Water-soluble components of Pinus pinaster wood

Abstract

Aqueous fractionation of wood has been proposed as a suitable processing method for biorefineries. When treatments are performed under low severity conditions, water-soluble components (which could be detrimental in further processing stages) are removed, whereas polysaccharides, lignin, and other water-insoluble constituents remain in solid phase with little alteration. In order to explore the presence of added-value products in aqueous extracts from Pinus pinaster wood, different samples (heartwood and sapwood with and without knots) were extracted with water at 130 to 140 ºC, and the resulting solutions were assayed for yield and composition (by GC-FID, GC-MS, and HPLC). The major extract components, such as polysaccharide-derived products, simple phenolics, stilbenes, lignans, flavonoids, organic acids, jubaviones, steryl esters, and triglycerides, were identified and quantified. In order to assess a possible application of the extracts, their antioxidant activity was measured using the Trolox Equivalent Antioxidant Capacity assay.

Full Article

Water-Soluble Components of Pinus pinaster Wood

Enma Conde,^a,b Wenwen Fang,^b Jarl Hemming,^b Stefan Willför,^bAndrés Moure,^a Herminia Domínguez,^a and Juan Carlos Parajó ^a,*

Aqueous fractionation of wood has been proposed as a suitable processing method for biorefineries. When treatments are performed under low severity conditions, water-soluble components (which could be detrimental in further processing stages) are removed, whereas polysaccharides, lignin, and other water-insoluble constituents remain in solid phase with little alteration. In order to explore the presence of added-value products in aqueous extracts from Pinus pinaster wood, different samples (heartwood and sapwood with and without knots) were extracted with water at 130 to 140 ºC, and the resulting solutions were assayed for yield and composition (by GC-FID, GC-MS, and HPLC). The major extract components, such as polysaccharide-derived products, simple phenolics, stilbenes, lignans, flavonoids, organic acids, jubaviones, steryl esters, and triglycerides, were identified and quantified. In order to assess a possible application of the extracts, their antioxidant activity was measured using the Trolox Equivalent Antioxidant Capacity assay.

Keywords: Pinus pinaster; Wood; Water extraction; Stilbenes; Flavonoids; Lignans

Contact information: a: Chemical Engineering Department, University of Vigo, As Lagoas, 32004, Ourense, Spain / CITI-University of Vigo, Parque Tecnolóxico de Galicia, Rúa Galicia nº 2, 32900, Ourense, Spain; b: Process Chemistry Centre, Åbo Akademi University, Porthansgatan 3, FI-20500, Turku, Finland; *Corresponding author: jcparajo@uvigo.es

INTRODUCTION

The selective separation of wood structural components (cellulose, hemicelluloses, and lignin) is a basic principle of biorefineries. For this purpose, wood processing with hot, compressed water (also known as autohydrolysis or hydrothermal treatments) under conditions of intermediate severity (usually at 160 to 210 ºC) lead to hemicellulose decomposition, which enables the separation of this fraction from cellulose and lignin (which remain in solid phase) (Gullón et al. 2012). Autohydrolysis has been proposed as a method for wood fractionation (Yáñez et al. 2009) since the resulting solids, which are almost free from hemicelluloses, can be further processed (for example, by delignification or enzymatic hydrolysis) to achieve a separate utilization of the three structural wood components (Gullón et al. 2012).

Besides hemicellulose-derived saccharides, the liquors from hydrothermal processing of wood contain other components (such as low molecular weight phenols or lipophilic compounds) that may be detrimental for the further purification and utilization of the soluble hemicellulose-derived saccharides. Because of this, an aqueous extraction under low severity conditions has been carried out prior to the hydrothermal stage in order to remove extractives (González-Muñoz et al. 2011 and 2012; Rivas et al. 2012). In this context, a wood biorefinery involving water extraction (for extractive removal) followed by further hydrothermal processing (for hemicellulose solubilization) could be better implemented if the aqueous extracts contain value-added compounds that could contribute to the profitability of the whole process. For this purpose, reliable data on the composition of the aqueous extracts are necessary. In the same way, an experimental evaluation of the antioxidant activity of extracts is of interest in order to assess their potential for other key applications.

