Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species

Phytochemistry xxx (2015) xxx–xxx

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Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species Sauro Bianchi a,⇑, Ivana Kroslakova b, Ron Janzon c, Ingo Mayer a, Bodo Saake c, Frédéric Pichelin a a

Bern University of Applied Sciences, Architecture Wood and Civil Engineering, Solothurnstrasse 102, 2502 Biel, Switzerland Zurich University of Applied Sciences, Institute of Chemistry and Biological Chemistry, Einsiedlerstrasse 31, 8820 Wädenswil, Switzerland c University of Hamburg, Department of Chemical Wood Technology, Leuschnerstrabe 91b, 21031 Hamburg, Germany b

a r t i c l e

i n f o

Article history: Received 24 April 2015 Received in revised form 20 October 2015 Accepted 23 October 2015 Available online xxxx Keywords: Abies alba Larix decidua Picea abies Pseudotsuga menziesii Pinus sylvestris Softwood bark extracts Thiolysis MALDI-TOF MS Condensed tannins Carbohydrates

a b s t r a c t Condensed tannins extracted from European softwood bark are recognized as alternatives to synthetic phenolics. The extraction is generally performed in hot water, leading to simultaneous extraction of other bark constituents such as carbohydrates, phenolic monomers and salts. Characterization of the extract’s composition and identification of the extracted tannins’ molecular structure are needed to better identify potential applications. Bark from Silver fir (Abies alba [Mill.]), European larch (Larix decidua [Mill.]), Norway spruce (Picea abies [Karst.]), Douglas fir (Pseudotsuga menziesii [Mirb.]) and Scots pine (Pinus sylvestris [L.]) were extracted in water at 60 °C. The amounts of phenolic monomers, condensed tannins, carbohydrates, and inorganic compounds in the extract were determined. The molecular structures of condensed tannins and carbohydrates were also investigated (HPLC-UV combined with thiolysis, MALDI-TOF mass spectrometry, anion exchange chromatography). Distinct extract compositions and tannin structures were found in each of the analysed species. Procyanidins were the most ubiquitous tannins. The presence of phenolic glucosides in the tannin oligomers was suggested. Polysaccharides such as arabinans, arabinogalactans and glucans represented an important fraction of all extracts. Compared to traditionally used species (Mimosa and Quebracho) higher viscosities as well as faster chemical reactivities are expected in the analysed species. The most promising species for a bark tannin extraction was found to be larch, while the least encouraging results were detected in pine. A better knowledge of the interaction between the various extracted compounds is deemed an important matter for investigation in the context of industrial applications of such extracts. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Condensed tannins, or proanthocyanidins, are natural polyphenolic oligomers made of flavan-3-ol units. They are recognized as suitable natural substitutes in the formulation of wood adhesives (Yazaki and Collins, 1994; Roffael et al. 2000; Pichelin et al., 2006; Pizzi, 2008), foamed resins (Lacoste et al., 2013) and heavy metal removal systems (Palma et al., 2003). Industrially used tannins are mostly extracted from the bark of Black wattle (Acacia mearnsii [De Wild.]) and the heartwood of Quebracho (Schinopsis lorentzii [Engl.]). The bark of softwood species has also been reported as a valuable source of condensed tannins (Porter, 1989; Foo and Karchesy, 1989; Matthews et al., 1997a; Karonen et al., 2004; Krogell et al., 2012). In Switzerland, 425,000 m3 of bark

⇑ Corresponding author.

was produced in 2013, the majority of which was burned for energy production (BAFU, 2014). Thus, softwood bark represents an important source of condensed tannins in Switzerland. In particular, Silver fir (Abies alba [Mill.]), Norway spruce (Picea abies [Karst.]), Scots pine (Pinus sylvestris [L.]), European larch (Larix decidua [Mill.]) and Douglas fir (Pseudotsuga menziesii [Mirb.]) are species of special interest, representing more than 95% of the total Swiss softwood growing stock (BAFU, 2014). These species are also among the most common softwoods in Central and Northern Europe, where round softwood production in 2013 was close to 250 million m3. As bark corresponds to 10% of the log volume, 25 millions m3 of it is thus theoretically available in Europe. Properties of condensed tannins are significantly influenced by the molecular structure of their flavan-3-ol units (Fig. 1) and by their degree of polymerization. Oligomers made of catechin or gallocatechin units showed up to tenfold shorter gelling times in the condensation reaction with

E-mail address: [email protected] (S. Bianchi). http://dx.doi.org/10.1016/j.phytochem.2015.10.006 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

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S. Bianchi et al. / Phytochemistry xxx (2015) xxx–xxx

on diverse softwood species covering both the analysis of the condensed tannins structure and the characterization of the coextracts, is still lacking. In this study the composition of hot water extracts from five European softwood barks (Silver fir, European larch, Norway spruce, Douglas fir and Scots pine) were investigated. The presence of total phenolics; tannins; inorganic compounds; free and bound carbohydrates in the extracts were assessed. The molecular structure of extracted tannins and carbohydrates was analysed by liquid chromatography and mass spectrometry.

