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Industrial Crops and Products 61 (2014) 430–437

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Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

nalysis of the structure of condensed tannins in water extracts fromark tissues of Norway spruce (Picea abies [Karst.]) and Silver fir (Abieslba [Mill.]) using MALDI-TOF mass spectrometry

auro Bianchia,∗, Alexia N. Gloessb, Ivana Kroslakovab, Ingo Mayera, Frédéric Pichelina

Bern University of Applied Sciences, Architecture Wood and Civil Engineering, Solothurnstrasse 102, CH-2502 Biel, SwitzerlandZurich University of Applied Sciences, Institute of Chemistry and Biological Chemistry, Einsiedlerstrasse 31, CH-8820 Wädenswil, Switzerland

r t i c l e i n f o

rticle history:eceived 28 March 2014eceived in revised form 8 July 2014ccepted 18 July 2014

eywords:bies albaicea abiesarkondensed tannins

a b s t r a c t

Condensed tannins extracted from the bark of softwoods have been proven to be suitable compoundsin the formulation of environmentally friendly adhesives and resins. Their chemical structure has beenshown to significantly influence their properties and possible applications. Condensed tannins extractedfrom the bark of Norway spruce (Picea abies [Karst.]) and Silver fir (Abies alba [Mill.]) still lack a detailcharacterization of their chemical structure.

In an effort to address this deficiency, barks from these species were collected and extracted in water at60 ◦C. The dried extracts were analyzed by MALDI-TOF mass spectrometry to identify the building blocksand to determine the degree of polymerization of the tannin oligomers. The condensed tannins extractedfrom spruce bark at the used conditions were mainly composed of procyanidins with a polymerization

ALDI-TOF degree up to 13. Silver fir extracts revealed a predominance of prodelphinidins with a polymerizationdegree up to 9. The presence of less common building blocks such as stilbene glucosides and flavan-3-ols gallates was also hinted. Different curing times and viscosities in resin formulations are expectedbetween the two studied species, as well as in comparison to the most known and available tannins fromtropical species.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The condensed tannins, or proanthocyanidins, are polyphenolicompounds present in bark, heartwood, roots, leaves and fruits ofeveral plant species (Haslam, 1989). Aside from their traditionalse in the leather industry, water extracted condensed tannins haveeen proven as suitable natural sourced chemicals for the formu-

ation of formaldehyde-free wood adhesives (Pichelin et al., 1996,006; Kim and Kim, 2004; Pizzi, 2006), foamed materials (Tondi andizzi, 2009; Lacoste et al., 2013) and rigid composites (Sauget et al.,013). Condensed tannins extracted from tropical species such aslack wattle (Acacia mearnsii [De Wild.]) and Quebracho (Schinopsis

orentzii [Engl.]), and from the softwood Radiata Pine (Pinus radi-

ta [D. Don]) are the most available and industrially used productsPizzi and Merlin, 1981; Pizzi, 1982; Sealy-Fisher and Pizzi, 1992;izzi et al., 1993; Pizzi and Stephanou, 1994; Valenzuela et al.,

∗ Corresponding author. Tel.: +41 32 344 02 79; fax: +41 32 344 03 91.E-mail addresses: sauro.bianchi@bfh.ch, saurbian@gmail.com (S. Bianchi).

ttp://dx.doi.org/10.1016/j.indcrop.2014.07.038926-6690/© 2014 Elsevier B.V. All rights reserved.

2012). The bark of European softwoods has also been recognizedas a source of condensed tannins (Porter, 1989; Matthews et al.,1997; Jerez et al., 2009; Navarrete et al., 2010; Krogell et al., 2012;Ucar et al., 2013; Chupin et al., 2013; Abdalla et al., 2014), whichhave been proven to be effective in wood-adhesive formulations(Liiri et al., 1982; Dix and Marutzky, 1987; Yazaki and Collins, 1994;Pizzi, 1998; Roffael et al., 2000; Bertaud et al., 2012). Today, soft-wood bark is considered a side-product of the wood industry and isgenerally disposed of as fuel or in horticulture. Extraction of tanninfrom European softwood bark could therefore represent a usefulvalorization of such a resource.

