Natural resources and bioeconomy studies 73/2017 Wood extractives of Finnish pine, spruce and birch – availability and optimal sources of compounds A literature review Johanna Routa, Hanna Brännström, Perttu Anttila, Marko Mäkinen, Janne Jänis and Antti Asikainen Natural resources and bioeconomy studies 73/2017 Wood extractives of Finnish pine, spruce and birch – availability and optimal sources of compounds A literature review Johanna Routa, Hanna Brännström, Perttu Anttila, Marko Mäkinen, Janne Jänis and Antti Asikainen Natural Resources Institute Finland, Helsinki 2017 Routa, J., Brännström, H., Anttila, P., Mäkinen, M., Jänis, J. and Asikainen, A. 2017. Wood extractives of Finnish pine, spruce and birch – availability and optimal sources of compounds : A literature review. Natural resources and bioeconomy studies 73/2017. Natural Resources Institute Finland, Helsinki. 55 p. ISBN: 978-952-326-494-6 (Print) ISBN: 978-952-326-495-3 (Online) ISSN 2342-7647 (Print) ISSN 2342-7639 (Online) URN: http://urn.fi/URN:ISBN:978-952-326-495-3 Copyright: Natural Resources Institute Finland (Luke) Authors: Johanna Routa, Hanna Brännström, Perttu Anttila, Marko Mäkinen, Janne Jänis and Antti Asikainen Publisher: Natural Resources Institute Finland (Luke), Helsinki 2017 Year of publication: 2017 Cover photo: Johanna Routa Printing house and: publishing sales: Juvenes Print, http://luke.juvenesprint.fi Natural resources and bioeconomy studies 73/2017 3 Summary Johanna Routa1, Hanna Brännström1, Perttu Anttila1, Marko Mäkinen2, Janne Jänis2 and Antti Asikainen1 1Natural Resources Institute Finland 2University of Eastern Finland, Department of Chemistry Forest-based biomass is one of the main sources of energy and feedstock for industrial production of bio-materials, bio-chemicals, bio-fuels, and other novel bio-refinery products in Finland. Apart from its main chemical constituents (cellulose, hemicellulose and lignin), wood also contains a wide variety of low molecular mass compounds known as extractives. These compounds can be separated from insoluble wood constituents by simple solvent extraction using polar or non-polar solvents. The aim of enhancing efficiency in the utilization of feedstock through product diversification and improved recovery has led to an increased interest in studying individual chemical components of wood. In particular, extractive compounds provide potential functionalities for several types of industrial chemicals and well-being and health products for consumers. The extractives content within a tree and between trees in the same stand is not uniform and may also vary according to the age of the trees, the season, the site and provenance. Differences in the amounts of extractives from one geographic location to the next can be found, but the differ- ences between plots and parts of a single tree are clearer. The effects of different factors (e.g. location, the part of the tree, age, the site type, time of har- vesting, or storage time) on the quantity of extractives have been well studied, but no review sum- marizing the effects of the aforementioned parameters on individual extractive compounds exists. A comprehensive review (or a set of more detailed summaries)concerning the extractive compositions of the overall tree biomass (incl. not only the stem, but also the bark, foliage, and stump-root sys- tem) of most common Finnish industrial wood species has not been available until now. Often scien- tific articles are concentrated on studying certain parts of trees, while some examine the geographic origins or site, or they might focus on even more specific issues such as certain groups of compounds in certain parts of the tree. In addition, the study material in some studies is very limited. In this re- port the results of the studies on extractives of Norway spruce, Scots pine, and silver and white birch have been summarized to facilitate the identification of the best suitable biomass assortments for sourcing future biorefineries and their production lines. Keywords: extractives, secondary metabolites, Norway spruce, Scots pine, silver birch, white birch, value added chemicals, forest industry, circular economy, biorefining/biorefinery Natural resources and bioeconomy studies 73/2017 4 Tiivistelmä Metsäbiomassa on yksi biomateriaalien, biokemikaalien ja muiden uusien biotuotteiden sekä bio- energian pääraaka-aineista. Pääosa metsäbiomassasta koostuu erilaisesta puuaineksesta. Puussa on sen pääkomponenttien (selluloosa, hemiselluloosa ja ligniini) lisäksi erilaisia pienimolekyylisiä uuteai- neita, jotka voidaan nimensä mukaisesti erottaa puusta uuttamalla. Nykyisin metsäbiomassan hyö- dyntämisastetta pyritään kasvattamaan ja käyttöä monipuolistamaan, minkä vuoksi yksittäisiin uu- teaineyhdisteisiin kohdistuva kiinnostus lisääntyy. Näitä uuteaineyhdisteitä voidaan hyödyntää erityi- sesti lähtöaineina hyvinvointi- ja terveystuotteiden ja erilaisten teollisuuskemikaalien valmistuksessa. Uuteaineiden koostumus ja määrä puun sisällä ja puuyksilöiden välillä vaihtelee runsaasti, ja li- säksi puun ikä, korjuuajankohta, geneettinen alkuperä ja kasvupaikka vaikuttavat uuteaineiden mää- rään. Uuteaineiden määrä vaihtelee maantieteellisen sijainnin mukaan, mutta erot ovat paljon sel- vemmät puun eri osien välillä ja yksittäisten metsikköjen välillä. Eri tekijöiden vaikutusta uuteaineiden määrään on tutkittu paljon, mutta yhteenvetoa näiden tekijöi- den vaikutuksesta ei ole tehty. Kattavia tai yksityiskohtaisia yhteenvetoja koskien uuteaineiden mää- riä puun eri osissa (ei vain runkopuussa, vaan myös kuoressa, lehvästössä, kantopuussa ja juurissa) ei ole tehty yleisimmillä suomalaisilla puulajeilla. Tieteelliset artikkelit ovat usein keskittyneet tiettyyn puun osaan, tai yhteen tiettyyn yhdisteryhmään tietyssä osassa puuta. Lisäksi tutkitut aineistot ovat olleet usein pieniä. Tämä raportti on yhteenveto kuusen, männyn ja koivun uuteaineiden tutkimustu- loksista. Sen tulosten toivotaan edesauttavan biojalostukseen ja sen prosesseihin parhaiten soveltu- vien biomassaositteiden tunnistamisessa. Asiasanat: uuteaineet, kuusi, mänty, rauduskoivu, hieskoivu, arvokemikaalit, metsäteollisuus, kierto- talous, biojalostus/biojalostamo Natural resources and bioeconomy studies 73/2017 5 Contents 1. Introduction ................................................................................................................... 6 2. Expert interviews............................................................................................................ 8 3. Extractives ...................................................................................................................... 9 4. Variation of chemical composition ................................................................................ 11 4.1. Different parts of the tree ...................................................................................................... 11 4.1.1. Stem: Heartwood and sapwood .......................................................................................... 11 4.1.2. Knots ................................................................................................................................... 11 4.1.3. Bark ..................................................................................................................................... 12 4.1.4. Stumps and roots ................................................................................................................ 13 4.2. Effect of tree age .................................................................................................................... 14 4.3. The effect of latitude ............................................................................................................. 15 4.4. Effect of fertilization .............................................................................................................. 18 4.5. Seasonal variation .................................................................................................................. 19 4.6. Effect of storage ..................................................................................................................... 20 4.6.1. Birch .................................................................................................................................... 21 4.6.2. Spruce ................................................................................................................................. 23 4.6.3. Pine ..................................................................................................................................... 25 5. Distribution and content of extractives in tree biomass ................................................. 27 6. Valuable bioactive extractives in common Finnish wood species and their properties .... 31 6.1. Phenolic extractives ............................................................................................................... 32 6.1.1. Stilbenes .............................................................................................................................. 33 6.1.2. Flavonoids ........................................................................................................................... 34 6.1.3. Tannins ................................................................................................................................ 36 6.1.4. Lignans ................................................................................................................................ 37 6.2. Terpenes and terpenoids ....................................................................................................... 38 6.3. Fats, waxes and their components ........................................................................................ 41 6.4. Suberin and its components .................................................................................................. 42 7. Applications utilizing extractives from wood-based biomass .......................................... 43 8. Conclusions .................................................................................................................. 45 Natural resources and bioeconomy studies 73/2017 6 1. Introduction The aim of forest management worldwide has changed over the years. In Finland and the other Nor- dic countries, the shift has been towards management practices aiming at producing both quality timber in addition to other forest products and services (Routa et al. 2013). The product palette is currently broadening to include novel biorefinery products, liquid biofuels, wild forest products and a multitude of forest related ecosystem services. This is obviously reflected in forest management schemes as well. One of the management options available is to implement pre-commercial and commercial forest thinnings to promote the volume growth and formation of high quality wood in the remaining trees before final harvesting of the trees. This practice combined with other manage- ment options, such as increased initial stand density and forest fertilization, increases the generation of forest biomass collectively including energy wood (logging residues, small diameter stem wood and stumps) (Alam et al. 2010). Trees from thinning operations have been used primarily as a raw material in the pulp, paper and paperboard industries. In addition, small-diameter logs for mechani- cal wood processing can be recovered as well as biomass for the production of wood energy for households, heating plants and combined heat and power plants. Forest-based biomass is the largest single source of energy and a very potential feedstock for in- dustrial production of different bio-materials, bio-chemicals, bio-fuels, and other novel bio-refinery products in Finland. Interest in studying the individual chemical components of wood with the aim of enhancing efficiency in feedstock utilization through product diversification and improved recovery has been increasing. Apart from its main chemical constituents (cellulose, hemicellulose, and