2. Results and discussion 2.1. Extraction yield and extract composition

Fig. 1. Molecular structures of the most common flavan-3-ols units in the condensed tannins.

formaldehyde than oligomers made of fisetinidol or robinetinidol units (Pizzi and Stephanou, 1994; Garnier et al., 2002). Differences in reaction rates have been identified even between the stereoisomers catechin and epicatechin (Takagaki et al., 2000). Higher viscosities and shorter gelling times have also been correlated with higher degrees of polymerization (Garnier et al., 2001). Likewise, an enhancement of the heavy metal chelating ability has been reported with increasing degrees of hydroxylation and polymerization (Yoneda and Nakatsubo, 1998). The purity of the extracted tannins represents yet another critical point. Bark extraction is typically performed with hot water. This method also yields the extraction of smaller phenolic compounds, carbohydrates and inorganic salts. Condensed tannins are therefore only a component of bark extracts, which chemical and physical characteristics could be significantly affected by their actual composition (Pizzi, 2008). The presence of polysaccharides is of particular concern, as the latter are related to an increase in the viscosity of resin formulations (Weissman, 1985; Garnier et al., 2001). A detailed characterization of bark extracts is therefore important to better understand their properties and limitations. Condensed tannins extracted from softwood bark have generally been associated with procyanidins (PC) and prodelphinidins (PD), oligomers composed mainly of catechin and gallocatechin units, respectively (Porter, 1989; Foo and Karchesy, 1989; Matthews et al., 1997a; Karonen et al., 2004; Navarrete et al., 2010). Nevertheless, the occurrence of building blocks other than simple flavan-3-ols, such as flavan-3-ol esters, glucosides, stilbenes and lignans has been suggested (Zhang and Gellerstedt, 2008; Ucar et al., 2013; Bianchi et al., 2014). Bark polysaccharides, apart from cellulose, have been described as galactoglucomannans and arabinomethylglucuronoxylans (Timell, 1961; Dietrichs et al., 1978), but the presence of glucans (callose), arabinans and pectins has also been reported (Timell, 1964; Fu et al., 1972; Fu and Timell, 1972; Weissman, 1981, 1985; Krogell et al., 2012; Le Normand et al., 2014). With the exception of spruce (Kemppainen et al., 2014), softwood bark extracts have so far been characterized with a focus on selected components (e.g. condensed tannins, distinct phenolic monomers, polysaccharides) using different and often not comparable sample preparation or analysis methods. A systematic study

The total extraction yield of softwood barks ranged from 26.9 to 120.2 g/kg of dry bark, corresponding to pine and Silver fir, respectively (Table 1). Similar yields have been reported for hot water extractions of European softwood bark (Weissman, 1981, 1985; Bertaud et al., 2012; Kemppainen et al., 2014). In these studies, consistent differences in yield were observed not only among different bark species, but also within the same species. Dissimilarity in the extraction temperature, time, water to bark ratio, size of the bark particles and bark storage conditions before extraction are factors that may account for observed variations. The measured extraction yields were considerably lower than those of the most common commercial tannin species (e.g. Mimosa, Quebracho), which range between 150 and 330 g/kg dry material (Sealy-Fisher and Pizzi, 1992; Hoong et al., 2011). All bark extracts showed the presence of phenolic compounds, carbohydrates, and inorganic compounds (Table 1). Total extracted phenolics, assessed by Folin–Ciocalteau assay, represented between 13.0% and 46.7% of the extract, corresponding to total phenolic yields between 3.5 and 42.8 g of epicatechin equivalent (gECE) per kg of dry bark (Table 1). Pine showed the lowest yield while larch showed the highest. Other studies on European softwood bark extracts have reported values in the same range (Jerez et al., 2009; Yesil-Celiktas et al., 2009; Bertaud et al., 2012). Previously performed Folin–Ciocalteau assays on commercial Mimosa and Quebracho extracts showed total phenolic concentrations equal to 40.7% and 42.1%, respectively. With the exception of larch, therefore, all the investigated softwood species showed a lower concentration of phenolics than commercial extracts. More detailed information on the phenolic extracts was obtained after separation by solid phase extraction (SPE) of the crude extract in the following three fractions:
F0 = carbohydrates and phenolic acids;
F1 = phenolic monomers;
F2 = phenolic oligomers (e.g. condensed tannins). The Folin–Ciocalteau assay on the single fractions showed that most of the phenolic compounds were re-collected in F1 and F2 (Table 1), indicating the presence of both phenolic monomers and tannins in all species. The phenolic acids (F0) contributed only minimally to the total extracted phenolics across all species. A high ratio of condensed tannins was indicated for spruce and pine, which showed higher phenolic concentrations in F2 than in F1. Conversely, a prevalence of phenolic monomers appears in Douglas fir extracts. The impact of phenolic monomers on the chemical and physical characteristics of the extracts hasn’t yet been thoroughly investigated. Though they are generally regarded as simple extract constituents that merely dilute the tannin concentration in the extracts (Pizzi, 2008), they may also play a role in condensation reactions.