Softwoods are the dominant lignocellulosic materials in the Northern hemisphere (Galbe and Zacchi 2002). Pinus pinaster is a fast-growing species and is drought- and salt-resistant (Berthier et al. 2001). This species is abundant in the North-West of Spain, as well as in other Atlantic and Mediterranean regions.

The fractionation of pine wood with hot, compressed water or steam has been considered in the literature, which is oriented either to the removal of extractives or to the manufacture of soluble saccharides from hemicelluloses (Shahbazi et al. 2005; González-Muñoz et al. 2011 and 2012; Rivas et al. 2012; Yoon et al. 2008; Koell and Lenhardt 1987). The profitability of implementing stages to recover extractive-derived products in a biorefinery has been questioned (Van Ree and Annevelink 2007); however, it would be feasible if value-added products are present in the feedstock (or in process streams) above a given threshold, and can be separated efficiently. In the case of pine woods, phenolic stilbenes, flavonoids, and lignans are potential targets for biorefineries owing to their biological properties.

A wide scope of applications has been suggested or pinosylvins including anti-fungal and antibacterial agents (Lindberg et al. 2004; Lee et al. 2005; Celimene et al. 1999; Gref et al. 2000; Venäläinen et al. 2004) with activity towards Listeria monocytogenes (Gözü et al. 2010), cytotoxicity against a murine hepatic carcinoma cell line (Välimaa et al. 2007), antimetastatic activity (Park et al. 2012), antiinflammatory action based on the reduction of blood reactive species (Jančinová et al. 2012; Bauerova et al. 2011), and angiogenic effects (Kimura and Sumiyoshi 2011).

Flavonoids are known to exert biological, nutraceutical, and clinical effects (Maimoona et al. 2011), including in vitro antioxidant, anti-allergic, anti-inflammatory, anti-microbial, anti-cancer, and anti-diarrheal activities. Flavonoids can also be involved in plant defense mechanisms. Specifically, antioxidant activity has been reported for pinobanksin and pinocembrin (Neacsu et al. 2007), whereas the ability to modulate inflammatory responses in vitro has been claimed for the latter (Soromou et al. 2012). Pinocembrin protects neurons against beta-amyloid-induced toxicity (Liu et al. 2012) and has been predicted to have a number of biological activities, including anti-HIV action (Maridass et al. 2008). Other reported properties include bacteriostatic and antifungal activities (Villanueva et al. 1970; Shain and Miller 1982) and the ability to trigger the mitochondrial apoptosis in colon cancer cells (Kumar et al. 2007). Both pinocembrin and pinobanksin possess antimutagenic properties, in particular against ofloxacin-induced mutagenicity in Euglena gracilis; whereas pinobanksin is able to inhibit the peroxidation of low density lipoprotein and to scavenge peroxyl radicals (Ondrias et al. 1997; Neacsu et al. 2007). Taxifolin exerts a number of protective and anticancer effects (Lee et al. 2007; Luo et al. 2008; Rogovskii et al. 2010), and enhances the antibiotic activity in combined therapies (An et al. 2011). Antimicrobial activity (Ango et al. 2012), the ability to reduce reactive oxygen species (ROS) formation in polymorphonuclear cells (Kang et al. 2010), and activity to reduce lipid peroxidation (Redzynia et al. 2009) have been reported for dihydrokaempferol.

Lignans occurring in softwoods possess chemopreventive properties (Lampe 2003), present antioxidant and antitumor activities, cause neuroprotective effects (Li et al. 2012), and can be employed cytotoxic antimicrobial agents (Willför et al. 2004). On the other hand, the associations between lignans and decreased risk of cardiovascular disease are promising, but they are not yet well established, perhaps due to low lignan intakes in habitual Western diets (Peterson et al. 2010). Nortrachelogenin has been proposed as a potential anti-malarial drug (Kebenei et al. 2011). The risk of certain types of breast cancer in premenopausal women is lowered by pinoresinol ingestion (Brown 2012). Pinoresinol presents activity against both human pathogenic fungi (Hwang et al. 2010) and Gram-positive bacteria (Céspedes et al. 2006). Secoisolariciresinol exhibited a significant antifungal activity on fungi of white rooting and wood staining (Céspedes et al, 2006).