lignin), wood also contains a wide variety of low molecular mass compounds known as extractives. These compounds can be separated from the insoluble wood constituents by simple solvent extraction using polar or non-polar solvents. In particular, wood extractives provide potential functionalities for several types of platform and specialty chemicals, such as pharmaceutical or nutritional products, cosmetics, beverages, wood adhesives, paints, wood protection agents, plant-protective products and detergents (Roitto et al. 2016). Valuable compounds can be obtained directly from different parts of standing trees (Mantau & Saal 2010), or as by-products of forest industries (Saal 2010). Spe- cialty chemicals have a potentially high value in various raw materials and products for techno- chemical industries and a multitude of consumer products. The demand for nature-derived ingredi- ents to replace synthetic chemicals is increasing and markets for natural health products are growing (Royer et al. 2012). In addition to cellulose and lignin, the most important chemical factors affecting the quality of wood are extractives, since their presence affects the processing and use of wood. In use, either in the soil, above ground or immersed in water, wood is exposed to attack from a number of bacteria, fungi, insects and marine organisms (Zabel & Morrell 1992). The major contribution of extractives in the use of solid wood is in their natural resistance against fungi and insects they impart.The extrac- tives also add to the density, geometric stability and some mechanical properties of wood (e.g. Hillis 1971, Zobel & van Buijtenen 1989, Hakkila and Verkasalo 2009). The chemical composition of wood cannot be defined precisely for a given tree species or even for a single tree (Alen 2011). The wood chemical composition of an individual tree can be influenced by many factors and variations exist between tree part (bark, stem, or branch), the type of stemwood (sapwood or heartwood) and the growth conditions, among other factors (Alen 2000, 2011). In addition, the age of a tree has effects on the extractives of the tree; generally, old trees contain more extractives in their heartwood than young trees (Hillis 1962, Uprichard and Lloyd 1980). While differences in the chemical composition of major cell wall components of the three (cellulose, hemicelluloses and lignin) vary only sparingly between different tree species, there is a great diversity in the extractives throughout species (Umezawa 2000). The extractives content within an individual tree or and between trees in same stand is not uniform (Solhaug 1990, Bergström et al. 1999, Ekeberg et al. 2006) and it may considerably vary according to age, season, provenance and Natural resources and bioeconomy studies 73/2017 7 site (Solhaug 1990, Toscano et al. 1991, Lindberg et al. 1992). Differences in the amounts of extrac- tives in different geographic locations can also be found (Wilför et al. 2003a, Kaakinen 2007, Piispanen 2008, Hakkila & Verkasalo 2009, Roitto et al 2016), but the differences between plots and parts of single trees are clearer. This has effects on the supply of raw materials, sampling and sorting if the aim is to procure raw materials containing specific extractives or to avoid certain extractives in the raw materials. In pulping, the presence of extractives is detrimental (Martınez-Inigo et al. 1999, Pereira et al. 2003). Usually, the extractives affect the bleachability of pulps negatively, causing lower brightness and higher brightness reversion of kraft pulp or lead to increased consumption of bleaching chemi- cals (Jewell et al. 1991).On the other hand, tall oil and turpentine are commercially important side- products of softwood kraft pulping (Alén 2000). Tall oil has wide variety of uses in industry (see chap- ter 7). For example, fats are valuable resources for producing fuels such as biodiesel while resins are suitable for producing glues and inks (Demirbas 2011). Wood knots which contain more extractives than clear wood, are particularly detrimental to pulping processes and pulp quality; and they should preferably be separated before pulping (Willför et al. 2004). The forest industries use substantial amounts of wood resources annually in Finland. In total 67 million m3 of wood were utilized in 2016 (Official Statistics of Finland 2017a). As a result of this utili- zation, there are significant amounts of side streams such as bark, branches and tree stumps. The consumption of bark in energy use in Finland was about 7.3 million m3 in 2016. Additionally, 2.5 mil- lion m3 of logging residues and 0.8 million m3 of tree stumps were used in energy production in 2016 (Official Statistics of Finland 2017b). The aims of this report are 1) to identify the most interesting wood extractives for biorefining, 2) to assess the factors influencing the variation of their concentrations and 3) to summarize the data on concentrations. A number of selected experts were interviewed to identify the extractives with the highest utilization potential. A literature review was then carried out to determine the average concentrations of extractives and their variation due to a variety of factors. The main emphasis was on the industrially most important Finnish tree species, namely Scots pine (Pinus sylvestris), Norway spruce (Picea abies), silver birch (Betula pendula) and white birch (Betula pubescens). Natural resources and bioeconomy studies 73/2017 8 2. Expert interviews A total of eight experts (listed in the Acknowledgements) representing both research and industry were interviewed to determine the most interesting extractives and other compounds now and in the future from the perspective of research and development activities and industrial utilization. The experts were asked whether it was possible for them to name some individual extractives that occur in forest biomass and which of the extractives would be interesting for industrial-scale utilization. They were also asked for specific information on how different factors (e.g. geographic location, part of tree, age, site type, time of harvesting, or storage time) affect the yield of extractives. The experts emphasized some extractive groups, but also some interesting single compounds. Stilbenes, tannins, lignans and terpenes were mentioned in every interview. Additionally, pinosylvin, pinosylvin monomethyl ether (stilbens), hydroxymatairesinol (lignans), betulin (triterpenoid), α-/β- pinene (monoterpenes), resin acids (diterpenoids) and fatty acids were all listed. In addition, galac- toglucomannan (hemicellulose) and xylans (hemicellulose) were mentioned as interesting com- pounds, althought they are not extractives. The concern of chemical instability and decomposition of extractives during the storage of wood-based raw materials was also raised. Amounts of hydrophilic and lipophilic extractives de- crease significantly during storage, as do lipophilic extractives. Therefore, it is important to plan sup- ply chains taking storage losses into consideration. The scale of operations was also mentioned as a risk factor. If extractives are utilized on a large scale in the secondary flow of a pulp mill, it does not mean much if the concentration is a little bit higher of some individual compounds of extractives from wood from some particular areas. Howev- er, on a small-scale use, when a company has specialized in selected valuable products, for example pharmaceutical or nutritional products, the choice of raw material sources is very important. Accord- ing to one of the experts, it may be possible to separate selected extractives more accurately with special methods (e.g. hot water extraction was mentioned) retaining the original composition of the compounds. Furthermore, the experts emphasized the very large inter-tree variation in concentra- tion and variation in spatial distribution of extractives. Natural resources and bioeconomy studies 73/2017 9 3. Extractives Extractives are numerous low molecular mass non-structural compounds present in wood that can be extracted using polar and non-polar solvents (Hillis 1971, Alen 2000, Taylor 2002). They include compounds such as alkaloids, waxes, fats, proteins, phenolics, gums, pectins, resins, terpenes, and essential oils (Nascimento et al. 2013). Most of the extractives are secondary metabolites, which are compounds that play other roles in a tree than those involved in growth and cell development, namely protection of the tree against pathogens or other biotic attacks (Barnett and Jeronimidis 2003). Morphological sites and main func- tions of different wood extractives are shown in Table 1. The secondary metabolites are preferential- ly deposited in the inner part of the stem, in the heartwood. In the outer part of the stem, the sap- wood includes recently divided cell layers, where molecules used in the metabolic synthesis of the cell wall are found as extractives. Some species have specialized defense structures to protect them- selves from attack, either physical or biotic. For example, resin canals in softwoods are particularly developed in pines that produce resin, which is a mixture of terpenoid compounds which are also important extractives in the wood of these trees (Table 1). A high content of extractives and their nature may be involved in the defense strategy of trees against biotic and abiotic injuries. Extractives represent a small proportion of wood, typically less than 10% of the dry mass, with the exception of tropical woods, where the proportion can be higher. There are considerable differences between extractives in softwoods and hardwoods, and also between tree species. Resin acids occur only in softwoods and the proportions between individual resin acids differ between tree species. The fatty acid composition also differs between softwoods and hardwoods and also between tree species (Holmbom 1999). Table 1. Morphological sites and main functions of wood extractives (Holmbom 1999). Location in wood Major compound classes Main function in the tree Resin canals (oleoresin) Resin acids, monoterpenoids, other terpenoids Protection Parenchyma cells Fats, fatty acids, steryl esters, sterols Physiological food reserve, cell membrane components Heartwood Phenolic substances Protection Cambium and growth zone Glycosides, Sugars, starch, proteins Biosynthesis, food reserve Ascending water Sap Inorganics Photosynthesis, biosynthesis Natural resources and bioeconomy studies 73/2017 10 Extractives of wood can be classified into the different groups as shown in Table 2, i.e. aliphatic and alicyclic compounds, phenolic compounds and other compounds. The composition of extractives varies widely from species to species, and the total amount of extractives in a given species depends on the growth conditions. For example, the typical content of extractives in Scots pine, Norway spruce and silver birch stem wood are, respectively, in the range of 2.5% to 4.5%, 1.0% to 2.0%, and 1.0% to 3.5%, of the wood on a dry basis (Alen 2011). Table 2. Classification of organic extractives in wood (Alen 2011). Aliphatic and alicyclic com- pounds Phenolic compounds Other compounds Terpenes and terpenoids (in- cluding resin acids and ster- oids) Esters of fatty acids (fats and waxes) Fatty acids Alkanes Simple phenols Stilbenes Lignans Isoflavones Flavonoids Condensed tannins Hydrolyzable tannins Sugars Cyclitols Tropolones Amino acids Alkaloids Coumarins Quinones However, many initial extractives suitable for potential applications are either rather volatile or chemically unstable. As it was mentioned in the expert interviews, the amount of many extractives starts to decrease immediately after tree felling and this degradation continues during storage (Ek- man 2000, Lappi et al. 2014a). This also means that the chemical composition of the extractives- based fraction changes as a function of time (Lappi et al. 2014a, b, c, Alén 2000). All chemical and biochemical reactions are largely influenced by the conditions prevailing during wood storage and they are markedly faster when the