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

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S. Bianchi et al. / Phytochemistry xxx (2015) xxx–xxx Table 1 Total extraction yield and composition of bark water extracts. All values are means of n measurements. Total yield (n = 12)

Phenolic compoundsa (n = 3) b

Silver fir European larch Norway spruce Douglas fir Scots pine a b c d e f g h

g/kg dry bark % dry extract g/kg dry bark % dry extract g/kg dry bark % dry extract g/kg dry bark % dry extract g/kg dry bark % dry extract

120.2 – 91.7 – 54.5 – 75.7 – 26.9 –

c

Carbohydrates (n = 1) d

e

f

Total

Acids

Monomers

Oligomers

Total

33.5 27.9% 42.8 46.7% 18.6 34.1% 23.3 30.8% 3.5 13.0%

2.0 1.7% 3.6 3.9% 2.2 4.0% 0.4 0.5% 0.1 0.4%

14.0 11.6% 18.4 20.1% 5.8 10.6% 13.1 17.3% 1.3 4.8%

17.4 14.5% 18.6 20.3% 9.9 18.2% 9.1 12.0% 1.9 7.1%

34.9 29.1% 19.2 20.9% 16.5 30.3% 31.4 41.5% 12.3 45.7%

Free

g

19.8 16.5% 10.4 11.3% 2.1 3.9% 17.8 23.5% 1.4 5.2%

Bound 15.1 12.6% 8.8 9.6% 14.4 26.4% 13.6 18.0% 10.9 40.5%

Inorganic compounds (n = 3) h

4.6 3.8% 4.2 4.6% 1.7 3.1% 1.1 1.5% 1.4 5.2%

All phenolic compounds are expressed as grams of ()-epicatechin equivalents (gECE). Folin–Ciocalteau assay on extracts. Folin–Ciocalteau assay on fraction F0, eluted from SPE cartridge with water. Folin–Ciocalteau assay on fraction F1, eluted from SPE cartridge with ethylacetate (it could contain also some small oligomers like dimers or trimers). Folin–Ciocalteau assay on fraction F2, eluted from SPE cartridge with methanol. Measured by Borat-HPAEC on extracts after acid hydrolysis. Free mono- and oligosaccharides, measured by HPAEC-PAD and Borat-HPAEC on the non-hydrolysed fraction F0. Polysaccharides and phenolic glycosyl residuals, calculated by the difference between total and free carbohydrates.

Total carbohydrates evaluated after hydrolysis of the extracts were found to represent between 20.9% and 45.7% of the extracted compounds (Table 1), a significant portion of the extract. In Silver fir, Douglas fir and pine, carbohydrates constituted the largest portion of the extract. Similar results were reported in previous studies (Weissman, 1985; Kemppainen et al., 2014). The amount of free mono- and oligosaccharides (free carbohydrates), evaluated by analysis of non-hydrolysed F0 fractions, varied broadly across species (Table 1). In Silver fir, larch and Douglas fir the free carbohydrate constituted the majority of carbohydrates extracted, with extraction yields ranging from 10.4 to19.8 g/kg dry bark. By contrast, spruce and pine showed a negligible amount of such compounds and almost 10 times lower yields (1.4–2.1 g/kg dry bark, respectively). These substantial differences across species may be related not only to species characteristics but also to a different degradation or leaching of free carbohydrates after the tree felling. Studying the impact of bark storage time and conditions on extracted monosaccharide concentrations is necessary to better understand the extent to which detected differences are associated to a tree species’ intrinsic characteristics. Bound carbohydrates (polysaccharides and glycosyl residuals associated with phenolic compounds) were calculated as the difference between total and free carbohydrates. Their extraction resulted in yields ranging from 8.8 to 15.1 g/kg (Table 1). This range was slightly narrower than those of other extracted compounds, suggesting a comparable extractability of polysaccharides across the diverse softwood species. The considerable fraction of carbohydrates in extracts represents a noteworthy difference between softwood bark extracts and Mimosa and Quebracho extracts. The analysis on commercial samples of these products showed total carbohydrate concentrations of 6.8% and 9.0%, respectively, significantly lower than in studied species. Inorganic compounds (ashes) were detected only in minor concentrations (Table 1) and represented less than 5% of the extracts. Their presence is most likely associated with soluble salts from the bark, and with potassium salts in particular (Krogell et al., 2012). The sum of total phenolics, total carbohydrates and inorganic compounds ranged from 60.8% (Silver fir) to 73.8% (Douglas fir) of the total extraction yield. A substantial fraction of the extracts remains unidentified. However, it should be considered that the Folin–Ciocalteau assay represents only an estimation of the actual concentration of phenolics in the extracts, quantified as epicatechin equivalents. The assay does not consider the variation in

response to the colorimetric reaction across different phenolic compounds (Singelton et al., 1999; Everette et al., 2010). In particular, for condensed tannins, an underestimation of their actual amount is expected due to steric unavailability of some phenolic groups to the assay reaction (Kraus et al., 2003). The presence of other water soluble extracts (e.g. aliphatic acids from the degradation of cutin and suberin) should also be considered (Yazaki and Aung, 1988). 2.2. Phenolic monomers The occurrence of phenolic monomers in the studied bark extracts was evidenced by the presence of well-defined absorption peaks in the UV chromatograms of the extracts before thiolysis. The example of spruce is reported in Fig. 2. After fractionation, peaks were identified almost exclusively in F1, confirming the association of this fraction with the phenolic monomer portion of the extract. The UV spectra of these peaks generally showed strong absorption at 290 nm and 325 nm, characteristics of typical softwood bark extractives, e.g. taxifolin, stilbenes and stilbenes glucosides

Fig. 2. HPLC-UV chromatographs (kabs = 280 nm) of the crude Spruce extract and its fractions F0, F1 and F2 (solid line = before thiolysis, dotted line = after thiolysis). The numbered peaks were associated to: 1 = catechin; 2 = catechin thioether; 3 = epicatechin; 4 = epicatechin thioether; 5,6,7,8 = diverse phenolic monomers.