Chemically, the condensed tannins have been identified asoligomers made of flavan-3-ol monomeric units linked by C Cbonds. The mean degree of polymerization of the extractedoligomers has been reported between 2 and 15 (Porter, 1992). Themost common monomers were identified as fisetinidol, robine-

tinidol, catechin, gallocatechin and their correspondent epimers(Fig. 1). These monomers represent the dominant building blocksof the oligomers profisetinidin, prorobinetinidin, procyanidin andprodelphinidin, respectively. The presence of methyl or glucosyl

S. Bianchi et al. / Industrial Crops and

Fisetinidol(MW = 274 .3 Da)

HOHO

HO

HO

O HO HO H

O H

O H

O H

O H

O H

O H

O H

OO

O

Robinetinidol(MW = 290 .3 Da)

Catechin(MW = 290 .3 Da)

HO

O HO H

O H

O H

O

HO

Gallocatechin(MW = 306 .3 Da)

A

A

A

A

B

BB

B

Fn

scZ

cctsmsyp1a

t(egwp4b2bf2p

st(oUi

toumto

tion threshold. Peptide calibration standard II (Bruker Daltonics,

ig. 1. Structural formula of the most common flavan-3-ol units of condensed tan-ins.

ubstitutes, gallic ester and copolimerization with other phenolicompounds was also reported (Hergert, 1992; Ferreira et al., 2005;hang and Gellerstedt, 2008).

The hydroxylation pattern and the degree of polymerizationould be considered the most important factors for the chemi-al and physical properties of condensed tannins. An increase inhe number of hydroxyl groups on the A-ring of the monomersignificantly reduces the gelling time of tannin-formaldehyde for-ulations (Pizzi and Stephanou, 1994; Garnier et al., 2002). A

imilar but less marked effect was reported for increasing hydrox-lation at the B-ring (Garnier et al., 2002). A higher degree ofolymerization results in higher viscosities (Kim and Mainwaring,995), shorter gelling times (Pizzi and Stephanou, 1994) and fasterutocondensation reactions (Masson et al., 1997).

Detailed information on the chemical structure of the barkannins from Norway spruce (Picea abies [Karst.]) and Silver firAbies alba [Mill.]) is limited. These species are of particular inter-st in Switzerland, as they represent 44.1% and 14.8% of the forestrowing stock, respectively (Brändli, 2010). Spruce bark tanninsere identified mainly as procyanidins, with rare occurrences ofrodelphinidins, having a mean polymerization degree equal to.6 (Matthews et al., 1997). Similar results were reported for rootark (Pan and Lundgren, 1995) and needle extracts (Behrens et al.,003). The likelihood of lignans, stilbenes and their derivativeseing present in the oligomeric structure of condensed tanninsrom spruce bark was also reported (Steynberg, 1983; Zhang et al.,001). No characterization of condensed tannins from Silver firhloem or xylem tissues has yet been reported in the literature.

Matrix assisted laser desorption–ionization time of flight masspectrometry (MALDI-TOF MS) is an established method for inves-igating the chemical structure of condensed tannin from plantsPasch et al., 2001; Monagas et al., 2010). MALDI-TOF MS spectraf softwood bark extracts (Jerez et al., 2009; Navarrete et al., 2010;car et al., 2013; Abdalla et al., 2014) were successfully used to

dentify the oligomeric structure of condensed tannins.In this paper, MALDI-TOF mass spectrometry was used to inves-

igate water extracts of Norway spruce and Silver fir barks. The mainbjectives were the identification of the type of the monomericnits in the extracted condensed tannins and identification of their

aximum observable degree of polymerization. The results were

hen compared with the structures of condensed tannins fromther species.