wood is stored in the form of chips instead of logs. The volatile extractives in the wood tissue of different tree species are unique and may be used for species identification (Wajs et al. 2007). Natural resources and bioeconomy studies 73/2017 11 4. Variation of chemical composition 4.1. Different parts of the tree 4.1.1. Stem: Heartwood and sapwood The formation of heartwood is strongly dependent on the age of the tree. Heartwood differs from sapwood in anatomical and physiological features. Heartwood is darker in color and denser and stiff- er than sapwood in some species (Taylor et al. 2002, Spicer 2005), while heartwood contains more extractives than sapwood (Rydholm 1967, Yanchuk et al. 1988, Campbell et al. 1990). Norway spruce sapwood contains more lipophilic extractives than heartwood (Ekman 1979, Ber- taud and Holmbom 2004). This is mainly due to the larger quantity of triglycerides. The qualitative composition of volatile organic compounds (VOCs) is similar in spruce sapwood and heartwood in the study of Wajs et al. (2006). Higher amounts of terpenes have been found in the heartwood than in the sapwood of southern pines while fatty acids and resin acids have been found more in the heartwood of radiata pine (Up- richard and Lloyd 1980, Ingram et al 2000). The total resin acid concentration in Scots pine heart- wood is much higher than in the sapwood, but the composition of resin acids is similar (Martinex- Inigo 1999, Turtola 2002). In a study by Arshadi et al. (2013), Scots pine heartwood contained up to five times more extractives than sapwood. Furthermore, resin acids were mainly associated with heartwood, but fatty acids were found more in sapwood. Additionally, the quantity of stilbenes (e.g. pinosylvins and pinosylvin monomethyl ester) in Scots pine heartwood is higher than in the sapwood (Wilför 2003b, Ekeberg et al 2006, Siwale 2015). The total extractive content of birch stemwood is low but it seems to rise from the base toward the top (Roitto et al. 2016). Piispanen & Saranpää (2004) found that the concentration of fatty acids and steryl esters were highest in samples close to the pith. The heartwood samples of goat willow (Salix caprea) stemwood contained 5–20 times more hy- drophilic extractives (most abundant catechin) than the sapwood samples (Pohjamo et al. 2003). The heartwood and sapwood samples of common aspen (Populus tremula) and quaking aspen (Populus tremuloides) contained similar amounts of extractives (Pietarinen et al. 2006a). 4.1.2. Knots Internal knots (i.e. branch bases inside tree stems) of several tree species have a remarkably high amount of phenolic substances, being therefore a valuable source of bioactive compounds. Softwood knots contain extremely large amounts of phenolic extractives (Willför et al. 2002, Holmbom et al. 2003, Willför et al. 2003a, b, c). Norway spruce knots contain as much as 6–24% of lignans, with 7- hydroxymatairesinol (HMR) as the predominant (70–85%) compound (Holmbom et al. 2003, Willför et al. 2002, 2003a, Kebbi-Bendeker et al. 2015). Some other spruce species also contain HMR as the main lignan, but some spruce species also have other dominating lignans. The content of lignans in knot heartwood can be 30–500 times higher than in the stem heartwood. (Willför et al. 2003a). The variation in the amount of lignan is large from knot to knot, both in an individual tree and from tree to tree (Willför 2003 a). Lignans occur also in the knots of pines, although in lower amounts than in the knots of spruces or firs (Holmbom et al. 2003). The concentration of extractives in the knotwood of Scots pines has been found to be higher than that in the stem heartwood (Erdtman and Rennerfelt 1944, Boutelje 1966, Willför et al. 2003a, Hovelstad et al. 2006), even up to four- to five-fold in a study by Karppanen et al. (2007). Scots pine knots have been found to contain 0.4–3% of lignans and 1–2.95% of stilbenes (Ekman et al. 2002, Natural resources and bioeconomy studies 73/2017 12 Willför 2003b, Hovelstad et al. 2006). Furthermore, Scots pine knots also contain large amounts of lipophilic extractives (4.5–32%) mainly resin acids (Willför 2003b). Lignans have also been identified in the knots of some hardwoods, although flavonoids are more abundant in hardwoods. The concentrations of hydrophilic extractives in willow have been found to be 2–10 times higher in the knots than in the stemwood (Pohjamo et al. 2003). Aspen (Populus trem- ula) knots have been found to be a rich source of bioactive flavonoids, and the quantity of hydro- philic extractives is larger in the knots than in the stemwood (Pietarinen et al. 2006a). The lignan content gradually decreases in the radial direction from the knot pith towards the outer wood, finally reaching the same level as in the surrounding stemwood (Willför et al. 2003a). In the branches, the lignan content also decreases in the radial direction from the branch pith out- wards, and approaches the same level as it is in the stemwood 20 cm outside the stem (Willför et al. 2005). The fact that the knots contain considerable amounts of lignans, unlike the surrounding wood tissue can be of technical and industrial importance. The knots can be recovered in a pulp mill before pulping and the lignans can then be extracted and utilized as active ingredients in health-promoting foods or pharmaceuticals, or as natural antioxidants in various applications (Willför et al. 2005). The HMR concentration is also the highest in the knots close to the starting point of the living crown and in knots that are in connection to living branches (Piispanen at al 2008, Willför 2003a). Snow loading on the branches can also affect the lignan concentrations of the knots, causing me- chanical loading in branch wood (Piispanen at al 2008). Branches and thus knots seem to possess a chemical response to mechanical loads such as wind and snow, especially in the case of softwoods, which do not lose their leaves during winter. The extractive content in the knots of broad-leaved species (hardwoods) is lower than in conif- erous species (softwoods) (Pohjamo et al. 2003, Pietarinen et al. 2006 a, b). The knotwood of hard- woods contains mainly flavonoids, tannins and/or sugars (Kebbi-Bendeker et al. 2015). 4.1.3. Bark Bark represents typically 10–15% of total weight of tree stems (Fengel and Wegener 1984, Sipi 2006, Krogell et al. 2012). Large amounts of bark waste are produced in the pulp and paper industry and in sawmilling operations. Bark is currently used mainly for producing heat and power. The extraction of valuable components before combustion is an interesting option for better utilization of tree bark. Bark contains 2–6 times more extractives than stemwood does. The total content of both lipophilic and hydrophilic extractives usually corresponds to 20–40% of the dry weight of bark (Sjöström 1993). For this reason, bark can be considered a potential raw material for the production of a range of val- ue-added products, e.g. platform and specialty chemicals, cosmetics, dietary supplements, biopoly- mers, bioplastics, foams/emulsions, coatings, pharmaceuticals (drugs or drug precursors). Bark is both morphologically and chemically very heterogeneous, and also differs considerably between wood species; not only between hardwoods and softwoods, but also between individual hardwood and softwood species (Fengel and Wegener 1984, Sakai 2001). Bark is built up of two ma- jor parts: the inner bark and outer bark. The proportion of inner and outer bark varies between spe- cies and according to the age of the tree, and it depends also on the height of the tree. The overall composition of inner and outer tree bark differs considerably. The quantity of lipophilic extractives has been found to be about two times larger in the outer bark of Norway spruce compared to the inner bark (Ånäs et al. 1983, Krogell et al 2012). The overall compositions of inner and outer bark of Norway spruce differ significantly: the inner bark contains large amounts of hydrophilic extractives whereas the outer bark contains more lipophilic extractives (Krogell et al 2012). Resin acids, fatty acids, steryl esters, diterpenoids and triglyseriders are the dom- inant compounds in Norway spruce bark (Ånäs et al. 1983, Krogell 2012). Stilbene glucosides, iso- rhapontin, astringin and piceid may account for up to 7.2% of Norway spruce bark dry weight (Man- Natural resources and bioeconomy studies 73/2017 13 nila and Talvitie 1992, Krogell et al. 2012, Jyske 2014). According to Latva-Mäenpää (2017) the aver- age concentrations of major stilbenoid glucosides (isorhapontin and astringin) are mainly localized in the inner bark of stumps and roots, and the concentrations are 30–200 times higher in the inner bark compared to the outer bark. Unique component in bark that is not present in wood is the suberin (Krogell et al 2012). Another example regarding the common softwood species, P. sylvestris and P. abies, are the condensed tannins that are present in bark but not in wood (see table 6). Resin acids and fatty acids are dominant compounds in Scots pine bark (Valentín et al. 2010). Bark extract have also been found to contain smaller amounts of other lipophilic compounds as well as some phenolic compounds such as stilbene glycosides, lignans and flavonoids (Pan and Lundgren 1996). The lipophilic compounds include fatty acids, fatty alcohols, oxidised resin acids, and sterols (Valentín et al. 2010, Belt 2013). Pine bark has also been found to be rich in condensed tannins (Ka- ronen et al. 2004a, Matthews et al. 1997). The highest concentrations of extractives in birch wood are found in bark (Roitto et al. 2016). The concentrations of individual compounds are quite low in birch bark (less than 1%) except for betulinol (up to 12.7%) (Roitto et al. 2016). Birch bark chemistry differs considerably from that of spruce and pine. For example, the outer bark of silver birch trees contains about 40% extractives, 45% suberin, 9% lignin, 4% hemicelluloses and 2% cellulose (Pinto et al. 2009). The extractives mainly comprise different triterpenoids, especially betulin and different betulin derivatives, accounting for as much as 30% of the dry weight (Ekman 1983). The birch outer bark contains only about 2 mg/g of phenolics, mainly comprising esterified hydroxycinnamic acids, whereas the inner bark has very high phenolics contents (Kähkönen 1999). Suberin itself is an interesting cross-linked co-polymer with polyaliphatic and polyaromatic domains, mainly made of the esterified long- and mid-chain hy- droxy/epoxy fatty acids and fatty diacids as well as a few phenolic constituents (Pinto 2009). While bark triterpenes possess considerably pharmaceutical potential, suberinic fatty acids are interesting building blocks for novel biopolymers and coating materials (Alakurtti 2006, 2013, Dzubak 2006, Gandini 2006). 