This article deals with the aqueous extraction of Pinus pinaster wood samples obtained at different positions of selected trees. Extractions were performed at 130 to 140 ºC, and extracts were assayed for yield, composition, and antioxidant activity. The experimental data provide information on the types and amounts of major extract components, as well as their potential antioxidant activities.

EXPERIMENTAL

Materials

Three healthy 30-year-old Pinus pinaster trees were felled near Ourense (NW Spain). Disks (5 cm height) were cut and the samples listed in Table 1 were milled, air-dried, milled to pass a 10-mesh screen, and stored at room temperature until use.

Table 1. Wood Samples and Nomenclature

Methods

Aqueous extraction

Samples were extracted with water in a batch pressurized reactor equipped with a temperature controller (Parr Instr. Co., Moline, IL). Samples were heated in the reactor to the treatment temperatures of 130 or 140 ºC for prescribed times, and afterwards cooled immediately. Treatments were performed at a liquid:solid ratio (LSR) of 10:1 g:g (oven-dry solid basis). The maximum temperature of treatments was chosen on the basis of literature data (González-Muñoz et al. 2012) and preliminary experimental results, as a compromise between high yield in soluble material and limited hemicellulose decomposition. After cooling, extracts were recovered by filtration and assayed for extraction yield, composition, and antioxidant activity.

Yield measurements and analytical methods

The aqueous extraction yield was measured gravimetrically by oven-drying at 105 ºC until constant weight. The compositional and antioxidant activity results are referred to the sample content of non-volatile components. Monosaccharides in extracts were determined by high performance liquid chromatography equipped with refractive index detector (HPLC-RI) as described by Garrote et al. (1999). Oligosaccharides were quantified as monosaccharides (using the same HPLC-RI method) by measuring the increase in sugar concentration caused by a quantitative acidic post-hydrolysis (Garrote et al. 1999). For measuring the concentrations of lipophilic and hydrophilic compounds in aqueous extracts, the samples were freeze-dried and re-extracted with acetone before silylation. Quantification and identification of components was done with GC-FID and GC-MS, respectively. Stilbenes, flavonoids, lignans, simple phenolics, juvabiones, resin acid, and free fatty acids were analyzed using a 25 m x 0.20 mm i.d. column coated with crosslinked methyl polysiloxane (HP-1, 0.11 µm film thickness) using heneicosanoic acid as an internal standard (Ekman and Holmbom 1989). Analysis of total steryl esters and triglycerides was performed on a short 6 m x 0.53 mm i.d. column (HP-1, 0.15 µm film thickness) using cholesteryl heptadecanoate and 1,3-dipalmiotyl-2-oleyl glycerol as internal standards (Örså and Holmbom 1994). The practical limit of quantification of the individual compounds was 1/100 of the internal standard amount in each sample, but compounds detected in smaller amounts were also identified. Identification of individual components was performed by GC-MS analysis of the silylated components using the HP-1 column cited above.

Trolox Equivalent Antioxidant Activity (TEAC)

The assay, based on the scavenging of 2,2´-azinobis (3-ethylbenzothiazoline-6-sulfonate) radical (denoted ABTS), was performed according to Re et al. (1999). The ABTS radical cation (ABTS^•+) was produced by reacting a 7 mM ABTS stock solution with 2.45 mM potassium persulfate. Results are expressed as Trolox equivalents using a standard curve (0 to 1.6 mM). Assays were performed in triplicate.

Average values and deviations

The data determined for yield, composition, and antioxidant activity for the corresponding samples of trees A, B, and C are reported in terms of average values and standard deviations. The standard deviations are largely due to between-tree variations, in agreement with the data reported for Pinus sylvestris in an extensive study (Willför et al. 2003a).