Products 61 (2014) 430–437 431

2. Materials and methods

2.1. Bark tissues

Bark flakes from spruce and Silver fir were collected at the begin-ning of March 2012 from harvested logs in a forest nearby Biel(Switzerland). The forest altitude was about 600 m above mean sealevel. All logs were felled during January and February 2012 and hadbreast-high girths between 40 and 60 cm. The barks were removedusing a handheld tool within the lowest 3 m of the stem height.Approximately 3 kg of wet bark was collected from 3 to 4 differentlogs for each species. The bark flakes were stored airtight at −20 ◦C,on the same day as felling, until sample preparation.

2.2. Tannin extraction

The deep-frozen bark flakes were manually cut into smallerchips, freeze-dried, and reduced to fine particles with a laboratoryhammer-mill (mesh width = 3 mm). The samples were then storedairtight, protected from light and kept at room temperature untilextraction.

Extractions were performed with deionized water at 60 ◦C. AnAccelerated Solvent Extraction device (Dionex ASE® 200) was used,operating with the following setting: 5 min for heating and pressur-izing up at 10 MPa, 5 min at static temperature and pressure, finalflushing of the extract through a cellulose filter. These mild condi-tions were chosen in order to avoid any possible modification of thecondensed tannin during extraction. Approximately 2.0 g of milledbark and 18 mL of deionized water (including the final flushing)were used for each extraction. The extract was thereafter immedi-ately freeze-dried, stored airtight, protected from light and kept atroom temperature until analysis. For each species, 12 extractionswere performed and the obtained dried extracts were combined inone sample.

Total extraction yield was calculated as weight ratio betweenthe freeze-dried extract and the initial dry weight of the bark.

2.3. MALDI-TOF mass spectrometry

Prior to analysis 2.5 mg of freeze-dried tannin extracts weredissolved in 1 mL of aqueous acetone (50%) and sonicated for30 min. The matrix solution was prepared by dissolving 10 mgof 2,5-dihydroxybenzoic acid (Sigma–Aldrich) in pure acetone(Sigma–Aldrich). 10 �L of tannin solution was mixed with 10 �Lof matrix solution and spiked with 1 �L of KCl (10 g/L in milliQwater) to enhance the ion formation. Finally, 5 �L of the mixturewas deposited on the stainless steel plate and allowed to dry atroom temperature.

The high concentration of added K+ cations was chosen to sup-press the formation of other cations naturally occurring in the bark(Xiang and Lin, 2006). Ca, K and Mn salts have been in fact widelyreported as typical inorganic compounds in bark tissues, with con-centrations between 1.1 and 5.8 g/kg of dry bark (Krizaj and Stupar,1996; Rothpfeffer and Karltun, 2007). Moreover, hot water extrac-tions were shown as able to completely leach these ions from thebark (Krogell et al., 2012).

The measurements were carried out with a MALDI-TOF massspectrometer (Reflex III, Bruker Daltonics, Germany) equipped witha nitrogen laser (337 nm) using FlexControl 3.0 software (BrukerDaltonics, Germany) monitoring mass range 300–5000 Da in linearpositive mode. The laser intensity was set slightly above the abla-

Germany) containing nine different peptides was used for exter-nal mass calibration covering range from 757.9 to 3149.6 Da. Eachcollected spectrum represents a sum of 700 laser shots.

432 S. Bianchi et al. / Industrial Crops and Products 61 (2014) 430–437

0.0

0.5

1.0

1.5

2.0

2.5

500 10 00 15 00 20 00 30 00 35 00 40 00 m/z2500

906.

210

22.2

1428

.0

1194

.611

39.6

1310

.9 1483

.015

99.5 17

71.4

1887

.7 2059

.621

76.1

2348

.324

63.6

2636

.4

2752

.3

2924

.2

3040

.2

3212

.5

3329

.8

3501

.2

3617

.3

3788

.31715

.9

2005

.0

1256

.7

1544

.8

1832

.1

867.

9

705.

6

445.

6

0.0

0.5

1.0

1.5

2.0

2.5

x104

Inte

ns. [

a.u.

]300 400 500 600 800 900

m/z700

381.

538

9.6

403.