4.1.4. Stumps and roots Stumps are an underutilized source of renewable forest biomass which are rich in chemical composi- tion in comparison to stemwood. The roots and stumps of Norway spruce and Scots pine trees are a vast source of biomass potentially containing many bioactive polyphenolic extractives (Latva- Mäenpää 2017), but this biomass is currently used for low-value energy production only. With al- ready existing harvesting techniques, roots and stumps of conifer trees could be used as a source of commercially valuable biochemicals (Latva-Mäenpää 2017). The main extractives found in the root bark of Norway spruce are astringin and isorhapontin, as well as smaller amounts of piceid (Latva‐Mäenpää et al. 2013, Mulat et al. 2014). The bark of fine roots of Norway spruce has been found to contain between 15% and 29% of extractives (Matthews et al. 1997, Richter et al. 2007). Latva‐ Mäenpää et al. (2013) found that root bark has a much higher extractive content than the wood of roots; the root zone nearest the stem had the highest amounts of extractives in the bark and the zone closest to the root tip had the highest amounts of extractives in the wood. While isorhapontin was the major stilbenoid found in the bark in all three of the root zones, astringin was the major compound found in the stump bark. Within the wood they mainly found saccharides, such as fructose, glucose and sucrose, as well as fatty acids and resin acids, such as pimaric acid, abietic acid and dehydroabietic acid (Latva‐Mäenpaa et al. 2013). Latva-Mäenpää et al. (2014) found relatively high concentrations of lignans in the root neck of Norway spruce (approx- imately 10% of total dry weight). According to Hakkila (1989), the stump-root system of a 100-year-old pine showed larger con- centrations of extractives than a 25-year-old pine. Accordingly, an order of magnitude regarding higher yields of extractions obtained with different solvents (methanol-benzene, acetone, ether, Natural resources and bioeconomy studies 73/2017 14 water) was observed compared to younger trees. This in turn might indicate that there are differ- ences in the relative compositions of extractive fractions of trees due to the different maturity. The concentration of acetone-extractives is highest in the stump and in the roots near the stump with high amounts of heartwood, and the concentration of extractives decreases towards the ends of the roots (Hakkila 1989). The composition of pine stump extractives is presented in Fig. 6 in Chapter 4.6.3. Proportion of extractives in different parts of common Finnish wood species according to differ- ent studies are presented in table 3. Table 3. Proportion of extractives in different parts of common Finnish wood species according to different studies, as dry mass percentages. Picea abies Pinus sylvestris Betula pendula Betula pubescens Stemwood Sapwood Heartwood 1.0-4.51-11 1.7-2.712-13 1.1-1.812-13 1-6.82,5,6,8,11,14 3.112,14 5.1-5,3512,14 0.8-55,6,8,11,20 0.8-6.75,6,8,20 Branchwood Knots 6.8-13,75-8 8.4-14.15,6,8 24.6 18 1.7-7.65,6,8,20 1.9-9.75,6,8,20 Stump 1.9-3.66,16,17 6.5-18.76,17,19 3.66 5.86 Rootwood 2.4-6.56,16,17 4.2-6.46,17,19 5.8-12.15,6 7.96 All bark Inner bark Outer bark 23.5-28.35-8 17.3-38.75,6,8 19.1-29.25,6,8 16-25.95,6,8 15.4-41.95,6,8 16.4-20.85,6,8 8-30.75,6,8,20 14.3-18.95,6,8 32.1-56.95,6,8 10.9-335,6,8,20 9.6-22.55,6,8 31.4-57.8 5,6,8 Foliage 37.8-43.35,6 38.6-40.65,6 28.8-33.42,4 32.4-32.56,8 1Anttonen at al 2002, 2Holmbom and Ekman 1978, 3Kaakinen at al. 2007, 4Kaakinen at al. 2009, 5Nurmi 1993, 6Nurmi 1997, 7Rhen 2004, 8Voipio 1992, 9Kimland 10Fengel 11Alen 2000, 12Shain 1971, 13Hakkila and Verkasalo 2009, 14Kilpeläinen et al. 2003, 15Martinez-Inigo et al. 1999, 16Latva-Mäenpää et al. 2014, 17Hakkila 1975, 18Belt 2013, 19Latva-Mäenpää 2017, 20Roitto 2016, 4.2. Effect of tree age Tree age has an effect on the extractives content of wood biomass, as shown in Tables 4 and 5. The stemwood of a 100-year-old pine shows larger concentrations of extractives than a 25-year-old pine (Table 4). Additionally, the order of magnitude of the yields of extractions with different solvents was different than in a younger tree. Table 4. The proportion of extractives in the stemwood of a 25- and 100-year-old Pinus Sylvestris when using different solvents in the extraction (Hakkila 1989). Average proportion of extractives (% w/w) Solvent Methanol- benzene Acetone Ether Hot water Cold water 25-year old tree 1.9 1.5 0.9 2.3 1.0 100-year old tree 3.5 2.9 2.1 3.4 2.1 Natural resources and bioeconomy studies 73/2017 15 Older trees contain more extractives in the heartwood than young trees (Hillis 1962, Uprichard and Lloyd 1980, Kasmani et al. 2011, Seyed Mohammadi et al. 2011). The formation of heartwood is strongly dependent of the age of tree, and heartwood contains more extractives than sapwood (Yan- chuk et al. 1988, Rydholm 1967, Campbell et al. 1990). The stump center of a 100-year old pine is particularly rich in extractives (Table 5). Table 5. The proportion of extractives in the stump-root system of a 25- and 100-year-old Pinus sylvestris when using different solvents in the extraction (Hakkila 1989). Proportion of extractives (%) Solvent Methanol- benzene Acetone Ether Hot water Cold water 25-year old tree Root collar 1.7 2.4 1.6 Stump center 2.0 1.4 1.5 2.1 1.1 Taproot 1.3 1.0 1.4 0.8 Large laterals 1.9 1.6 1.4 2.0 1.0 Average 2.0 1.6 1.3 2.0 1.1 100-year old tree Root collar 6.6 5.9 4.9 3.6 1.5 Stump center 9.7 9.1 8.2 5.2 2.5 Taproot 6.9 6.6 4.7 2.7 0.9 Large laterals 2.8 2.0 1.6 2.2 1.2 Average 6.5 5.9 4.8 3.4 1.5 The proportion of extractives in the inner and outer bark varies between species and according to the age of the tree, and depends also on the height and position in a tree (Pasztory et al 2016). For example, older trees have higher average amounts of stilbene glucosides than younger trees in the bark of Norway spruce trees (Jyske et al 2014). The flavonoid composition of Scots pine trees seems also strongly dependent on the age of the tree (Oleszek et al. 2002). This applies also to the resin acid and total phenolics content of the needles. It has also been found that mature needles have higher concentrations of extractives compared to young needles for the afore-mentioned compounds (Nerg et al. 1994). 4.3. The effect of latitude Willför (2003a) found lignan concentrations in spruce knotwood to be higher in northern Finland than in southern Finland. This is natural because the growth conditions are generally more unfavora- ble for Norway spruce close to the Arctic tree line than in more southern sites. It seems that these unfavorable environmental conditions could cause higher lignan production in spruce trees, which might be useful for the trees during occasional periods of environmental stresses, such as when the trees could be under microbial attack (Piispanen et al. 2008). Spruce trees in northern Finland have been observed to contain more than twice as many lignans in their knots than in southern Finland (Holmbom 2011). Kaakinen et al. (2007) confirmed this by finding that the stemwood of Norway spruce had 54% higher concentrations of extractives in a northern site than in a southern site. Hakkila and Verkasalo (2009) reported higher amounts of acetone extractives in pine and spruce stem heartwood and sapwood in northern Finland than in southern Finland. Hakkila (1968) also found a higher total quantity of extractives in pine and spruce stemwood in northern Finland. Natural resources and bioeconomy studies 73/2017 16 Siwale (2015) indicated strongly that the formation of vanillic acid in both young and mature Scots pine stem woods is affected by different climatic conditions due to the geographic location, and concluded that poor conditions favor the formation of vanillic acid. The mean concentration of vanillic acid in samples from first-thinning harvests increased from southern Finland (0.0069 µg/mg) to northern Lapland (0.01 µg/mg) and a significant difference was found between these two extreme locations. The concentrations of pinosylvin and pinosylvin monomethyl ester were also significantly affected by geographic location, and samples from middle Finland clearly had the lowest concentra- tions with the means being significantly lower than those in the samples from other locations. Roitto et al. (2016) found considerable north-south differences in the concentrations of individu- al extractive compounds of birch in different climatic regions. They observed that gallocatechins and neolignans were generally more abundant in the north while (+) catechin and some lignans were more abundant in the central or southern regions (Figure 1). According to Oleszek et al. (2002) the relationship between the flavonoid level in pine needles and the latitude of the population origin is very evident. This can be explained by the protecting function of flavonoids against UV radiation. Thus, at higher latitudes the lower light intensity does not force the plants to produce metabolically costly protective flavonoids. Additionally, the taxifolin content of needles has been detected to vary according to the elevation (Nerg et al. 1994). It has also been found that the resin acid concentration of sun-exposed needles is significantly higher than in shaded needles. According to a study by Nerg et al. (1994) the concentrations of total monoterpenes and total phenolics in seedlings of Pinus syl- vestris were the lowest in the most northern sampling locations. Siwale (2015) showed for Scots pine that in southern Finland site soil fertility has a significant ef- fect on the concentrations of pinosylvin, its glycoside and one of the neolignans, but the exact effect was not the same for all compounds. Site soil fertility affected the concentration of some phenolic compounds in Scots pine stemwood, but the exact effect was inconsistent. Latva-Mäenpää et al. (2013) compared stilbene glucosides in Norway spruce roots and stumps grown on either peatland or mineral soil sites, observing that the isorhapontin concentration was especially higher in extracts obtained from the mineral soil than in those from peatland. Natural resources and bioeconomy studies 73/2017 17 Figure 1. Examples of variation in the concentration of individual phenolic compounds: (+) catechin and an unidentified neolignan 6 in outer part of stemwood in Betula pendula and B. pubescens by geographic region in Finland. The values are the mean values obtained from multiple trees (Roitto et al. 2016). Natural resources and bioeconomy studies 73/2017 18 However, the amounts of extractives display high between-tree variation, although the studies have often been made with very limited data, consisting of only few sample trees. For this reason, more studies are required to confirm conclusions about the effects of geographic location. Although there are not many studies of tree species in Finland, the effect of latitude has been studied more thoroughly on berries. Recent studies have shown that bilberry populations growing at northern lati- tudes contain significantly higher amounts of flavonoids, in particular anthocyanins, in comparison to the southern populations (Lätti et al. 2008, Uleberg et al 2009, Åkerström et al. 2010). Moreover, higher amounts of anthocyanins were found in bilberry fruits grown in controlled conditions in a phytotrone in 24 h natural daylight, mimicking the light conditions of Arctic summers (Uleberg et al. 2012). 4.4. Effect of fertilization Fertilization treatment (for both macro and micro nutrients) has not been found to have any statisti- cally significant effect on the total concentration of terpenes in Norway spruce stem wood (Anttonen et al. 2002). However, concentrations of acetone soluble extractives (mainly fatty acids, resin acids, non-structural carbohydrates and phenolics) were significantly higher in fertilized plots than control plots in their study. Production of secondary metabolites predominates in nutrient limiting situa- tions, and has been shown in several studies where low nitrogen availability has resulted in the ac- cumulation of phenolics in needles (Muzika 1993, Holopainen et al. 1995, Kainulainen et al. 1996). In Norway spruce knotwood, nitrogen fertilization was not found to cause any significant change in lignan concentration (Piispanen et al. 2008) (Figure 2). The effects of nitrogen fertilization on wood chemistry in Norway spruce were mild and they dif- fered between locations in a study by Kaakinen et al. (2007). However, the amounts of extractives in stemwood were higher in fertilized plots than in control plots. In contrast, Kostiainen et al. (2004) found no changes in Norway spruce wood in the concentrations of extractives caused by fertilization (macro and micro nutrients). In a study by Viiri et al. (2001), nitrogen fertilization resulted in lower concentrations of terpenes and stilbene aglycones in Norway spruce, but phosphorus fertilization led to a minor reduction in the concentration of total terpenes. The combination of nitrogen, phosphorus and potassium fertilization increased the total amount of terpenes. In a study by Turtola et al. (2002) on Scots pines, nitrogen fertilization did not have any signifi- cant effect on the concentrations of resin acids in the heartwood, but in the sapwood the concentra- tions of individual resin acids, such as abietic and dehydroabietic acids decreased significantly in one of the locations. Nitrogen fertilization did not cause any significant changes in xylem monoterpene or heartwood resin acid concentrations of Scots pine (Thorin and Nommik 1974, Turtola et al. 2002). Heijari et al. (2005) studied the resistance of Scots pine wood to brown-rot fungi after forest fer- tilization (nitrogen, calcium, phosphorus) and found that fertilization generally increased the concen- tration of extractives in the wood. They noted, however, that while in some cases fertilization in- creased the wood resistance to brown-rot fungi, in other cases it decreased. Natural resources and bioeconomy studies 73/2017 19 Figure 2. Concentrations of knotwood lignans according to the size of knot in two experimental sites with two treatments of fertilization (control and N-fertilized) (Piispanen et al. 2008). 