RESULTS AND DISCUSSION

Wood Processing and Aqueous Extraction Yield

Extraction of Pinus pinaster wood at 130 ºC has been proposed to remove water-soluble extractives while causing little hemicellulose decomposition (González-Muñoz et al. 2011 and 2012; Rivas et al. 2012). Based on preliminary experimental data, extractions were also performed at 140 ºC in order to assess the expected benefits (increase in extraction yield) and disadvantages (increased hemicellulose solubilization) on a quantitative basis. The results in Table 2 showed significant differences between heartwood samples (HW, DK_HW and LK_HW, with average extraction yields in the range 80.7-84.5 g/kg g oven-dry wood) and sapwood samples (SW, DK_SW, and LK_SW, for which the extraction yield ranged from 12.8 up to 17.6 g/kg oven-dry wood). The extraction yield scarcely varied when the treatment temperature was increased from 130 to 140 ºC, confirming that limited incremental hemicellulose decomposition took place.

The major components contributing to the solid yield were soluble saccharides and non-saccharide compounds, as discussed in the following sections. In addition, the extraction treatment also efficiently removed inorganic salts and metals, which can be a great advantage in later upstream process stages.

Table 2. Aqueous Extraction Yields

Water-Soluble Saccharides

According to the literature (González-Muñoz et al. 2012), the hemicelluloses of Pinus pinaster wood show the following distribution of non-glucose anhydrosugars (expressed as % wt., oven-dry wood basis): mannosyl units, 10.5; xylosyl units, 4.30; galactosyl units, 2.39; and arabinosyl units, 1.71 (total, 18.9 % wt or 189 g/kg). Looking at the extraction yields shown in Table 2 and considering the extractive content (the ethanol-soluble fraction accounts for 28.4 g/kg, according to the same literature reference), it can be inferred that a significant part of the aqueous extracts must come from hemicelluloses (at least, with the heartwood samples). This was confirmed by the experimental data in Table 3, which lists the contents of samples in monosaccharides and in oligo- or polymeric saccharides. Whereas only small amounts of monosaccharides were observed in all cases (below 94 g monosaccharides/kg extract), oligo- and polymeric saccharides accounted for 366 to 459 g/kg extract from samples SW, DK_SW, and LK_SW, and for 780-964 g/kg extract from samples HW, DK_HW, and LK_HW. According to these findings, the separation of saccharides from non-saccharide com-pounds seems to be necessary in order to make profitable use of the extracts. The necessary refining steps would lead to purified oligo- and/or polymeric-saccharides derived from the hemicelluloses, for which applications as immunostimulatory agents, prebiotics, or radical-scavengers have been reported (Rivas et al. 2012; Ebringerova et al. 2008).

Table 3. Hemicellulose-Derived Products in Water Extracts

Phenolic Compounds

A variety of phenolic compounds were present in the extracts, including simple phenolics, phenolic stilbenoids, flavonoids, and lignans. The presence of similar phenolic fractions dominated by stilbenes, lignans, and flavonoids has earlier been reported for other pine species (Willför et al. 2003a and 2007; Pietarinen et al. 2006a). In the samples obtained in this work, the most abundant simple phenolics were isoferulic and 3,4-dihydroxycinnamic acids, which reached concentrations up to 2.3 to 1.3 g/kg in extracts from the sample DK_SW. Among the rest of the compounds, the most abundant simple phenolics were vanillin, 3-hydroxybenzoic acid, 4-hydroxycinnamic acid, pinitol, and coniferyl alcohol, which appeared at average concentrations in the range 1.3 to 0.5 g/kg in extracts from samples DK_SW and/or LK_SW, DK_SW, and at lower concentrations in the rest. Vanillic alcohol, 4-hydroxybenzyl alcohol, vanillic acid, dihydroconiferyl alcohol, 3,4-dihydroxybenzoic acid, and 1-guaiacylglycerol were also found in some extracts, all of them at concentrations below 0.4 g/kg extract.