542

9.7

445.

645

9.6 52

3.4

543.

656

3.5

617.

5

705.

6

867.

985

0.4

906.

2

733.

5

x104

Inte

ns. [

a.u.

]

3.0

617.

5 733.

5

F . The pd

3

3

draeKRaed(iDpes

3

soa(iw

as

ig. 2. MALDI-TOF mass spectrum of spruce bark extract in the range 300–5000 Daetail.

. Results and discussion

.1. Extraction yield

Total extraction yields equal to 3.3% and 10.1% (referred to thery bark weight) were measured for spruce and Silver fir bark,espectively. These values, and in particular the one of spruce,re lower than those reported in the literature for softwood barkxtractions (Dix and Marutzky, 1983; Sealy-Fisher and Pizzi, 1992;önig and Roffael, 2003; Vázquez et al., 2001; Bertaud et al., 2012).elatively low extraction temperature (60 ◦C) and the absence ofny chemicals such as sulphites, carbonates or hydroxides in thextraction media are most likely the main causes of the observedifferences. Spruce bark extraction performed in water at 75 ◦CBertaud et al., 2012) showed a total extraction yield of approx-mately 3%, comparable with the results achieved in this study.ifferences in the extraction yield could also be associated to theroportion of inner and outer bark (Matthews et al., 1997; Krogellt al., 2012), age of the bark (Matthews et al., 1997), and particleize (Vázquez et al., 2001).

.2. Norway spruce

The MALDI-TOF mass spectrum of the spruce bark extract (Fig. 2)howed, in particular for masses higher than 900 Da, the presencef several regular and repetitive patterns of peaks, which could bessociated with specific oligomer series. In the lower mass range300–900 Da) intense and irregularly spaced peaks were observednstead. The presence of a considerable amount of low molecular

eight extractables is therefore hinted.Mass spectra of the extract collected without the addition of KCl

s cationization support (not reported here) resulted in identicalpectra as the one with KCl. The equivalence of the spectra with and

ortion of the mass spectrum in the range between 300 and 900 Da is shown in the

without KCl suggests that concentration of K+ ions in the extractis high enough to participate in the MALDI ion formation. Massspectra misinterpretations could therefore occur when using lowconcentrations of a cationization support different from K+ salts.

Scrutiny of the mass spectra in the range 300–900 Da (Fig. 2)revealed the presence of intense peaks at 429.7, 445.6 and 459.6 Da.The peaks could possibly be associated to the three stilbene gluco-sides piceid (390.4 + K+ = 429.4 Da), astringin (406.4 + K+ = 445.4 Da)and isorhapontin (420.4 + K+ = 459.4 Da), respectively. These com-pounds have been recognized as very typical extractives of sprucebark (Mannila and Talvitie, 1992; Krogell et al., 2012; Latva-Mäenpää et al., 2013). The highest intensity of the 445.6 Dapeak indicates astringin as the predominant among them. Thepeak at 850.4 Da could be interpreted as an astringin dimer(2 × (406.4 − 1H) + K+ = 849.8 Da).

Other intense series of peaks could be observed in the massranges 380–410 Da, 520–580 Da, at 705.6 Da and at 867.9 Da. Thesepeaks could not be in general associated to any typical extractablefrom softwood barks (e.g. flavonoids, lignans). Among them a peakseries with a repetition unit mass of approximately 162 Da was rec-ognized (381.5, 543.6, 705.6 and 867.9 Da). It could be tentativelyinterpreted as a series of oligosaccharides (e.g. sucrose, raffinose,stachyose, etc.) from the dimer (342.3 + K+ = 381.3 Da) up to thepentamer (828.6 + K+ = 867.6 Da).