4.5. Seasonal variation The products of photosynthesis of trees are stored as reserves in living cells, and the sugar content is typically high in the winter and low in the summer, whereas the starch content is high in the spring and in the autumn and low throughout the rest of the year (Höll 1985). Large seasonal variation in the amounts of soluble sugar and starch were detected in the sec- ondary xylem of young silver birch trees (Piispanen & Saranpää 2001, Höll 1981). In a study by Piispanen & Saranpää (2001) the amount of sucrose decreased gradually during the spring until July, while it stayed near the minimum level in September and increased dramatically in November. In coniferous trees (e.g. Scots pines in Fischer and Höll 1992) the changes in soluble sugars are less pronounced than in silver birch. Hoch et al. (2003) found no significant seasonal variation in storage lipids in the sapwood of several coniferous and deciduous trees. Additionally, in a study by Piispanen & Saranpää (2004) the seasonal variation of storage lipids was low in silver birch trees. Free fatty acid concentrations in silver birch trees have been found to be the highest during mid- summer and September, and they increase during the spring (Piispanen & Saranpää 2004). The tri- acylglycerol concentration of silver birch have been found to be slightly but not significantly higher during the dormant period in November and in March than during summer and spring. The concentration and fatty acid composition of triacylglycerols have been found to remain sta- ble in Scots pine sapwood throughout the year (Saranpää & Nyberg 1987, Fischer & Höll 1992). No seasonal trend in the distribution pattern or concentration of pinosylvin in Scots pine trees was found in a study by Berström et al 1999. In contrast, Höll & Priebe (1985) reported marked seasonal variation in triacylglycerols in the wood of elm trees (Tilia cordata). According to Höll (1985) Norway spruce stem wood contains higher concentrations of soluble sugars in the cold period than in the spring and summer months. According to Solhaug (1990) stil- bene glucoside concentrations in Norway spruce bark tend to increase during the summer and early autumn. The free sugars in spruce bark are at their maximum level after the growing season and at their lowest in May/June (Weissman, 1984). In their study on young seedlings of Scots pine trees, Nerg et al. (1994) detected remarkable sea- sonal variation of terpene, resin acid and total phenolic concentrations. The overall phenolic concen- tration was higher in the spring than in the autumn, whereas concentrations of some individual resin acids (levopimaric and dehydroabietic acids) were higher in the autumn. The total monoterpene con- centration was higher in the spring. Natural resources and bioeconomy studies 73/2017 20 4.6. Effect of storage Many initial extractives suitable for potential raw material applications are either rather volatile or chemically unstable. The extractives content starts to decrease immediately after tree felling and this degradation continues during storage (Alén 2000, Assarsson et al. 1963, Jirjis & Theander 1990, Ek- man 2000, Lappi et al.2014a). This also means that the chemical composition of the extractives- based fraction changes gradually. The major chemical changes in the resin during wood storage can be divided into three types: (1) rapid hydrolysis of triglycerides accompanied by slower hydrolysis of waxes, especially steryl esters, (2) oxidation/degradation/polymerization of resin acids, unsaturated fatty acids and to some extent other unsaturated compounds, and (3) evaporation of volatile terpenoids, mainly monoterpenes (Ekman 2000). The rate of all these reactions increases with increasing temperature. It has to be pointed out that not only storage, but also other phases of the wood handling pro- cess affect the extractives content of the biomass material, as well as the composition of the extrac- tives fraction. Several factors determine the nature and rate of change in the properties of wood resin (Ekman 2000, Rupar & Sanati 2005). These factors include the particular types of harvesting, transportation, storage and the inventory-control systems of the wood used at the mill. They also depend on the tree species, type of material, age of the material, time in storage, physical form of the wood, and the weather and other environmental conditions in all phases of the wood-handling process. The parallel effects of different factors related to the storage of wood-based biomass on the chemical compounds and their concentration is summarized as follows: Weather and other environmental conditions: High temperatures generally facilitate chemical reactions (Ekman 2000). Transition metal ions and light generally accelerate auto-oxidation reactions (Alén 2000). For example, according to Zahri et al. (2007), UV light induces the degradation of phe- nolic compounds present in oak extract. It is known that stilbenes are sensitive to daylight. The pho- to-induced chemical reactions of compounds belonging to this compound group have been studied, e.g., by Zhang and Gellerstedt (1994). The key step in these reactions seems to be an electron-proton transfer reaction leading to the formation of a stilbene phenoxyl radical. Increasing ventilation and thereby increasing the access of air and oxygen in the chip pile further speeds up evaporation and oxidation reactions (Ekman 2000). Some extractives are water soluble (hydrophilic), which means that both rainfall and water-debarking at the mill will leach some compounds of extractives from the biomass. Different tree species contain varying concentrations and types of water-soluble com- pounds in their wood and their leaching rates vary as well (Hedmark and Scholz 2008). The com- pounds of extractives that are generally found in woodyard runoff include phenolic compounds, resin acids and short chain fatty acids. According to Rupar and Sanati (2005), there seems to be correlation between the amount of precipitation and the emission levels of terpenes into the air. They also con- cluded that this phenomenon is more obvious for bark or wood chips than for forest residues, be- cause bark and wood chips are more sensitive to precipitation owing to the smaller particle size of the material, and for bark, owing to the porosity of the material. Tree species: different tree species have different extractives contents (see chapters 4.6.1-4.6.3) and the composition of this fraction which affects the chemical decomposition reactions during stor- age Harvesting: the time of the year, environmental conditions and whether it is the first-thinning or final felling (for the effect of tree age on the composition of wood extractives, see chapter 4.2) have an effect on chemical reactions occurring after the tree is felled. Transportation: If an open carriage is used during train or truck transport it leaves the material vulnerable to weather conditions, or generally to environmental conditions during the transport. When transportation in water is used, the situation is totally different as water protects wood from damage caused by fungi or insects (Kinnari 2002). Additionally, the chemical reactions in water are Natural resources and bioeconomy studies 73/2017 21 different (see the text related to the storage below). Average delivery times from the logging site to the sawmill or pulp mill in Finland are approximately 2–3 weeks for sawn wood, 4 weeks for spruce pulpwood and 8–13 weeks for pine and birch pulp wood (Lukkari et al. 2004). Road transportation takes only hours or days, whereas transportation in ships or barges takes approximately one week. Storage: Outside or inside storage and seasoning in water are all utilized when supplying wood to the mills. Environmental storage conditions (e.g., temperature and precipitation) and the duration of storage after harvesting the wood are important factors determining the amount and composition of wood resins after the storage period. It is well known that the hydrolysis of glycerides leading to free fatty acids and glycerol proceeds faster when the conditions for wood storage are wet instead of dry (Alén 2000). On the other hand, water and a high moisture content of wood protect it from dam- age caused by fungi or insects (Kinnari 2002). This is particularly important during the storage of wood logs in water in the summer (Alén 2000). During seasoning there is an increase in the amount of fatty acids and a reduction in neutral resin components (i.e., hydrocarbons, waxes, glycerides and higher alcohols) which is ascribed mostly to the saponifiable substances (Assarsson & Åkerlund 1967). Unsaponifiables have shown only a small decrease. The only chemical reaction taking place when wood is seasoned under water is the hydrolysis of glycerides to fatty acids. No changes indica- tive of metabolism or autoxidation have been observed (Assarsson & Åkerlund 1966). Type of biomass material: As it was mentioned before, the porosity of material among other factors might have effects on the chemical reactions of extractives, because evaporation of volatile terpenes is faster for more porous material (Rupar & Sanati 2005). Not only the tree species, but also different parts of the wood have different extractives contents and the compositions of extractive fractions (see chapters 4.6.1–4.6.3). This affects the chemical decomposition reactions of extractives during storage and wood handling as described above. Physical form of the wood: Chemical reactions are markedly faster when the wood is stored in the form of chips instead of logs (Alén 2000). Promberger et al. (2004) concluded that the faster de- terioration of compounds in wood chips is due to the larger surface area that makes substances more easily accessible. As an example, it has been reported that the degree of hydrolysis of triglycer- ides after eight weeks of outdoor chip storage was about the same as round-wood storage for one year (Ekman 2000). Living cells, which are contained in the bark, foliage, and wood, remain viable for some time when the tree is cut (Fuller 1985). These cells remain viable for long periods of time when the wood is stored in log form. When wood is chipped and placed in a pile, the living parenchyma cells respire in an attempt to heal the tree. Oxygen is consumed and heat is released. This heat gen- eration provides good growth conditions for bacteria which feed on the extractives in the wood. Af- ter a period of 7–14 days, it is not unusual that the temperature in a chip pile has reached about 50 °C (Nurmi 1999). The rate of heating is influenced by the rate of the pile construction and the fresh- ness of the wood. Additionally, the tree species and season of the year affect the temperature build- up. Pulpwood chip storage piles contain a myriad of microorganisms that develop in the wood one after another (Ekman 2000). The predominant microorganisms in chip piles are thermal-tolerant fungi and bacteria. Instead, chip piles rarely experience attacks by brown-rot fungi which are com- mon in piles of round-wood. Microorganisms in round-wood stored under continuous sprays of wa- ter are very