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

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S. Bianchi et al. / Phytochemistry xxx (2015) xxx–xxx

(Barton and Gadner, 1958; Pan, 1989; Yesil-Celiktas et al., 2009; Jerez et al., 2009; Ucar et al., 2013; Latva-Mäenpää et al., 2013). HPLC-MS mass spectra of such peaks and MALDI-TOF spectra in the mass range between 300 and 900 Da (data not shown) also identified masses that matched with the above-mentioned compounds (Bianchi et al., 2014). 2.3. Condensed tannins After thiolysis of the extracts, the development in the HPLC-UV chromatograms of new peaks associated with catechin, epicatechin, gallocatechin, epicatechin and their respective thioethers was observed, as shown in Fig. 2 for spruce. Such peaks evinced the presence of PC and PD in the extracts. A predominance of PC units was found in most samples. PD units represented the majority only in Silver fir, and were also detected in small amounts in larch (Table 2). For PC a cis-configuration (epicatechin, epigallocatechin and their thioethers) was dominant, while for PD the trans-configuration (catechin, gallocatechin and their thioethers) was most common (Table 2). The results are in agreement with those of Matthews et al. (1997a) on methanol–water extracts from different softwood barks. The actual concentration of cis units might be slightly overestimated due to epimerization of the flavan-3ols during thiolysis (Matthews et al., 1997b). Preliminary tests showed that roughly 20% of free catechin monomer was converted to epicatechin. MALDI-TOF analysis of spruce and Silver fir extracts reported by Bianchi et al. (2014) confirmed the HPLC results. Spruce samples showed a mass spectrum with a main repetition unit of approximately 288 Da, while Silver fir samples spectra displayed a main repetition unit of 304 Da. These masses could be associated with a catechin (or epicatechin) unit (290.3–2H = 288.3 Da) and a gallocatechin (or epigallocatechin) unit (306.3–2H = 304.3 Da), respectively. The predominance of PC in spruce and of PD in Silver fir was therefore supported. The MALDI-TOF mass spectrum of larch (Fig. 4) resembled that of spruce. A main oligomer series with a repetition unit corresponding to a catechin monomer could be observed and described by the Eq. (1).

M þ Kþ ¼ x  288:3 þ 2:0 þ 39:1

ð1Þ

where x is the number of catechin units, 2.0 are the two hydrogen end units, and 39.1 the mass of the potassium ion. The presence of PC in larch extract was therefore indicated, in agreement with the HPLC results (Table 2).

Table 2 Procyanidins (PC) and prodelphinidins (PD) detected by thiolysis and subsequent HPLC-UV in the hot water extracts of European softwood barks. Species Silver fir

European larch Norway spruce Douglas fir Scots pine

Extract F1 F2 Extract F1 F2 Extract F1 F2 Extract F1 F2 Extract F1 F2

Total % dry extract

PC:PD %:%

cis:trans %:%

mDP

13.0 5.6 7.5 12.1 4.4 6.8 6.6 1.0 4.8 6.1 1.1 4.6 6.4 0.9 5.2

21:79 16:84 19:81 90:10 83:17 100:0 100:0 100:0 100:0 100:0 100:0 100:0 100:0 100:0 100:0

31:69 37:63 39:61 71:29 64:36 74:26 76:24 67:33 79:21 75:25 70:30 77:23 82:18 79:21 82:18

3.5 3.1 3.6 5.6 4.1 6.5 6.2 3.5 8.2 4.4 2.6 4.6 6.7 2.7 8.4

Thiolysis yield

52%

33%

26%

38%

72%

Fig. 3. Molecular structure of astringin and a flavan-3-ol glucoside, likely building blocks in the tannin oligomer structure of softwood bark extracts.