Above 600 Da, two main peak series were identified: the mostintense series s1 (617.5, 906.2, 1194.6, 1483.0, 1771.4 Da, etc.), andthe slightly less intense series s2 (733.5, 1022.2, 1310.9, 1599.5,1887.7 Da, etc.). Both of these series displayed a repetition unitmass of approximately 288 Da and their peaks were detectable up

to 4000 Da. In the series s2, substructures with mass increments of16 Da adjacent to the dominant peak were also observed. Furtherpeak series, having much lower intensities than series s1 and s2,were also recognized in the mass range between 1100 and 2000 Da

S. Bianchi et al. / Industrial Crops and Products 61 (2014) 430–437 433

288.4 s1

s2

s3

s4

288.4

404.9288.6 288.2303.3

405.0288.4 287.9304.5

404.5288.1 287.7304.8

1000 11 00 12 00 13 00 15 00 16 00 17 00 m/z00020091008100410.0

2.0

4.0

6.0

8.0

10.0In

tens

. [a.

u.] x

103

F 00 and 2000 Da, with the identified repeating mass units. The presence of four differento tely 405 Da is highlighted.

(vrso

wsh(Awsdh(at

s14adabGtor

Astringin(MW = 406.4 Da)

O H

O H

O H

O H

O HO

OHO

HO

ig. 3. MALDI-TOF mass spectrum of spruce bark extract in the range between 10ligomeric series (s1, s2, s3 and s4) with a common shift among them of approxima

Fig. 3): the series s3 (1139.6, 1428.0, 1715.9, 2005.0 Da) and theery low intense series s4 (1256.7, 1544.8, 1832.1 Da). The mainepeating unit mass for these series was approximately 288 Da,imilar as in the series s1 and s2. Substructures at mass incrementsf 16 Da from the dominant peak were also identified.

The mass of the main repetition unit for all series matchesith the mass of both (epi)catechin and (epi)robinetinidol exten-

ion units of condensed tannins (288.3 Da). However, flavan-3-olsaving a monohydroxylated A-ring like (epi)robinetinidol andepi)fisetinidol are predominantly restricted to Leguminosae andnacardiaceae species (Porter, 1989). The 288 Da repetition massas then more confidently attributed to an (epi)catechin exten-

ion unit. The substructures at a mass increment of 16 Da from theominant peak were interpreted as differences in the number ofydroxyl groups attached to the monomeric units. Combinations ofepi)catechin and (epi)gallocatechin extension units were thereforessumed to be present in the structure of the analyzed condensedannins from spruce bark.

A common shift factor of approximately 405 Da among theeries was detected, e.g. 1310.9–906.2 = 404.7 Da (series s2–s1),428.0–1022.2 = 405.8 Da (series s3–s2) and 1544.8–1139.6 =05.2 Da (series s4–s3). This value matches with the mass ofstringin (Fig. 4) when considered covalently bonded within a con-ensed tannin molecule (406.4 − 2H = 404.4 Da). Stilbenes, lignansnd their derivatives have been described as potential buildinglocks in spruce bark tannins (Steynberg et al., 1983; Zhang and

ellerstedt, 2008), usually as terminal units. The high intensities of

he peaks at 445.6 and 850.4 indicate a substantial concentrationf astringin and astringin dimer in the extract. There is therefore aeasonable possibility of astringin units to be a part of the structure

Fig. 4. Structural formula of the stilbene glucoside astringin, which could matchwith the observed 404.4 Da mass unit (406.4 - 2H) observed in the MALDI-TOFspectra of spruce.

of the spruce bark tannins. More detailed analyses are needed toconfirm such a structure.

The interpretation of the mass spectrum of the spruce barkextracts was then performed according to Eq. (1):

M + K+ = x · 288.3 + y · 304.3 + z · 404.4 + 39 + 2 (1)

where x, y and z are the number of the respective mass units presentin the oligomer, 39 is the weight of the K+ ion, and 2 is the weight

of the two hydrogen atoms at the ends of the oligomer.

In Eq. (1), the possibility of a (epi)fisetinidol unit (272.3 Da) wasnot included. This flavan-3-ol is quite uncommon in softwoods(Porter, 1989), and no peaks were identified in the spectrum of

434 S. Bianchi et al. / Industrial Crops and Products 61 (2014) 430–437

Table 1Interpretation of the MALDI-TOF mass peaks for the oligomeric series s1 and s2 in spruce bark extracts.