different from those in dry piles. Every change in wood storage and handling technology (e.g. flotated and irrigated wood vs. dry wood, or storing stem wood vs. storing chips) will result in new substrate and environmental conditions for the development of wood-attacking microorgan- isms. 4.6.1. Birch Stemwood In birch round-wood storage, the dominating reaction is hydrolysis of triglycerides, resulting in the formation of free fatty acids (Ekman 2000), while steryl esters and unsaponifiables are generally sta- Natural resources and bioeconomy studies 73/2017 22 ble. Assarsson et al (1963) observed that the amount of free, unmodified fatty acids increased strongly at the beginning and reached a maximum level after 3–4 months in experiments with birch logs. After that period, the rate of reactions modifying free fatty acids (mostly oxidation) was greater than the ester hydrolysis, and, hence, the amount of unmodified free fatty acids in the resin de- creased. Assarsson et al (1963) found also that the rate of hydrolysis slowed down after 5–6 months storage of birch logs, after which period the amount of esterified fatty acids constituted less than 10% of the resin; in the beginning this fraction amounted about 70% of the resin. During chip storage the triglyceride hydrolysis is fast during the first couple of weeks (Ekman 2000). With longer storage times, the total amount of resin decreases as the fatty acids and steryl esters are degraded. Bark The total amount of birch bark extractives decreases notably and the chemical composition of this fraction changes during 24 weeks of storage (Figures 3 and 4) (Lappi et al. 2014a, b). Not only the amount of tri- and diglycerides but also the amount of betulin and other bioactive triterpenoids de- crease significantly during the storage of birch bark. Figure 3. Gravimetric amounts of extractives (Lappi et al. 2014c). Key: BB0 = birch bark sample taken before storage, BB1 = after 1 week, BB2 = after 2 weeks, BB4 = after 4 weeks, and BB24 = after 24 weeks. Similarly, SB0 = softwood bark sample taken before storage, SB1 = after 1 week, SB2 = after 2 weeks, SB4 = after 4 weeks, and SB24 = after 24 weeks. Fresh birch, pine, and spruce bark samples were used as references to determine the amount of losses of extractives before and during the pulp mill wet debarking. Natural resources and bioeconomy studies 73/2017 23 Figure 4. Results of extractive group analysis for birch bark. Key: BB0 = birch bark sample taken before stor- age, BB1 = after 1 week, BB2 = after 2 weeks, BB4 = after 4 weeks, and BB24 = after 24 weeks (Lappi et al. 2014c). 4.6.2. Spruce Stemwood According Assarsson et al. (1963), the total resin content in chipped spruce wood was almost con- stant for two weeks. Thereafter the resin content decreased. After four weeks the rate of hydrolysis reaction slowed down. In spruce wood chip seasoning, the amounts of triglycerides decreased by 90% and the amounts of waxes decreased by 70% in 3 months (Assarsson & Åkerlund 1967). The remaining esters might have been sterol esters to a large extent, as these are more difficult to hydro- lyze than glycerol esters. The amounts of free fatty acids increased strongly at the beginning and reached a maximum lev- el after one week in a spruce chip pile (Assarsson et al 1963). After that period, the rate of free fatty acid oxidation became greater than that of ester hydrolysis, hence, the amount of free fatty acids started to decrease. Assarsson et al (1963) also found that in a spruce chip pile, resin acids were not affected until af- ter two to four weeks seasoning. In a later study the amounts of resin acids had decreased by 60% after 3 months seasoning (Assarsson& Åkerlund 1967). The unsaponifiable compounds seemed to have remained fairly constant during the first eight weeks in the chip pile, whereafter a decrease was discernible. In the log storage of spruce wood, the unsaponifiable fraction was found to be almost unaffected (Assarsson et al 1963). Ekman (2000) studied the changes in the resin content of heartwood and sapwood of spruce during log storage. He detected that no triglyceride hydrolysis or other changes occurred in the heartwood, in contrast to sapwood. The resin content rapidly decreased in the sapwood but re- mained constant in the heartwood. Natural resources and bioeconomy studies 73/2017 24 Spruce wood samples taken from round-wood piles in a pulp mill woodyard on land in south- western Finland were analyzed (Ekman 2000). The resin content in logs that had been sprayed with water to avoid drying showed a very similar pattern of change as logs stored in water. As in the case of birch the most dominant chemical reaction was triglyceride hydrolysis, while total resin content decreased only slightly. In contrast, dry round-wood from piles without spraying with water showed a considerable decrease in the total amount of the wood resin. There was a decrease especially in the concentration of unsaturated fatty acids. Bark The amounts of extractives in the bark material which consisted of mixed softwoods (Norway spruce and Scots pine) decreased significantly during storage on land, roughly halving during the first four weeks (Lappi et al. 2014a, b, c). The total amounts of extractives decreased notably and the chemical composition of this fraction changed as well (Figs. 3 and 5) (Lappi et al. 2014a, b). Not only the amount of tri- and diglycerides decreased but so did the amounts of lignans. Figure 5. Results of extractives group analysis for softwood bark. Key: SB0= sample taken before storage, SB1= after 1 week, SB2= after 2 weeks, SB4= after 4 weeks, SB24=after 24 weeks (Lappi et al. 2014c). Lappi et al. (2014c) did not analyze stilbenes in the aforementioned study. However, it is known that the spruce bark stilbenes are sensitive to degradation (Holmbom 2011). Industrial bark at mill sites usually contains only 1–2% of stilbenes, sometimes even less. During debarking at the mills much of the stilbenes which are still left after storage are dissolved in the debarking process waters (Holmbom 2011). Natural resources and bioeconomy studies 73/2017 25 4.6.3. Pine Stemwood A drawback of seasoning Scots pine wood chips is that microbial degradation and oxidation reactions of fatty and resin acids directly decrease the yield of tall oil and turpentine which are by-products of kraft pulping (Ekman 2000). Fatty acids are oxidized much faster than resin acids during chip storage and at a faster rate than the amount of resin acids. Tall oil from fresh pine yields approximately equal amounts of fatty and resin acids. After four months storage, only 25% of the fatty acids remain, while 56% of the resin acids are retained. A few weeks of storing pine wood chips can lower the turpentine yield by about 50%. The losses are due to evaporation of volatile terpenes that are carried away in the convection of moist air currents that normally occur in the pile. Obviously, during round-wood storage, the turpentine components of the wood resin, especially the most volatile monoterpenes also evaporate, but at a much slower rate. According to one earlier study on radiata pine, if logs or chips remain wet during storage, triglyceride hydrolysis and other changes of the resin components occur primarily in the sapwood due to biological reactions (Ekman 2000). Stumps In stump wood of Scots pine trees, the changes in the chemical composition of the extractives frac- tion are more remarkable than the changes in the gravimetric extractives amount (Lappi et al.2014b). In a study by Lappi et. al (2o14b), stump material consisted of pine (Pinus sylvestris) stumps. The stumps were lifted and crushed and a storage pile (150 m3) built at UPM Pietarsaari Mill. The sam- pling frequency was initiated after 1, 2, 4, and 24 weeks of storage. Four different samples were tak- en at each sampling time, representing all areas of the pile. The average result based on the analysis of these samples is presented in Fig. 6. The total amount of stump-derived extractives decreased slightly during 24 weeks of storage, from 4.1% of dry solids to 3.6% of dry solids in the case of fresh stumps (Lappi et al.2014b). It has to be taken into account that although multiple samples were tak- en at each sampling time, some variation in the sample composition still may exist. However, it was concluded so that general trends on the behavior of extractives compounds of stumps could be not- ed based on these results. Natural resources and bioeconomy studies 73/2017 26 Figure 6. Results of an extractives group analysis for fresh stump and root biomass of Scots pine. trees. Key: FS0 = fresh stump sample taken before storage, FS1 = after 1 week, FS2 = after 2 weeks, FS4 = after 4 weeks, and FS24 = after 24 weeks (Lappi et al. 2014b). Natural resources and bioeconomy studies 73/2017 27 5. Distribution and content of extractives in tree biomass In this chapter the results from the studies on extractives of Norway spruce, Scots pine and silver and white birch trees have been summarized. It is rather challenging to compile data about the contents of individual extractives compounds in different parts of the tree. This is partly because the yields of extractives strongly depend on the extraction method and solvent (Co et al. 2012). The solvent used for extraction and the extraction method itself affect the gravimetric amount of the extractives frac- tion as well as its composition to some extent. Furthermore, the analysis techniques have some limi- tations, meaning that, for example, not all chemical compounds are detected by gas chromatography (GC). This might concern very low- or high-molecular weight compounds due to the restrictions caused by GC-column dimensions and/or GC-oven temperature program, or due to the retention of some compounds or part of them in a GC-column stationary phase. Thus, the true amounts of gravi- metric extractives differ from the amount determined by gas chromatography (GC). In addition, dif- ferent analysis methods (e.g., GC, HPLC and NMR) may result in differences in the analysis results. Thus, the amounts of extractives given in the following text cannot strictly be compared with each other, unless they are from the same references. However, they do provide general trends in the extractives contents of the different tree parts and information on the composition of those extracts. In Table 6, the variation in the amounts of different groups of extractives has been collated and reported regardless of the solvent used or the analytical method applied. The solubility of extractives varies greatly from one solvent to another. Extractives are typically removed from wood by solvent extraction, and the choice of solvent is a critical factor that determines the chemical composition and yield of the extract. Although significant differences can exist between specific extractives, hydro- philic extractives (phenolics) generally have better solubility in more polar solvents, while lipophilic extractives (terpenoids and aliphatic compounds) have better solubility in less polar solvents. Ace- tone is an exception in this rule, as it is known to dissolve both hydrophilic and lipophilic extractives with great efficiency (Harkin & Rowe 1971, Holmbom 1999). As for inorganic solvents, aqueous alkali is highly effective at dissolving otherwise insoluble components, mainly some phenolic acids and the insoluble fraction of condensed tannins, while water is a moderately effective solvent for most phe- nolics (Harkin & Rowe 1971, Fengel & Wegener 1989, Conde et al. 2013). Table 6. Range of variation of different groups of extractives (% w/w) in Norway spruce, Scots pine and Finnish birch species in stem heartwood, stem sapwood, knots, bark and tree stumps. Values have been reported re- gardless of solvent or method used. This information can be found in the literature. Norway spruce Extractive He ar tw oo d Sa pw oo d St em Kn ot w oo d Ba rk : 1) In ne r b ar k 2) O ut er b ar k St um ps Ro ot s Study Triglyserides 0.07 0.45 0.25 0.37- 0.96 Ånäs 1983, Anttonen et al. 2002, Bertaud and