A further similarity between spruce and larch MALDI-TOF mass spectra was the presence, in addition to the main peak series, of a low intensity peak series having a repetition unit of approximately 288.3 Da, but shifted by the main series of about 117 Da. This value doesn’t correspond to any known building block or moieties in tannin oligomers. However, if the shift is not considered from the first closest peaks but from the second, its value corresponds approximately to 405 Da. This shift matches with that of stilbene glucoside astringin (Fig. 3) when assumed covalently bound within a condensed tannin molecule (406.4 – 2H = 404.4 Da). The presence of stilbenes and their glucosides in the molecular structure of bark tannins from spruce species has been suggested in various studies (Steynberg et al., 1983; Hergert, 1989; Matthews et al., 1997b; Zhang and Gellerstedt, 2008), and determined using different analytical methods, including 2D 13C NMR. Further evidence of astringin units in the tannin structure of spruce and larch bark emerged from the thiolysis yield analysis (Table 2). The yield of the thiolysis reaction was calculated on fraction F2 as the ratio total released tannin units (from HPLC analysis) to total phenolics measured by Folin–Ciocalteau assay (under the assumption that F2 was almost exclusively composed of phenolic oligomers). Spruce and larch showed significantly lower thiolysis yield than other species. Reduced thiolysis yields have been correlated with the presence of non-flavan-3-ol units in tannins, and in particular of stilbene glucosides (Matthews et al., 1997b). The low thiolysis yields observed for spruce and larch could therefore also be related to the occurrence of such units in these species. The analysis of MALDI-TOF mass spectra of pine and Douglas fir proved more complex. For both species, an intense peak series with a repetition unit of about 132 Da was observed. The example of pine is shown in Fig. 5. This series could not be associated with any known phenolics in wood or bark. Navarrete et al. (2010) observed a similar mass spectrum for Pinus pinaster [Aiton] extracts and proposed a condensed tannin structure containing a peculiar deoxidized flavanol dimer with a mass equal to 528 Da (=4  132 Da). The association of this series to non-tannin oligomers, and more specifically with polysaccharides, is considered likely. Tests performed on polygalacturonic acid standard (not reported) showed that the sample preparation used and MALDITOF analysis conditions were in fact effective in polysaccharide detection. Partially masked by the most intense peak series, a low-intense sequence of peaks with a mass repetition unit of about 288 Da could also be observed in the mass spectra of pine (906.1, 1194.2, 1483.1, 1771.5, 2059.6, 2348.2, 2637.3 Da – Fig. 5) and Douglas fir (906.3, 1194.4, 1483.0, 1771.0, 2059.3, 2348.4 Da – data not shown). These series were almost identical to the PC series observed in larch and spruce, and could also be described by Eq.

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

S. Bianchi et al. / Phytochemistry xxx (2015) xxx–xxx

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Fig. 4. MALDI-TOF mass spectrum of European larch bark extract. In particular, three oligomer series are depicted and their repetition mass unit highlighted (bold = procyanidin; bold⁄ = procyanidin with astringin unit; italic = pentosan).

(1). The MALDI-TOF analysis indicated the presence of PC also in pine and Douglas fir, in agreement with the HPLC results (Table 2). In the Douglas fir spectrum, some additional mass peaks (778.6, 1067.3, 1229.7 and 1806.5 Da) were also associated with the occurrence of flavan-3-ol glycosides (Fig. 3). For example, the 1229.7 Da peak could be described as an oligomer made of a catechin and two catechin glucosides (288.3 + 2(288.3 + 162.2) + 2.0 + 39.1 = 1230.4 Da). The presence of flavan-3-ol glucosides could be responsible for the low thiolysis yield measured in Douglas fir extracts (Table 2). The mean degree of polymerization of the extracted tannin (mDP) was estimated with the molar ratio of the total to the terminal units detected by HPLC-UV after thiolysis, as described in Jerez et al. (2007). Pine, spruce and larch showed an mDP between 5.6 and 6.7, while Douglas and Silver fir had considerably lower mDP (Table 2). Matthews et al. (1997a) and Jerez et al. (2007) reported mDP values between 3.1 and 7.6 for softwood bark extracts, with analogous differences across species. Thiolysis was also performed on F2 fractions, yielding a sensibly higher mDP for pine and spruce than for other species (Table 2). The presence of longer tannin oligomers in these extracts was therefore suggested. Thiolysis on fraction F1 showed the presence of phenolic oligomers with an mDP ranging from 2.6 to 4.1 (Table 2), evincing the elution of some oligomers in this fraction generally associated to phenolic monomers. In the case of pure PC (spruce, Douglas fir, pine), the amount of oligomers eluted in F1 was below 20% of the total. When PD units were also detected (Silver fir, larch), the separation between oligomers and monomers

was less efficient, most likely due to differences in polarity of the compounds. The maximum observable polymerization degree of tannins in MALDI-TOF spectra ranged from 9 (Silver fir) to 14 (spruce), following variations among the species similar to those observed in the mDP measurements. The molecular structures of the tannin detected in the bark extracts of European softwood could therefore be generally described as PC with occurrence of less common building units like stilbene glucosides or flavan-3-ols glucosides. This structure differs substantially from those detected in tannins from tropical species and in particular Mimosa and Quebracho, which are associated to oligomers of robinetinidol and fisetinidol units with a maximum degree of polymerization between 8 and 10 (Pasch et al., 2001). The higher degree of hydroxylation and polymerization European tannins should bestow faster curing properties and improved heavy metal chelation on the latter. This is particularly true for Silver fir tannins, represented by the high hydroxylated prodelphinidins. 2.4. Carbohydrates composition Monosaccharides in softwood bark extracts were mostly represented by glucose and fructose (Table 3). Oligosaccharides (sucrose, raffinose and stachyose) were generally present in traces, with the exception of Silver fir, for which sucrose represents the main compound. A considerably high amount of free glucose was also extracted from Douglas fir bark.

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

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S. Bianchi et al. / Phytochemistry xxx (2015) xxx–xxx

Fig. 5. MALDI-TOF mass spectrum of Scots pine bark extract. In particular, three oligomer series are depicted, and their repetition mass unit highlighted (bold = procyanidin; italic = pentosan; standard = hexosan).