Polymerizationdegree (unit)

Series MALDI-TOFobserved peak (Da)

Calculated mass(Da)

Monomeric units

Catechin orrobinetinidola

(288.3 Da)

Gallocatechin(304.3 Da)

Unknownb

(404.4 Da)

2s1 617.5 617.6 2s2 733.5 733.7 1 1

3s1 906.2 905.9 3s2 1022.2 1022.0 2 1s2 1037.5 1038.0 1 1 1

4s1 1194.6 1194.2 4s2 1310.9 1310.3 3 1s2 1326.1 1326.3 2 1 1

5s1 1483.0 1482.5 5s2 1599.5 1598.6 4 1s2 1614.2 1614.6 3 1 1

6s1 1771.4 1770.8 6s2 1887.7 1886.9 5 1s2 1903.4 1902.9 4 1 1

7s1 2059.6 2059.1 7s2 2176.1 2175.2 6 1s2 2190.5 2191.2 5 1 1

8s1 2348.3 2347.4 8s2 2463.6 2463.5 7 1s2 2478.4 2479.5 6 1 1

9s1 2636.4 2635.7 9s2 2752.3 2751.8 8 1s2 2768.7 2767.8 7 1 1

10s1 2924.2 2924.0 10s2 3040.2 3040.1 9 1

11s1 3212.5 3212.3 11s2 3329.8 3328.4 10 1

12s1 3501.2 3500.6 12s2 3617.3 3616.7 11 1

13 s1 3788.3 3788.9 13

a The 288.3 Da repetition mass was more confidently attributed to a catechin unit as robinetinidol units are predominantly restricted to Leguminosae and Anacardiaceaes

in uni

sT

s

nacs

tdae(

ee

tqtt2

pecies.b The 404.4 Da repetition mass could be associated to a stilbene glucoside astring

pruce that would strictly require this mass for the interpretation.he presence of such a unit was then considered very unlikely.

The application of the Eq. (1) to the peaks of the two main series1 and s2 is reported in Table 1.

The minor series s3 and s4 could also be interpreted as combi-ations of (epi)catechin, (epi)gallocatechin units and two or threestringin units, respectively. The low intensity of these series indi-ated the low amount in the extracts of the s3 and s4 oligomereries.

The water extracted condensed tannins from spruce bark wereherefore identified as mainly procyanidins with a polymerizationegree of up to 13 units. The predominance of procyanidins is ingreement with the data reported by other studies on spruce barkxtract, in which however different analytical methods were usedPan and Lundgren 1995; Matthews et al., 1997).

Tannin oligomer lengths between 12 and 15 units were alsovidenced by MALDI-TOF analysis on Pinus spp. bark extracts (Jerezt al., 2009; Navarrete et al., 2010).

The identified tannin structure is considerably different fromhose of the most common and industrially used black wattle and

uebracho tannins. These species were in fact described as profisi-inidins and prorobinetinidins with a polymerization degree equalo or lower than 10 (Porter, 1989; Pasch et al., 2001; Hoong et al.,010).

t.

3.3. Silver fir

MALDI-TOF mass spectrum of Silver fir bark extract showed,similarly to that of spruce, the presence of intense and irregularlyspaced peaks in the region below 900 Da (Fig. 5). The large majorityof these peaks correspond in the mass value to the peaks observedin the spruce extract spectrum in the same range. In particular,a peak series (381.5, 543.6, 705.9 and 868.2 Da), which could bepotentially associated to different oligosaccharides, was observedin both species. No presence of the mass peaks associated to thestilbene glucosides was observed for Silver fir, confirming the char-acteristic presence of these compounds in spruce species.