Holmbom 2004, Krogell et al.2012, Lappi et al. 2014, Diglycerides 0.06- 0.59 Krogell et al. 2012 Natural resources and bioeconomy studies 73/2017 28 Monoglyserides 0.15 Lappi et al. 2014 Fatty acids 0.11- 0.17 0.06- 0.74 0.01 0.25 0.05- 0.91 Ånäs 1983, Anttonen et al. 2002, Willför et al. 2003, Bertaud & Holmbom 2004, Krogell et al.2012, Lappi et al. 2014 Steryl esters 0.11 0.14 0.16 0.10- 0.63 Anttonen et al. 2002, Ber- taud & Holmbom 2004, Lappi et al. 2014,Krogell et al. 2012, Anäs 1983 Sterols 0.02- 0.09 0.01- 0.11 0.01 0.10 0.02- 0.29 Ånäs 1983, Anttonen et al. 2002, Willför et al.2003, Bertaud & Holmbom 2004, Krogell et al.2012, Lappi et al.2014 Resin acids 0.18- 0.19 0.17- 0.22 0.28 0.04- 0.24 0.02- 0.291) 0.03- 0.292) 2 0.47 Ånäs 1983, Anttonen et al. 2002, Willför et al. 2003, Bertaud & Holmbom 2004, Hovelstad et al. 2006, Krogell et al. 2012, Latva-Mäenpää 2013 et al., Lappi et al. 2014 Stilbenes 0.07- 0.27 Hammerbacher et al. 2011, Krogell et al. 2012 Stilbene gluco- sides 2.4- 5.5 Krogell et al. 2012, Jyske et al. 2014 Lignans 0.05- 1.2 0.04- 0.05 0.06 7.9– 12.3 0.31 Holmbom et al. 2003, Willför et al. 2003, 2004, 2005, Piispanen et al. 2008, Lappi et al. 2014 Oliolignans 0.02 0.05 2.5– 2.87 Willför et al. 2003,2004 Flavanoids 0.14 Krogell et al. 2012 Tannins 10.7 Kemppainen et al. 2014 Condensed tannins 1.7- 1.91) 7.9- 8.02) Holmbom et al. 2007 Terpenes 0.03 Anttonen et al.2002 Sesquiterpenes 0.00 0.02 Wajs et al. 2006 Diterpenoids 0.05 0.53 0.005 -0.71) 0.005 - 0.322) Willför et al. 2005, Krogell et al. 2012 Aliphatic alco- hols 0.04 Krogell et al.2012 Natural resources and bioeconomy studies 73/2017 29 Scots pine Extractive He ar t- w oo d Sa pw oo d St em Kn ot w oo d Ba rk : 1) In ne r b ar k 2) O ut er ba rk St um ps Study Triglyserides 0.03- 0.1 0.73– 1.22 0.62 0.27 0.17 1) 3.34 2) 1.88 0.32 Ånäs 1983, Martinez-Inigo et al. 1999, Willför et al.2003, Lappi et al. 2014 Diglycerides 0.44 0.14 Lappi et al. 2014 Mono and di- glycerides 0.231) 0.552) Ånäs 1983 Monoglyserides 0.12 0.03 Lappi et al. 2014 Free Fatty acids 0.22– 1.8 0.03- 0.4 0.11– 0.94 0.42 0.06 1) 0.17 2) 0.55 0.90 Ånäs 1983, Martinez-Inigo et al. 1999, Fries et al. 2000, Piispanen & Saranpää 2002, Willför et al. 2003, Ekeberg et al. 2006, Karppanen et al. 2008, Tiitta et al. 2009, Arshadi et al. 2013, Lappi et al.2014 Steryl esters 0.05- 0.14 0.07- 0.17 0.15 0.25 0.09- 0.43 0.14 Ånäs 1983 Sterols 0.01 0.45 1) 0.3 2) 0.29 0.03 Fries et al. 2000, Lappi et al. 2014, Ånäs 1983 Recin acids 0.21– 6.34 0.26– 0.89 0.85– 1.8 7.97– 20.2 0.72 1) 0.24 2) 0.49 1.35 Ånäs 1983, Martinez-Inigo et al.1999, Fries et al. 2000, Turtola et al.2002, Harju et al. 2002, Willför et al. 2003, Ekeberg et al. 2006, Hovel- stad et al.2006, Karppanen et al. 2007, 2008, Leinonen et al. 2008, Tiitta et al. 2009, Arshadi et al. 2013, Lappi et al. 2014 Stilbenes 0.14– 1.67 4.59– 5.21 Fries et al. 2000, Hovelstad et al. 2006, Karppanen et al. 2007,2008, Partanen et al. 2011 Lignans 0.01 0.01 0.01 1.33 1.09 0.05 Lappi et al.2014, Willför et al.2013 Oligolignans 0.08 0.03 0.06 0.39 Willför et al. 2003 Condensed tan- nins 3.21- 8.51 Steinshamn 2014 Diterpenoids 0.05 0.53 0.0005- 0.2 Willför et al. 2005, Krogell et al. 2012 Aliphatic alcohols 0.04 Krogell et al. 2012 Soluble sugars 0.02- 0.03 Karppanen et al. 2008, Tiitta et al. 2009 Natural resources and bioeconomy studies 73/2017 30 Silver birch St em w oo d Br an ch es Ba rk Ro ot s Study Triacylglycerols 0.2- 0.68 0.4 0.8-1 Piispanen & Saranpää 2004, Lappi et al. 2014, Roitto et al 2016 Diglycerides 0.57 Lappi et al. 2014 Monoglyserides 0.51 Lappi et al. 2014 Fatty acids 0.03- 0.25 0.38 0.48– 0.57 Piispanen & Saranpää 2004, Lappi et al. 2014, Roitto et al 2016 Steryl esters 0.18– 0.50 0.41– 0.42 0.78– 1.16 Piispanen & Saranpää 2004, Lappi et al. 2014, Roitto et al 2016 Free sterols 3 Lappi et al. 2014 Sterols 0.10– 0.11 0.5- 0.7 Roitto et al. 2016 Lignans 0.06 0.18 0.29– 1.86 Lappi et al. 2014, Roitto et al. 2016 Flavonoids 0.55– 1.43 1.14 Sutela et al. 2009, 2014 Phenolic glycosides 1.68– 2.84 Sutela 2009, 2014 Procyanidin 0.005 Roitto et al. 2016 Betulin and other triterpenoids 0.98 Lappi et al. 2014 Condensed tannins 10.8 13.05 Sutela et al.2009 Total Cinnamic acids 0.17 Sutela et al. 2014 Natural resources and bioeconomy studies 73/2017 31 6. Valuable bioactive extractives in common Finnish wood species and their properties Bioactive molecules are readily available through eco-friendly extraction processes using mild organic or aqueous solvents (Royer et al. 2012). Controlled and optimized isolation of extractives, e.g., from wood or forest residues prior to their use as combustible material represents an essential path lead- ing to added intrinsic value. Extracts may be used in various applications, e.g., pharmaceuticals, agri- food additives, cosmeceuticals, and nutraceuticals. Worldwide demand for bioactive molecules of natural origin has increased sharply in recent years. The natural health products (NHP) market has shown remarkable growth in developed countries. In Figure 7 routes to obtaining valuable bioactive extractives are summarized. More about the replacement of fossil fuel based chemicals and chemi- cals from edible oils with, e.g., tall oil based compounds are discussed in Chapter 7. Figure 7. Routes to obtaining valuable bioactive extractives. Currently utilized routes are illustrated with green arrows (modified from Holmbom 2010). Red arrows are used for the routes that currently are not commercially utilized. However, some research related to these routes is ongoing. Residues like bark represent attractive sources of bioactive molecules (Royer et al. 2012). Bark is a low-value by-product which is a readily available renewable resource, amounting generally to 7– 13% of the total volume of the tree (Xavier et al. 2012). Bark generally contains a high concentration of extractives, a number of which possess unique biological and therapeutic properties (Royer et al. 2012). Bark contains, for example, polyphenols which have beneficial properties such as antioxidant, anticancer, bactericidal, fungicidal, antispasmodic, sedative, analgesic and anti-inflammatory quali- ties. Both bioactivities of pure extractives (Pietarinen et al. 2006a), knotwood extracts (Välimaa et al. 2007, Pietarinen et al. 2006a, b, Metsämuuronen & Siren 2014) and bark extracts (Pietarinen et al. 2006a) have been studied. Various bark extracts have been used in traditional medicine, such as Chi- Natural resources and bioeconomy studies 73/2017 32 nese and Indian medicine, for the treatment of many diseases (Holmbom 2011). Resins scraped from the bark of old Norway spruce trees have been used as a resin salve in Northern Sweden and Finland for centuries. Recently this old invention was rediscovered in Finland and a commercial resin salve possessing antifungal and antibacterial properties was developed and introduced to the market. This is effective against pressure ulcers, burns and wound infections. In the following a number of extractives from wood biomass which have been found interesting due to their bioactivity potential are presented. However, not all such extractives have been listed and new ones can be found. 6.1. Phenolic extractives Plant phenolics have been suggested to provide multiple advantageous health and well-being effects, including maintenance of health and protection from diseases such as cancer and coronary heart disease (Karonen et al. 2004b). It is also possible to use them as natural food additives, since they influence the quality and stability of foods by acting as flavourants, colourants and antioxidants. A large variety of low-molecular phenolic compounds are found in trees, especially in the heart- wood, bark and knots whereas only traces occur in sapwood (Holmbom 2011). Compounds belonging to phenolics represent a wide variety of chemical structures, featuring a benzene ring as a core struc- ture to which one or more hydroxyl group(s) is/are attached, either free or substituted with various specific chemical functions (e.g. alkyl groups in ethers and esters, or mono/disaccharides in phenol glycosides) (Royer et al. 2012). These compounds can be grouped into several families, such as cou- marins, lignans, stilbenes, flavonoids, phenolic acids, tannins (grouped into condensed and hydrolys- able tannins), xanthones, quinones etc. They range from monomers to polymers and include various types of complexes. Such a wide variety of structures explains the impressive range of physico- chemical and biological activities recorded, and which come from their significant chemical reactivity. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators and singlet oxygen quenchers (Kähkönen et al. 1999). Addi- tionally, they provide metal chelation potential. Another key factor with regards to their biopotential is their ability to interact with cellular proteins; in fact, polyphenols can function as activators or in- hibitors of numerous cellular enzymes (Royer et al. 2012). Polyphenols have the ability to scavenge free radicals (strong antioxidant activity), block enzy- matic processes and stop fungal growth (Royer et al. 2012). They also contribute to antiseptic and antibacterial properties and have a positive incidence on product preservation, be it cosmetics, foods or pharmaceuticals. Their free-radical scavenging properties coupled with the antioxidant and anti- inflammatory properties are linked to the prevention of certain diseases that implicate oxidative stress and cellular ageing, cardiovascular and degenerative conditions: osteoporosis, cancer, arthritis and type II diabetes. The amount of extractable phenolic compounds in the heartwood of Norway spruce knots can be close to 30% (w/w), but on average it is around 15% (w/w) (Willför et al. 2003a, b). The amounts of phenolic compounds in knot heartwood are usually 50–100 times that found in the stemwood. The amounts of extractable phenolic compounds in the heartwood of Scots pine knots can be as high as 10% (w/w) (Willför et al. 2003a, c). In a study where the antioxidant activities of phenolic extracts from edible and nonedible Finnish plant materials were examined by their inhibition of methyl lino- leate oxidation, Scots pine bark was ranked among the most potent plant sources for natural phenol- ic antioxidants (Kähkönen et al. 1999). Several phenolic compounds such as phenolic acid glycosides, stilbene glycosides, lignans and flavonoids have been previously isolated from the inner bark of Scots pine trees, and the bark of Norway spruce trees (Krogell et al 2012). Scots pine bark has been found rich in procyanidins as well (Karonen et al. 2004b). Natural resources and bioeconomy studies 73/2017 33 6.1.1. Stilbenes Coniferous trees contain relatively large amounts of stilbene compounds (Mannila & Talvitie 1992). In Norway spruce trees they are concentrated in the bark of the tree. The total amount of isorhapon- tigenin, piceatannol, resvatrol and their glucosides is 1–6% of the dry weight. Stilbenoids in Norway spruce bark are mainly in glucosidic form (Latva-Mäenpää 2017). Stilbenoids of Scots pine trees are mainly localized in the heartwood region of the tree (Latva-Mäenpää 2017). Stilbenes, or stilbenoids, have been shown to be potential anti-inflammatory, anti-cancer, anti- oxidant, anti-ageing and chemoprotective agents (Mulat et al. 2014). In Table 7 some interesting stilbenes which are found in common Nordic softwoods are presented with the information about their properties for activities/functionalities. Table 7. Interesting bioactive stilbenes in common Nordic softwood species (Picea abies, Pinus sylvestris) Compound Properties Tree species and part References Piceatannol antimicrobial, anticancer, cardioprotec- tive, allelopathic, antioxidant, artemicide, fungicide, pesticide, P. abies: bark P. sylvestris Reinhold et al. 1980, Mannila & Talvitie 1992, Wolter et al. 2002, Latva-Mäenpää et al. 2014, 2017 Lim & Koffas 2010, Duke 2017 Siwale 2015 Resvatrol antimicrobial, anti-cancer, antiviral, car- diovascular and neuroprotective, extends the life-span of cells