysaccharide starch was reported for the sapwood and wood condensate of beech, respectively (Dietrichs, 1964b; Irmouli et al., 2002).Sugar alcohols, i.e. erytritol, arabitol, sorbitol, mannitol, xylitol and glycerol, as well as inositols were qualitatively evaluated in extracts of wound-associated tissues and knots of beech (Zule and Može, 2003;Vek et al., 2014).In addition to sugar alcohols, the occurrence of several sugar acids (ribonic, threonic, gluconic and glucuronic acid) has been proven for the methanol extracts of stem wood by means of GC/MS (Figure 4).Our recent chromatographic study has also shown the presence of lowmolecular carboxylic acids in the extracts of both stem wood and knots of beech, i.e. malic, malonic and citric acid (Figure 5) (Vek et al., 2014).
Numerous simple phenols have been extracted from beech wood as well.Challinor (1996) applied gas chromatography coupled with mass spectrometry for the qualitative evaluation of extractives in the wood shavings/turnings of intact beech.As the characteristic compounds in the tetramethylammonium hydroxide extracts, 5-hydroxy, 2-hydroxymethyl pyranone (triv.kojic acid), 3,4-dimethoxybenzaldehyde, 3,4-dimethoxybenzoic acid methyl ester, 3,4,5-trimethoxybenzaldehyde and 3,4,5-trimethoxybenzoic acid methyl ester were found.From thermal treated wood, i.e. steamed and dried beech wood, a sinapyl alcohol, coniferyl alcohol, 2,6-dimethoxybenzoquinone, protocatechuic acid, vanillic acid, vanillin, syringic acid, coniferaldehyde, siringalydehide synapic acid, gallic acid, p-hy- Moreover, vanillin, vanillic acid and syringic acid are also known as the constituents of phenolic fraction of a depolymerized suberin in beech (Perra et al., 1993).In addition to simple phenols, the presence of aromatic dimeric structures, i.e. lignin-type compounds, was also proven in beech wood extracts (Koch et al., 2003;Vek et al., 2014).These dimers made up of coniferyl and sinapyl alcohol were referred to as the precursors of a lignin biosynthesis accordingly to Koch et al. (2003), but they can represent another group of phenolic extractives, called lignans.Lignans were found in extracts of beech knot by Lindberg et al. (2004).Furthermore, they reported about the presence of small amounts of stilbenes, generally known as bioactive compounds with the 1,2-diphenylethene structure (Lindberg et al., 2004).Some of simple phenolic compounds that have been shown as the extractives of beech wood are presented in Figure 6.
Flavonoids are commonly referred to as both the most important and abundant group of extractives of beech tissue (Brignolas et al., 1995;Pearce, 1996;Torelli, 2003;Vek, 2013).Catechin is the most frequently reported fl avonoid that can be extracted from the beech wood (Figure 7).A few research groups reported on the presence of catechin in the sapwood and discoloured wood (Kubel and Weissmann, 1988;Baum and Schwarze, 2002;Koch et al., 2003;Zule and Može, 2003;Hofmann et al., 2004;2008).Catechin was also found in the extracts of thermally treated and decayed beech wood (Mounguengui et al., 2007;Lekounougou et al., 2008).It was reported that the amount of catechin in discoloured tissues decreases due to its participation in the process of formation of colour chromophores (Hofmann et al., 2004).It was also suggested that, during thermal treatment of beech wood, the condensation of catechin contributes to a brown stain pig-ment, whereas other extractives, e.g. a 2,6-dimethoxybenzoquinone (Figure 6) and taxifolin (Figure 7), contribute to the fi nal colour of discoloured tissues (Koch et al., 2003).Therefore, the discolouration mechanism in beech wood can be understood as a condensation of catechin monomers to polymer forms.Besides wood, the presence of fl avonoids has also been shown for the extracts of other parts of beech tree, e. Phenolic compounds frequently occur as glycosides as the core aglycone in plant tissues.According to Sherwood and Bonello (2013), the sugar unit of phenolic glycoside serves to improve solubility for storage in cell organs, e.g.vacuoles.Some research groups described the presence of fl avonoid glycosides as characteristic for beech extracts (Figure 8).Catechin glucoside and taxifolin glucoside were found in the extracts of both normal and thermally treated beech wood (Kubel and