Above 600 Da, a single main repetitive pattern of peaks wasobserved (Fig. 5). This pattern was assigned to the predominantoligomer series, labeled f1 (649.9, 938.1, 1242.5, 1546.7, 1851.3 Da,etc.). The mass of main repetition unit was approximately 304 Da(Fig. 6). Numerous and evident substructures at mass differencesof 16 Da were detected close to the dominant peak. In the massrange between 1000 and 2000 Da (Fig. 6) a series with much lowerintensity was observed (series f2 = 1073.9, 1378.2, 1682.4 Da). A

main repetition unit equal to 304 Da and substructures at massdifferences of 16 Da were identified for this series too.

As well as in the case of spruce, mass spectra detected with-out the addition of any cationization support (not reported) well

S. Bianchi et al. / Industrial Crops and Products 61 (2014) 430–437 435

500 100 0 150 0 200 0 300 0 350 0 400 0m/z

25000.0

0.5

1.0

1.5

2.0

2.5

x104

Inte

ns. [

a.u.

]

00–50

oTw

3o

Fo

Fig. 5. MALDI-TOF mass spectrum of Silver fir bark extract in the range 3

verlapped with those obtained using KCl as cationization support.hus, the presence of a relevant amount of K+ ions in the extractsas hinted for the Silver fir sample too.

The mass of the detected main repetition unit matched with the04.3 Da molecular mass of an (epi)gallocatechin unit. The numer-us substructure peaks at a still high intensity were associated

0.0

4.0

2.0

6.0

8.0

Inte

ns. [

a.u.

]x1

03

1002100110001009

304.3288.3

304.2288.0

ig. 6. MALDI-TOF mass spectrum of Silver fir bark extract in the range between 900 anligomeric series (f1 and f2) with a shift of approximately 152 Da is highlighted.

00 Da. The lower mass range between 300 and 900 Da is shown in detail.

to a significant presence of (epi)catechin units (288.3 Da) in thecondensed tannin molecular structure.

A shift of approximately 152 Da between series f1 and f2 (e.g.

1073.9–922.2 = 151.7 Da, 1378.2–1226.9 = 151.3 Da) was detected.This value matched with the mass of a gallate moiety (152.1 Da).The occurence of (epi)catechin or (epi)gallocatechin gallate units

0051003 m/z00610041

f1

f2

304.1288.4

151.9

d 1600 Da, with the identified repeating mass units. The presence of two different

436 S. Bianchi et al. / Industrial Crops and Products 61 (2014) 430–437

Table 2Interpretation of the MALDI-TOF mass peaks for series f1 and f2 of Silver fir bark extract.

Polymerizationdegree (units)

Series MALDI-TOFobserved peak (Da)

Calculated mass(Da)

Monomeric units

Catechin orrobinetinodola

(288.3 Da)

Gallocatechin(304.3 Da)

Gallate moiety(152.1 Da)

2 f1 649.9 649.6 2

3

f1 922.2 921.9 2 1f1 938.1 937.9 1 2f1 954.2 959.9 3f2 1073.9 1074.0 2 1 1f2 1090.2 1090.0 1 2 1

4

f1 1226.9 1226.2 2 2f1 1242.5 1242.2 1 3f1 1258.5 1258.2 4f2 1378.2 1378.3 2 2 1f2 1394.4 1394.3 1 3 1

5

f1 1515.4 1514.5 3 2f1 1530.9 1530.5 2 3f1 1546.7 1546.5 1 4f1 1562.6 1562.5 5f2 1666.6 1666.6 3 2 1f2 1682.4 1682.6 2 3 1

6

f1 1819.7 1818.8 3 3f1 1835.3 1834.8 2 4f1 1851.3 1850.8 1 5f1 1867.1 1866.8 6

7

f1 2123.7 2123.1 3 4f1 2139.5 2138.1 2 5f1 2155.4 2155.1 1 6f1 2171.1 2171.1 7

8

f1 2427.6 2427.4 3 5f1 2443.3 2443.4 2 6f1 2459.3 2459.4 1 7f1 2475.5 2475.4 8

9f1 2747.5 2747.7 2 7f1 2762.8 2763.7 1 8

as ros

wp

be

M

AE

f

ttsg1pst(eha1

a The 288 Da repetition mass was more confidently attributed to a catechin unitpecies.

as therefore hinted. Due to the very low intensity of the signal theresence of such monomeric units was considered quite scarce.