Phenolic stilbenoids (pinosylvin and its derivative pinosylvin monomethyl ether) were found in extracts at concentrations up to 0.5 and 0.4 g/kg, respectively (see Table 4). Studies have reported the occurrence and the extraction of pinosylvins from conifers such as Picea abies, Pinus sylvestris (Willför et al. 2003a, 2003b, 2004 and 2007; Pietarinen et al. 2006a; Hovelstad et al. 2006), Pinus thunbergii (Kokubo et al. 1990), Pinus strobus (Geraldo de Carvalho et al. 1996), Pinus radiata (Hillis and Inoue 1968), Pinus contorta (Loman 1970), Pinus sibirica, and Pinus cembra (Willför et al. 2003c). The occurrence of pinosylvins has also been reported for Pinus jeffreyi (Anderson 1956), Pinus resinosa (Simard et al. 2008), and Pinus griffithii (Mahesh and Seshadri 1954). In comparison, scarce information has been reported on the composition of Pinus pinaster wood. Pioneering studies byAlvarez Novoa et al. (1950) and Hata (1955) reported on the isolation of pinosylvin monomethyl ether from Pinus pinaster heartwood using a multistage extraction method.

According to the results in Table 4, both pinosylvin and pinosylvin monomethyl ether were more abundant in samples containing knots, and in most cases, the content of pinosylvin monomethyl ether was equal to or slightly higher than that of pinosylvin. Increased amounts of stilbenes in knotwood compared to stemwood have been reported for Pinus sylvestris (Willför et al. 2003a) and Pinus radiata (Hillis and Inoue 1968; Pietarinen et al. 2006a). Mass ratios of pinosylvin monomethyl ether/pinosylvin in the range 1.1 to 1.4 (near the values determined in this work) have been reported for Pinus sylvestris(Hovelstad et al. 2006).

Flavonoids, characterized by a C₆–C₃–C₆ structure, are common phenolics in trees. In the water extracts obtained in this study, pinobanksins (including the dihydroflavonol pinobanksin and its derivative pinobanksin-3-acetate) were the most abundant flavonoids, reaching average concentrations in the range 1.4-2.0 and 2.0-2.3 g/kg in extracts from samples DK_HW and DK_SW, respectively. The presence of the flavanone pinocembrin (which can be converted into pinobanksin by hydroxylation), was also noticed, reaching higher average concentrations (0.5 to 1.6 g/kg extract) in samples containing knots than in HW or SW samples (for which the concentration range was 0.1 to 0.2 g/kg extract). Pinobanksin and pinocembrin were earlier identified in Pinus pinaster, Pinus radiata, Pinus banksiana, Pinus contorta, Pinus griffithii, Pinus resinosa, Pinus parviflora, and Pinus morrisonicola (Hata 1955; Alvarez-Novoa et al. 1950; Hillis and Inoue 1968; Willför et al. 2003c; Sinclair and Dymond 1973; Lindberg et al. 2004; Neacsu et al. 2007; Loman 1970; Simard et al. 2008; Mahesh and Seshadri 1954; Fang et al. 1987; Pietarinen et al, 2006a), but not in wood samples from Pinus strobus (Geraldo de Carvalho 1996). Pinocembrin was present in extracts from Pinus sylvestris and Pinus jeffreyi (Willför et al. 2003a; Anderson 1956). Pinobanksin was also found in Pinus resinosa wood (Simard et al. 2008).

The results in Table 4 also show the presence of minor amounts of taxifolin and dihydrokaempferol, which have been previously identified in extracts from pine (Neacsu et al. 2007; Lutskii et al. 1971) and from other softwoods (Pietarinen et al. 2006a; Willför et al. 2003c) with antioxidant activity (Willför et al. 2003c).