i.e., anti-aging P. abies: bark P. sylvestris Välimaa et al. 2007, Holmbom 2011, Mannila & Talvitie 1992 Pinosylvin fungicide, antimicrobial, antioxidant, anticancer, anti-inflammatory, antifeed- ant, pesticide P. sylvestris: heartwood, knots Willför et al. 2003b Välimaa et al. 2007, Fang et al. 2013 Latva-Mäenpää 2017 Duke 2017 Siwale 2015 Pinosylvin monomethyl ether antimicrobial, anticancer, anti- inflammatory, antifungal P. sylvestris: heartwood, knots Willför et al. 2003b, Välimaa et al. 2007 Latva-Mäenpää 2017 Siwale 2015 Pinosylvin dimethyl ether antifungal P. sylvestris: heartwood, knots Willför et al. 2003b, Latva-Mäenpää 2017 Dihydropi- nosylvin antibacterial, fungicide, pesticide P. sylvestris Duke 2017 Astringin antibacterial, anticancer, antifungal, pes- ticide P. abies: nee- dles, bark Latva-Mäenpää et al. 2014, 2017 Slimestad & Hostett- mann 1996, Duke 2017 Natural resources and bioeconomy studies 73/2017 34 Isorhapontin antibacterial, anti-leukemia, cardio pro- tection, antifungal P. abies: nee- dles, bark Mannila & Talvitie 1992, Latva-Mäenpää et al. 2014,2017 Slimestad & Hostettmann 1996 Isorhaponti- genin antibacterial, antifungal P. abies: bark Mannila & Talvitie 1992, Latva-Mäenpää et al. 2014, 2017 Piceid antibacterial, antilipoperoxidant, antioxi- dant, fungicide, hepatoprotective, pesti- cide P. abies: nee- dles, bark Latva-Mäenpää et al. 2014, Slimestad & Hostett- mann 1996 Duke 2017 6.1.2. Flavonoids A considerable amount of research has been directed toward the activity of flavonoids as antioxi- dants and radical scavengers, as well as their anti-mutagenic and anti-carcinogenic properties (Rauha 2001). Flavonoids have been suggested to play a preventive role in the development of cancer and heart diseases (Kähkönen et al. 1999). Flavonoids are divided into several subgroups, and it is im- portant to keep in mind that the biological and chemical properties of flavonoids belonging to differ- ent subgroups can be quite different (Eklund 2004). Flavonoids are mainly present in plants as glyco- sides, whereas aglycones occur less frequently. Flavonoids can be found in the foliage of hardwoods and softwoods, and in the bark, at least in the rootbark of Norway spruce trees (Rauha 2001, Pan and Lundgren 1995). In Table 8 some interesting flavonoids and their properties for various activi- ties/functionalities are presented. Table 8. Interesting bioactive flavonoids in common Nordic wood species (Picea abies, Pinus sylvestris, Betula pendula and Betula pubescens) Compound Properties Tree species and part References apigenin Reduced risk of coronary heart disease, vasoprotective activity antiADD, antiaflatoxin, antiaggregant, antiaging, antiallergic, antiangiogenic, antiarrhythmic, antibacterial, anticancer antidermatitic, antiestrogenic, antiher- petic, antihistaminic, anti-HIV, anti- inflammatory, antimelanomic, antimuta- genic, antioxidant, antiperistaltic, anti- proliferant, antispasmodic, antistress, antithyroid, antiviral, anxiolytic, apoptot- ic, , choleretic, CNS-depressant, cytotoxic, differentiator, diuretic, DNA-Protective, estrogenic, hypotensive, inotropic, mus- culotropic, mutagenic, myorelaxant, pes- ticide, progestational, radioprotective, sedative, sunscreen, uterotrophic B. pubescens and B. pendu- la: leaves Rauha 2001, Valkama et al. 2003, Keinänen & Julkunen- Tiitto 1998, Lim & Koffas 2010, Duke 2017 Natural resources and bioeconomy studies 73/2017 35 acacetin protective potential against prostate can- cer allergenic, antiaflatoxin, antiallergic, anti- histaminic, antimalarial, antimutagenic, hepatoprotective, mutagenic, plasmodi- cide, protisticide B. pubescens and B. pendu- la: leaves Rauha 2001, Valkama et al. 2003, Keinänen & Julkunen-Tiitto 1998, Singh et al. 2005, Duke 2017 glycosides of kaempferol anticancer, antioxidant, anti- inflammatory, antiaflatoxin, antiaggre- gant, antiallergic, antibacterial, antifertili- ty, antigingivitic, antiherpetic, antihista- minic, anti-implantation, antilymphocytic, antimutagenic, antiperiodontic, an- tiplaque, antiradicular, antiseptic, anti- serotonin, antispasmodic, antistaphylo- coccic, antiulcer, antiviral, apoptotic, carcinogenic, choleretic, cytotoxic, diuret- ic, estrogenic, fungicide, hepatoprotec- tive, HIV-RT-Inhibitor, hypotensive, ino- tropic, mutagenic, natriuretic, neuropro- tective, pesticide, protisticide, teratolog- ic, uterotrophic, vasodilator B. pubescens and B. pendu- la: leaves P. abies: nee- dles P. sylvestris: needles Rauha 2001, Lim & Koffas 2010, Svahn 2015, Räisänen et al. 2008, Duke 2017 quercetin and glyco- sides of quercetin Reduced risk of coronary heart disease. Neuroprotective effects. Potent for treat- ing or preventing cancer due to its anti- mutagenic, anti-proliferative, antioxida- tive activities and its role in cellular re- ceptor interactions and modification of signal transduction. Anti-inflammatory. B. pubescens: leaves*) P. abies: nee- dles P. sylvestris: needles Rauha 2001, Oleszek et al. 2002, Ossipov et al. 1995, 1996, Keinä- nen & Julkunen-Tiitto 1998, Strack et al. 1989, Lim & Koffas 2010, Svahn 2015 glycosides of myricetin Reduced risk of coronary heart disease, anti-oxidant, pro-oxidant, anti- carcinogenic, mutagenic, antiviral, antidi- abetic, lowers cholesterol level, prevents formation of blood-cloths B. pubescens: leaves P. abies Rauha 2001, Ossipov et al. 1995, 1996, Kei- nänen & Julkunen- Tiitto 1998, Svahn 2015 isorhamnetin and glyco- sides of iso- rhamnetin antibacterial, antihistaminic, anti- inflammatory, antioxidant, antispasmod- ic, cancer-preventive, hepatoprotective, pesticide, vasodilator P. abies: nee- dles, rootbark P. sylvestris: needles*) Rauha 2001, Strack et al. 1989, Pan & Lund- gren 1995, Kähkönen et al. 1999, Duke 2017 naringenin and glyco- sides of naringenin anti-oxidant, anti-inflammatory, lowers cholesterol level anti-acetylcholinesterase, antiaflatoxin, antiaggregant, antialzheimeran, anti- amnesic, antibacterial, anticancer, antiesch- erichic, antiestrogenic, antifibrotic, anti- hepatotoxic, antiherpetic, anti-HIV, anti- inflammatory, antimutagenic, antiperistaltic, antiperoxidative, antiradicular, antisindhis, antispasmodic, antistaphylococcic, anti- tumor, , antiulcer, antiviral, aphidifuge, can- didicide, choleretic, embryotoxic, estrogen- ic, fungicide, fungistat, hepatoprotective, neuroprotective, pesticide, serotonin- inhibitor, teratogenic, uterotrophic B. pubescens: leaves P. abies*) Rauha 2001, Ossipov et al. 1995, 1996, Svahn 2015, Duke 2017 Natural resources and bioeconomy studies 73/2017 36 pinocembrin anesthetic, antibacterial, antileukemic, antiseptic, artemicide, candidicide, cyto- toxic, fungicide, pesticide P. sylvestris: knotwood, heartwood Rauha 2001, Willför et al. 2003b, Duke 2017 (+)-catechin antiaggregant, anticoagulant, anticom- plementary, anti-HIV, anti-inflammatory, antimutagenic, antioxidant, antiperoxi- dant, antiprostaglandin, antiradicular, antiulcer, cancer-preventive, cardiotonic, cytotoxic, dermatitigenic, hemostat, hepatoprotective, immunostimulant, neuroprotective, phagocytotic, vasocon- strictor B. pubescens, B. pendula: leaves, bark, stemwood P. abies: nee- dles, rootbark P. sylvestris: bark, nee- dles**) Rauha 2001, Ossipov et al. 1995, 1996, Kei- nänen & Julkunen- Tiitto 1998, Strack et al. 1989, Pan & Lund- gren 1995, Karonen et al. 2004a, Kähkönen et al. 1999, Roitto et al. 2016, Duke 2017 *) Only as glycosides, **) Also as glycoside 6.1.3. Tannins Tannins are polyphenolic compounds based either on flavan-3-ol monomers (condensed tannins, also called proanthocyanidins), or on gallic or hexahydroxydiphenic acid esters linked to a sugar moi- ety (hydrolysable tannins) (Kemppainen 2014). Tannins are the main components of softwood (P. abies and P. sylvestris) extracts (see table 6). The tannins in wood bark are mainly condensed tannins (Tamminen et al. 2017). Tannins also have bioactive properties, including antioxidative and radical-scavenging power, thus, they are also of great interest in nutrition and health (Holmbom 2011). The use of tannins is presently expanding into nutritional and pharmaceutical areas. Tannins are known to display antimi- crobial activity and they can also complex with the proteins in bacterial cell membranes. Tannins are used in pharmaceuticals which aim at curing bacterial intestinal infections. Additionally, tannins have been indicated to have antitumor and anticancer properties as well as antiviral effects. There has been interest in the antimutagenic and antitumorigenic properties of tannic acid especially (Rauha 2001). In Table 9 interesting tannins and their properties for activities/functionalities are presented. Table 9. Interesting bioactive tannins in common Nordic softwood species (Picea abies, Pinus sylvestris) Compound Properties Tree species and part References epigallocatechin- 3-gallate antiangiogenic, antitumor P. sylvestris Duke 2017 tannic acid allergenic, antianacarditic, antibacterial, anticariogenic, anticolitic, antidecubitic, antidermatotic, antidiarrheic, antidote, antidysenteric, antiencephalitic, antien- teritic, antifeedant, antigargantitic, an- tigingivitic, antihemorrhoidal, antiherpet- ic, anti-HIV, antimutagenic, antinitrosa- minic, antiobesity, antiophidic, antioxi- dant, antipharyngitic, antipolio, antirhini- tic, antiseptic, antistomatitic, antitonsili- tic, antiulcer, antiviral, astringent, cyto- toxic, detoxicant, emetic, flavor, hemo- stat, hepatotoxic, immunostimulant, pes- ticide P. abies: nee- dles Duke 2017, Adamczyk et al. 2011 Natural resources and bioeconomy studies 73/2017 37 gallic acid analgesic, antiadenovirus, antiallergenic, antianaphylactic, antiangiogenic, anti- asthmatic, antibacterial, antibronchitic, anticancer, c, antiescherichic, antifibrino- lytic, antiflu, antihepatotoxic, antiherpet- ic, antiHIV, anti-inflammatory, antileish- manic, antiMRSA, antimutagenic, an- tinitrosaminic, antioxidant, antiperiodon- titic, antiperoxidant, antipolio, antiprote- olytic, antiradicular, antiseptic, antistaph- ylococcic, antitumor, , antiviral, apoptot- ic, astringent, bacteristat, bronchodilator, candidicide, carcinogenic, choleretic, cytotoxic, gram(+)icide, gram(-)icide, hemostat, hepatoprotective, immuno- modulatory, immunostimulant, immuno- suppressant, insulin-sparing, myorelax- ant, nephrotoxic, pesticide, styptic P. sylvestris Duke 2017 6.1.4. Lignans Lignans have attracted much interest due to their broad range of biological activity (Eklund et al. 2004). The knots, i.e., the branch bases of softwood tree species are the richest sources of lignans. Lignan 7-hydroxymatairesinol (HMR), has been stated to have a positive influence on the develop- ment of breast, prostate and colon cancer which rely specifically on estrogens in order to progress (Holmbom 2011). Lignans also help to maintain good cardiovascular health and to moderate other estrogen dependent health problems (menopause, osteoporosis). Lignans and oligolignans are strong antioxidants and radical scavengers, however according to tests performed by Välimaa et al. (2007), these mechanisms are not directly associated with antimicrobial effects. In Table 10 interesting bio- active lignans and their properties for activities/functionalities are presented. Table 10. Interesting bioactive lignans in common Nordic softwood species (Picea abies, Pinus sylvestris) Compound Properties Tree species and part References matairesinol antileukemic, fungicide, insecticide- synergist P. abies: Knotwood, heartwood P. sylvestris: Knotwood, bark Willför et al. 2003a, b, Duke 2017, Karonen et al. 2004a, Fengel & Wegener 1989 hydroxyma- tairesinol chemopreventive effects, antioxidant, P. abies: Knotwood P. sylvestris: Knotwood Willför et al. 2003a, b, Eklund et al. 2004 matairesinol dimethyl ether P. abies: Knotwood Willför et al. 2003a Natural resources and bioeconomy studies 73/2017 38 pinoresinol antioxidant P. abies: Knotwood, heartwood P. sylvestris: bark Willför et al. 2003a, Duke 2017, Karonen et al. 2004a, Fengel & Wegener 1989 dimethyl pinoresinol P. abies: Knotwood Willför et al. 2003a nortrache- l