The interpretation of the Silver fir mass spectrum could thene performed with an equation similar to the ones used for sprucextract (Eq. (2)):

+ K+ = x · 288.3 + y · 304.3 + z · 152.1 + 39 + 2 (2)

s well as for spruce, no (epi)fisetinidol units were considered inq. (2).

Table 2 reports the application of the Eq. (2) for the series f1 and2.

The water extracted condensed tannins from Silver fir bark wereherefore identified mainly as prodelphinidins with a polymeriza-ion degree of up to 9 units. A preponderance of prodelphinidins inoftwood bark has so far been reported only for Pinus contorta [Dou-las] and the secondary phloem of Pinus patula [Schiede] (Porter,989). Within the Abies genus, the presence of about 30% of prodel-hinidins (Porter, 1989) in Abies firma [Siebold & Zucc.] and Abiesachalinensis [F. Schmidt] bark was indicated. More common washe detection of prodelphinidins in softwood needles and conesHemingway, 1989; Porter, 1989). The analyzed condensed tannins

xtracted from Silver fir bark were hence characterized by a higherydroxylation degree than other softwood bark tannins, whichre generally described as procyanidins (Porter, 1989; Matthews,997). The highest observed oligomer lengths were instead slightly

binetinidol units are predominantly restricted to Leguminosae and Anacardiaceae

shorter than those detected in other softwood bark extracts (Jerezet al., 2009; Navarrete et al., 2010).

The extracted condensed tannin from Silver fir bark showedthus a molecular structure significantly different from both theextracted spruce bark extracts and the industrial black wattle andquebracho tannins (Porter, 1989; Pasch et al., 2001; Hoong et al.,2010).

4. Conclusions

MALDI-TOF MS was successfully used for the analysis of thestructure of condensed tannins extracted from the bark of Norwayspruce and Silver fir. Differences in the dominant building blockas well as in the maximal observable oligomer length were identi-fied between the two species. Procyanidins with a polymerizationdegree of up to 13 units and prodelphinidins with a polymeriza-tion degree of up to 9 units were observed in spruce and Silverfir extracts, respectively. The likelihood, scarcely reported in theliterature, of stilbene glucosides and flavan-3-ol gallates as build-ing blocks of the condensed tannins was also hinted. The presenceof intense peaks in the lower mass range of the mass spectra sug-

gested the presence of a considerable amount of compounds havinga molecular weight below 900 Da in both extracts. These com-pounds could be associated to low molecular weight phenolics andoligosaccharides.

ps and

eboFthv

detaamm

A

Pt

R

A

B

B

B

C

D

D

F

G

H

H

H

H

J

K

K

K

K

of some pine and spruce species. Holz Roh. Werkst. 52, 307–310.Zhang, L., Gellerstedt, G., 2008. 2D heteronuclear (1H–13C) single quantum correla-

S. Bianchi et al. / Industrial Cro

The identified condensed tannin structures differed consid-rably from those of the most common and industrially usedlack wattle and quebracho tannins. In particular, slightly longerligomers and higher hydroxylation degrees were recognized.aster condensation reaction rates than industrial tannins arehen expected for the extracted oligomers. In particular, the highydroxylation degree observed in Silver fir bark tannins suggestsery fast condensation reactions for these extractables.

The identified molecular structures have to be related to con-ensed tannins extracted at the specific conditions used. The totalxtraction yields achieved in this study are in fact lower than thoseypically reported for softwood bark extractions. These are usu-lly performed at higher temperatures or with addition of chemicalids. The different process conditions could then potentially pro-ote the extraction of different oligomers or even modify theolecular structure of the extracts.

cknowledgments

The authors would like to thank the Swiss National Researchrogram “Resource Wood” (NRP66) for the financial support andhe Burgergemeinde Biel for providing the raw bark tissues.

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