Weissmann, 1988;Koch et al., 2003).Numerous glycosides, i.e. taxifolin-O-pentoside, taxifolin-O-hexoside, quercetin-O-pentoside and quercetin-O-hexoside were proven to occur in the methanol extracts of wood dust by Mammela (2001).The presence of glycosides was confi rmed in the extracts of beech bark as well.Thus, cis-coniferin, cis-syringin, Flavonoids can also occur in wood extracts as the aglicon polymers (Fengel and Wegener, 1989).The latter are known as the condensed tannins or proanthocyanidins.It has to be mentioned that the term proanthocyanidins describes biofl avonoids, leucoantocyanidins as well as condensed tannins.The building blocks of most proanthocyanidins are the fl avanols catechin and epicate-  Different antioxidants were identifi ed in the beech leaf extracts, i.e. vitamin C (ascorbic acid) and vitamin E (α-tocopherol) (Kunert and Ederer, 1985).Structural formulas for both compounds are presented in Figure 10.It was shown that vitamin E also occurs in the tissues at the bases of living and dead breaches of beech.Furthermore, it can be extracted from these tissues by means of non-polar solvent, i.e. cyclohexane (Vek et al., 2014).Further to the above, vitamin E could be classifi ed as the lipophilic extractive as well.
After all, some of characteristic hydrophilic extractives occurring in tissues of beech trees are biologically active compounds, which make this forest biomass interesting for extraction and further utilization.For instance, antifungal properties against some basidiomycetes were found for catechin and taxifolin by Malterud et al. (1985).Some years later, epicatechin was also reported as the compound with antifungal activity (Ardi et al., 1998; Baum and Schwarze, 2002).In addition to fl avonoids, aglycones of cis-coniferin and cis-syringin, i.e. coniferyl alcohol and syringic acid, identifi ed in the methanolic extracts of beech bark were reported as fungicides by Dubeler et al. (1997).Not only pure compounds, but also acetone extract of a beech knot, which was reported to be composed mainly of catechins and fl avonoids, showed evident antibacterial properties (Välimaa et al., 2007).Wood and bark extractives of various tree species have attracted much attention due to their possible application in various fi elds, including nutritional, cosmetic, medical and pharmaceutical industry, as natural biocides, feed additives or in industry of adhesives and leather production etc. Replacement of synthetic, artifi cial chemicals with more benign natural products, whose recovery has only little or no impact on human health and environment, is one of the important challenges in the fi eld of biorefi ning forest biomass (Holmbom, 2011).

ZAKLJUČAK
It can be summarized that successive extraction using the non-polar and polar solvent, respectively, represents the most frequently used and appropriate way of extraction of different beech material.The extractives can be obtained from grinded samples by using magnetic stirrer, whereas, more aggressive extraction techniques are recommended, e.g.conventional Soxhlet apparatus or various subcritical systems for accelerated extraction (Thurbide and Hughes, 2000;Vek, 2013).From the analytical aspect of view, chromatography in combination with mass spectrometry has been found as the convenient technique for the qualitative and quantitative analysis of extractives.Since stem-wood of beech is generally characterized by the relatively low amounts of extractable compounds, there is a strong need to recognize the appropriate source for extraction.Knots of trees have been presented more times as a very rich source of different polyphenols, perhaps the richest in all of nature as reported by Holmbom (2011) and beech is not an exception (Vek, 2013).Furthermore, a very important step for recovering the target compounds from different parts of a beech tree is the optimization of appropriate and effi cient extraction systems (i.e.extraction techniques and solvents) as it was recently demonstrated for beech bark (Hofmann et al. 2015).This short review revealed that the composition of low-molecular fraction of lipophilic and hydrophilic extractives is relatively well investigated, whereas the data on oligomeric fraction of extractable compounds are extremely sparse and require further research efforts.