Table 4. Stilbenoids and Flavonoids in Water Extracts

Lignans, derived from phenylpropanoid precursors, are a large class of secondary metabolites in vascular plants, particularly conifers, where they reach higher concentra-tions in knots (Willför et al. 2003b and 2005a). Table 5 lists the results achieved in this work. Nortrachelogenin, a compound also found in extracts from Pinus sylvestris knotwood and other softwood species (Willför et al. 2005b), was the most abundant component, reaching average concentrations in the range 7.9 to 55.7 g/kg in extracts from samples containing knots (in comparison with 0 to 0.3 g/kg determined for samples HW and SW). Pinoresinol, a lignan widely distributed in plants, was the second most abundant lignan in extracts, reaching higher concentrations (up to 16.6 mg/g extract) in sample DK_SW. In comparison, secoisolariciresinol and isolariciresinol (which are typical softwood lignans reaching increased concentrations in knots) (Willför et al. 2005b), were found at lower proportions (0.3 to 2.2 g/kg extract); whereas todolactol (identified as a component of industrially important trees of the Pinaceae family) (Willför et al. 2005b) was found at limited concentrations (0 to 0.2 g/kg).

Other Compounds

Other extract components include resin acids, fatty acids, juvabiones, steryl esters, and triglycerides.

Resin acids are biologically active compounds whose occurrence in conifers is well known, including in Pinus pinaster wood (Hemingway et al. 1973; Nascimento et al. 1995) and bark. The fatty acid content of Pinus pinaster wood is known to vary widely with the age, growth rate, and sample position in the tree (Hemingway et al. 1973), a pattern confirmed by the data in Table 6. The average contents of native resin acids were below the threshold 2.5 g/kg extract in samples HW, SW, and DK_HW, and averaged for 3.8 to 5.7 g/kg extract from samples DK_SW, LK_HW, and LK_SW. The major individual resin acids in these extracts were dehydroabietic acid (accounting for 1.3 to 2.0 g/kg) and abietic acid (0.7 to 1.2 g/kg); whereas neoabietic acid and palustric acid reached concentrations up to 0.9 and 0.8 g/kg. In comparison, pimaric and isopimaric acids presented maximum concentrations of 0.7 and 0.4 g/kg. Neoabietic acid and palustric acid, which reached limited contents in stemwood (0 to 0.3 g/kg in extracts from samples HW and SW), were not found in a previous study dealing with the composition of extracts from three Pinus pinaster Ait subspecies (Arrabal and Cortijo 1994). The high proportion of modified resin acids (including hydroxyabietic acid, 7-oxodehydroabietic acid, hydroxydehydroabietic acid, dihydroxy-dehydroabietic acid, and hydroxy-7-oxodehydroabietic acid) with respect to native resin acids was remarkable in extracts from samples DK_SW and LK_SW.

Table 5. Lignans in Water Extracts

Table 6. Other Components in Extracts from Pinus pinaster Wood

In comparison, the fatty acids (caprylic acid, palmitic acid, and oleic acid) presented limited average concentrations in extracts (0 to 1.2 g/kg, except in samples DK_SW and LK_SW, for which the concentrations fell in the range 1.9 to 3.2 g/kg).

Epijuvabione was present in extracts as the only member of the juvabione family, which includes sesquiterpenes commonly found in conifers involved in plant defense with insect-juvenilizing properties (Phillips et al. 2006; Bohlmann et al. 1998).

Finally, little added value seems to be achievable from steryl esters and triglycerides, owing to their limited concentrations in samples (0.1 to 0.6 and 0.2 to 1.5 kg/kg, respectively) and the complexity of the media.

Antioxidant Activity

From the experimental information summarized in this work it can be seen that the complex composition of the extracts would make the individual separation of a single compound difficult and that the manufacture of multicomponent, active concentrates could be a more favourable approach to their valorisation. This philosophy has been followed in literature studies, in which applications such as technical antioxidants, functional foods, pharmaceuticals, natural biocides, and wood preservatives have been suggested for crude extracts or concentrates (Moure et al. 2005; Willför et al. 2003c, 2005c; Pietarinen et al. 2006b).