Table 3 Composition of the carbohydrates in the bark extracts and fractions F1 and F2. The reported amount of Glu, Gal and Fru are the sums of free monosaccharides and their units in sucrose, raffinose and stachyose. Carbohydrates (g/kg dry bark)

Silver Fir

Free Bound

European larch

Free Bound

Picea abies

Free Bound

Douglas fir

Free Bound

Scots pine

Free Bound

Total Total F1 F2 Total Total F1 F2 Total Total F1 F2 Total Total F1 F2 Total Total F1 F2

Glc

Gal

Fru

Man

Ara

GalA

Suc

Raf

Sta

8.97 8.84 1.77 1.22 4.33 3.88 1.24 1.43 0.86 8.21 2.00 3.04 10.42 4.10 1.00 0.77 1.01 2.67 0.76 0.49

1.06 2.06 0.04 0.10 0.65 0.44 0.04 0.18 0.09 1.46 0.02 0.13 0.52 2.03 0.03 0.29 0.01 1.66 0.03 0.16

9.76 <0.01 <0.01 0.02 3.94 <0.01 <0.01 <0.01 0.87 <0.01 <0.01 <0.01 5.62 <0.01 <0.01 <0.01 0.24 0.39 <0.01 <0.01

<0.01 0.42 <0.01 0.07 0.03 0.53 0.01 0.11 0.01 0.41 <0.01 0.09 0.03 0.38 0.01 0.08 <0.01 0.78 0.01 0.09

<0.01 1.56 0.19 0.65 1.19 3.38 0.15 2.01 0.16 2.80 0.09 1.27 0.84 5.78 0.07 2.84 0.08 3.97 0.22 1.52

<0.01 0.69 – – 0.24 0.17 – – 0.11 0.72 – – <0.01 0.64 – – <0.01 0.65 – –

8.08 – – – 1.22 – – – 0.38 – – – 0.41 – – – 0.08 – – –

1.06 – – – 0.20 – – – 0.07 – – – 0.38 – – – <0.01 – – –

1.25 – – – 0.54 – – – <0.01 – – – 0.44 – – – <0.01 – – –

Bound carbohydrates consisted mostly of glucose, galactose, and arabinose in all species (Table 3). Mannose and galacturonic acid were detected in much smaller amounts, while rhamnose, xylose, mannitol, and galacturonic acid were found only in traces.

Silver fir extracts differed substantially from the others, showing more glucose and less arabinose. The carbohydrate residual composition suggests that the extracted polysaccharides were not typical softwood bark polyoses (e.g., galactoglucomannans and

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

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arabinomethylglucuronoxylans), but more likely pectin-like compounds such as arabinans, arabinogalactan and glucans (Timell, 1961; Fu and Timell, 1972; Krogell et al., 2012; Le Normand et al., 2014). The presence of starch was excluded: an iodine test on the bark extracts did not show any blue color development. Similar results were reported by Weissman (1981, 1985) on hydrolysed spruce and larch bark extracts. A higher occurrence of galacturonic acid residuals was reported by Le Normand et al. (2014) and Kemppainen et al. (2014) after methanolysis of spruce bark extract. A degradation of the galacturonic acid during the stronger hydrolysis conditions used in the present study could be related to the different concentrations detected in this and the previous studies. Hydrolysis on the fractions F1 and F2 show a significant amount of glucose residuals, which could be related to the occurrence of phenolic glucosides both as free monomers and in the structure of condensed tannins. In particular, this result is in agreement with the assumption, suggested by MALDI-TOF analysis, of stilbene glucosides in the structure of spruce and larch tannins and of flavan-3ol glucosides in that of Douglas fir tannins. Considerable amounts of arabinose residuals were detected in all F2 fractions after hydrolysis (Table 3). Carbohydrate analysis on the same fractions before hydrolysis didn’t detect any free monomers; by consequence the detected arabinose should either be bound in polysaccharides (arabinans) or as present as glycosyl, though phenolic arabinosides have rarely been reported in the literature. The presence of arabinans in the methanol-eluted fraction F2, commonly associated with phenolic oligomers, is considered possible. Arabinans are alcohol soluble polysaccharides and could be isolated from other carbohydrates by simple alcoholic extraction (Hara et al., 2013). To validate the presence of arabinans in the F2 fractions, a MALDI-TOF analysis was performed on the F2 fractions of pine and Douglas fir (data not reported). The mass spectra detected were almost identical to those of the respective crude extracts (Fig. 4), showing a main mass peak series with a 132 Da repetition unit and a less intense peak series corresponding to PC. The main peak series may belong to a polysaccharide made of pentose units (150.1 – H2O = 132.1 Da) like arabinans, and described by Eq. (2):

The presence of pectin in the extracts is expected to considerably affect their aqueous solution viscosity.

M þ Kþ ¼ x  132:1 þ 18:0 þ 39:1

4. Experimental

ð2Þ

where x is the number of arabinose units, 18.0 is the combined mass of the polysaccharide terminal units (OH + H), and 39.1 the mass of the potassium ion. The intense 132 Da sequenced series observed in the MALDITOF spectra of crude Douglas fir and pine bark extract (Fig. 5) could in turn be associated to arabinans. It has to be noted that the same series was detected in the larch extract spectrum (Fig. 4), albeit at much lower intensities. The presence of arabinans was thus also possible in this sample, in agreement with the relevant concentration of arabinose residual detected in the larch fraction F2 (Table 3). Likewise, a low intense series with a repetition unit of approximately 162 Da in the MALDI-TOF mass spectrum of the pine crude extract (Fig. 5) suggests the presence of a polymer made of hexose units (180.1 – H2O = 162.2 Da). This hexosan series may relate to a glucan, and in particular callose, which is reported as largely present in pine bark (Fu and Timell, 1972). The MALDI-TOF analysis of the pine F2 fraction didn’t show any peak of this series, suggesting that the associated hexosan was most likely eluted before and this in the F0 fraction. The particularity of pine and Douglas fir extracts that show a pentosan peak series with a much higher intensity than those of tannins may be due to a higher ratio of bounded arabinose to phenolic oligomers observed for these species (Table 1 and Table 3).