In order to assess the value of extracts for a representative potential application, and considering that a number of the extract components are active antioxidants, experimental work was carried out to measure the antioxidant activity of extracts. Interestingly, the synergistic effects among active extract components may result in higher antioxidant activity than the one of the dominating compounds in pure form (Willför et al. 2003c; Pietarinen et al. 2006b).

In this study, the performance of extracts was measured using the Trolox Equivalent Antioxidant Capacity (TEAC) method, which led to the experimental results listed in Table 7. This assay was selected owing its suitability for assessing both the hydrophilic and lipophilic antioxidant capacities of the target compounds in multiple media. The antioxidant activities were higher in sapwood samples (0.24 to 0.93 g Trolox/ g extract) than in heartwood samples (0.09 to 0.28 g Trolox/ g extract). The highest radical scavenging capacity (0.92 to 0.93 g Trolox/ g extract) corresponded to the extract from sample DK_SW, and was consistent with its phenolic content (higher than the ones of other extracts). The experimental result corresponded to 17.6 to 20.9% of ABTS radical scavenging activity at 200 mg/L; an activity similar to the that observed for the extract from sample DK_HW sample (16.0 to 20.1%) but at much higher concentration (800 mg/L).

It can be noted that the antioxidant potential of the extracts are influenced not only by the individual contributions of the major phenolic compounds (stilbenes, flavonoids, lignans, simple phenols) and accompanying components (polysaccharide-derived and lipophilic compounds), but also by synergisms among them. In related studies, high radical scavenging capacities have also been reported for hydrophilic extracts from knots of softwoods and hardwoods (Willför et al. 2003c). As it has been shown for lignans, the strength of such compounds also rely on the capability to scavenge a large number of radicals (Eklund et al. 2005), although the reaction kinetics might be slower than for related synthetic compounds.

Table 7. TEAC of Extracts

CONCLUSIONS

1. Hemicellulose-derived saccharides (mainly of oligomeric or polymeric nature) were the major components of all extracts obtained in this work from Pinus pinaster wood by water extraction at 130 to 140 ^oC.
2. A variety of phenolic compounds were present in the extracts, including simple phenolics, phenolic stilbenoids, flavonoids, and lignans.
3. The most abundant simple phenolics were isoferulic and 3,4-dihydroxycinnamic acids.
4. Pinosylvin and its derivative pinosylvin monomethyl ether were the only phenolic stilbenoids found in the extracts.
5. The dihydroflavonol pinobanksin, its derivative pinobanksin-3-acetate, and the flavanone pinocembrin were found in extracts in measurable concentrations. Taxifolin and dihydrokaempferol were found at smaller concentrations.
6. The lignans identified were nortrachelogenin, pinoresinol, secoisolariciresinol and isolariciresinol.
7. Other components of extracts (resin acids, fatty acids, juvabiones, steryl esters, and triglycerides) seem to add little value to the extracts.
8. Extracts from samples containing knots presented remarkable radical scavenging activities.

ACKNOWLEDGMENTS

The authors are grateful to the Spanish “Ministry of Science and Innovation” for supporting this study, in the framework of the research project “Development and evaluation of processing methods for biorefineries” (reference CTQ2011-22972), and to Xunta de Galicia (INBIOMED project) for additional financial support. Both projects were partially funded by the FEDER Program of the European Union (“Unha maneira de facer Europa”). Ms. Sandra Rivas thanks the Ministry for her predoctoral grant. Dr. Enma Conde thanks the COST Action FP0901 and the Process Chemistry Centre – Åbo Akademi University for the funding received through the Short Term Scientific Mission 090412-016530 and the Johan Gadolin Scholarship, respectively. Docent Annika Smeds is acknowledged for help with the MS analyses. The research leading to these results has received funding from the WoodWisdom-Net Research Programme, which is a transnational R&D programme jointly funded by national funding organizations within the framework of the ERA-NET WoodWisdom-Net 2. This work was also part of the activities at the Process Chemistry Centre at Åbo Akademi University.

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Article submitted: January 9, 2013; Peer review completed: February 16, 2013; Revised version received and accepted: February 22, 2013; Published: February 28, 2013.