3. Conclusions The hot water extracts from the bark of Silver fir, European larch, Norway spruce, Douglas fir and Scots pine are composed only partially from condensed tannins. Relevant amounts of other co-extracts like phenolic monomers, monosaccharides and pectins were also detected in most samples. The relative ratio of the different compounds in the extracts varied widely across species, hinting at distinctive chemical and physical properties. Peculiar structures of the condensed tannins, containing building units different from the commonly reported flavan-3-ols, were also identified; such peculiarities could affect their behavior in resin formulations or as heavy metal scavengers. Carbohydrates were detected in considerable amounts in all samples, and in some cases (Silver fir, Douglas fir and pine) even represented the bulk of the extract. In particular, pine, and also Douglas fir, showed a high ratio of pectin to tannin, suggesting high viscosity and low reactivity for such extracts. This data, combined with a low total extraction yield, identified pine as the less suitable species for tannin extraction from among those investigated. On the contrary, larch was valued as the most promising, due to its high yield of tannin and relative low yield of pectins. Compared with the commercially available extracts from tropical species (Mimosa and Quebracho), the European softwood bark extracts studied show a considerably lower total extraction yield, poorer phenolic fraction and higher concentration of carbohydrates, hinting at possible limitations for their use. On the other hand the higher hydroxylation and longer polymerization degrees could still provide characteristics (e.g. faster curing time, improved metal chelation ability, etc.) that offer a useful advantage in some applications. The significant presence of non-tannin compounds in the extracts underlined the need for a better knowledge on possible effects and interactions in resins and materials based on tannin chemistry, and these beyond their simple dilution effect.

4.1. Bark tissue collection Bark flakes were collected in early of March 2012 (Silver fir, larch) and March 2013 (spruce, Douglas fir, pine) from harvested logs in a forest near Biel (Switzerland), at approximately 600 m above sea level. The soil of the forest was typical of the Jura Mountains, therefore mostly calcareous lithosol- and rendzina-type (Eggenberg and Kurz, 2000). For each species, bark flakes from at least three different logs were collected. According to the information provided by the harvesters, the logs were felled between 1 and 3 months before bark collection and ready for transportation to the sawmill. The collected samples were therefore considered having a representative quality on delivery to the sawmill. Breast-high diameters were between 40 and 60 cm. The bark was removed with handheld tools within the lowest 5 m of the stem height. No attempts were done to separate the inner and outer bark. The same day of collection, bark flakes were deep-frozen and stored in airtight conditions. 4.2. Extraction The deep-frozen bark flakes were freeze-dried, milled and extracted with water at 60 °C as reported previously in Bianchi et al. (2014). All extractions were performed using an Accelerated

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

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Solvent Extraction device (Dionex ASEÒ 200). The extracts were finally freeze-dried and stored in airtight conditions away from light. Commercial samples of Mimosa and Quebracho extracts were purchased from Silvateam S.p.A. (Italy). 4.3. Extract fractionation A modification of the procedure proposed by Sun et al. (1998) using solid phase extraction (SPE) cartridges was applied. The SPE cartridges (Agilent Bond-EluteÒ 10 mg) were preconditioned with methanol (Sigma Aldrich) and distilled water, in sequence. An aq. solution of the extract (1 g/L) was then loaded (15 mL) in the SPE cartridge. Sequential elutions with 15 mL of distilled water, 20 mL of ethylacetate and 20 mL of methanol, were then performed in order to gain the three different fractions F0, F1 and F2, respectively. To the collected F0, 1 mL of sodium azide solution (60 mg/L, Sigma Aldrich) was added to stop any biological activity, and the volume brought up to 30 mL. F1 and F2 were vacuum dried at 40 °C (Büchi Syncore). 4.4. Total phenolic assay The total amount of phenolic compounds was determined by the Folin–Ciocalteu method (Singelton et al., 1999). Measurements were performed thrice on extract aq. solutions (0.2 g/L), F0 (as prepared), F1 and F2 (after dispersion in 20 mL of water). Calibration was performed with ()-epicatechin (Sigma Aldrich). 4.5. Thiolysis followed by HPLC-UV Thiolysis was performed on extracts and fractions in acid methanol at 65 °C with cysteamine hydrochloride (Sigma Aldrich), as reported by Jerez et al. (2007), but extending the thiolysis time to 1 h and quenching the reaction with 1 mL of distilled water. The freeze-dried extracts (5 mg) and the dried F1 and F2 were dissolved in 5 mL of methanol. Fraction F0 was used as prepared. Non-thiolysed reference samples were prepared in the same way, but the thiolysis media was substituted with methanol. Reversed phased HPLC was performed on an Agilent 1100 system with a Cosmosil Protein-R ø4.6  250 mm column. An elution (1 mL/min) was made with [A] 0.1% of trifluoracetic acid (TFA) in water, and [B] 0.082% of TFA in acetonitrile:water (4:1 v/v). The following gradient was optimized in order to gain good separation of the flavan-3-ols and their thioethers: initial, 0%[B]; 0–5 min, from 0.0% to 7.5%[B]; 5–20 min, from 7.5% to 8.5%[B]; 20–30 min, from 8.5% to 13.5% [B]; 30–45 min, from 13.5% to 33.5%[B]. Column washing and reconditioning followed. Detection was made with a UV diode array at 280 nm. Identification and quantification of catechin, gallocatechin and their epimers were performed by comparison with analytical standards (Sigma Aldrich). Since no standards were available for the flavan-3-ols thioethers, they were identified by mass spectrometry using the same HPLC setting as described above, but on an Agilent 1290 Infinity HPLC system equipped with a mass detector (Agilent 6130 quadrupole MS). The quantification of the flavan-3-ol thioethers was performed assuming the same UV molar absorption factor of the corresponding flavan-3-ols.

the mass spectra due to the formation of molecular ions with different cations. Bark and bark extracts have been in fact reported as rich in Ca, K and Mn ions (Fengel and Wegener, 1983; Krogell et al., 2012) that could interfere during the MALDI ionization process (Xiang and Lin, 2006). The measurements were carried out with a MALDI-TOF mass spectrometer (Reflex III, Bruker Daltonics, Germany) equipped with a nitrogen laser (337 nm) in linear positive mode. The monitored mass range was between 300 and 5000 Da. Each collected spectrum represents a sum of 700 laser shots. 4.7. Carbohydrates analysis Extracted free carbohydrates (mono- and oligosaccharides), mannitol, 5-hydroxymethylfurfural (5-HMF) and uronic acids were determined on the water-eluted fraction F0. Detection of fructose (Fru), sucrose (Suc), stachyose (Sta) and raffinose (Raf) was performed by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) using a 4.0 mm Carbopac PA1 – 250 mm column (Thermo scientific). Isocratic elution (1.0 mL/min. – 20 min.) was made with 75 mM NaOH. The compounds were detected using an ED50 electrochemical detector equipped with a disposable gold electrode and an Ag/AgCl reference electrode (Thermo scientific). Mannitol, 5-HMF, galacturonic acid (GalA) and glucuronic acid (GlcA) were also detected by HPAEC-PAD as described in detail by Manns et al. (2014). Glucose (Glc), Xylose (Xyl), Mannose (Man), Galactose (Gal), Arabinose (Ara) and Rhamnose (Rha) were analysed by Borat anion exchange chromatography (Borat-HPAEC) with post-derivatization and detection at 560 nm as reported by Willför et al. (2009). Total extracted carbohydrates were assessed after acid hydrolysis of the extracts. The freeze-dried extracts (50 mg) were suspended in 5 mL of water and then 0.9 mL of H2SO4 1 M was added. The samples were then hydrolysed in an autoclave at 120 °C for 40 min. After cooling, the samples were brought to 50 mL with water and filtered on a No. 4 sintered glass crucible. The monomeric carbohydrate components of the hydrolysate, including mannitol, 5-HMF and the uronic acids, were afterwards detected by Borat-HPAEC and HPAEC-PAD. Bound carbohydrates (polysaccharides and glycosyl residuals) in the extracts were calculated by the difference between total and free carbohydrates. The presence of phenolic glycosides was analysed by acid hydrolysis of the fractions F1 and F2 followed by Borat-HPAEC. Hydrolysis was performed similarly to the extracts, but using 10 times smaller solution volumes. The possible elution of free carbohydrates in these fractions was checked by Borat-HPAEC on nonhydrolysed samples. 4.8. Thermogravimetry Total inorganic compounds in extracts were measured as residuals (ashes) of the extracts after heating to 550 °C. The sample (20 mg) was placed on the balance plate of a thermogravimetric system (Mettler TC10A/TC15), heated at a rate of 20 °C/min and then kept for 15 min at constant temperature. Measurements were performed three times for each bark species.

4.6. MALDI-TOF MS

Acknowledgments

MALDI-TOF mass spectrometry was performed on extracts and some F2 fractions as reported in Bianchi et al. (2014). Briefly, acetone solutions of the sample were mixed with the matrix (2,5-dihydroxybenzoic acid) and spiked with KCl. The addition of a high concentration of KCl was done to strongly favor the formation of molecular ions with K+ and avoid any misinterpretation of

The authors would like to thank the Swiss National Research Program ‘‘Resource Wood” (NRP66) for the financial support, the Burgergemeinde Biel for providing the raw bark, Christina Hinterleitner from the Bern University of Applied Sciences for her great support in the development and implementation of the analytical work, Alexia N. Gloess from the Zürich University of Applied

Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006

S. Bianchi et al. / Phytochemistry xxx (2015) xxx–xxx

Sciences for her assistance in the MALDI-TOF analysis, Matthias Knop and Prof. Florian Seebeck from the Department of Chemistry of the University of Basel for their kind availability in performing HPLC-MS measurements.

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Please cite this article in press as: Bianchi, S., et al. Characterization of condensed tannins and carbohydrates in hot water bark extracts of European softwood species. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.10.006