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Industrial Crops and Products 52 (2014) 23– 28

Contents lists available at ScienceDirect

Industrial Crops and Products

journa l h om epage: www.elsev ier .com/ locate / indcrop

hemical composition of the silver fir (Abies alba) bark extractbigenol® and its antioxidant activity

va Tavcar Benkovic a,∗, Tina Grohara, Dusan Zigonb, Urban Svajgerc, Damjan Janes a,amo Krefta, Borut Strukelj a

Faculty of Pharmacy, University of Ljubljana, Askerceva cesta 7, Ljubljana SI-1000, SloveniaJozef Stefan Institute, Department of Environmental Sciences, Jamova 39, Ljubljana SI-1000, SloveniaBlood Transfusion Centre of Slovenia, Slajmerjeva 6, Ljubljana SI-1000, Slovenia

r t i c l e i n f o

rticle history:eceived 17 July 2013eceived in revised form 2 October 2013ccepted 4 October 2013

eywords:

a b s t r a c t

Extracts from the bark of different conifer species are known to contain various polyphenols and possessinteresting pharmacological activities. So far the most extensive research was done on the antioxida-tive extract of the maritime pine (Pinus maritima) bark, which is widely used in food supplementsand cosmetic products. Here we have shown, that antioxidant activity of silver fir (Abies alba) barkextract is higher than of maritime pine bark extract in cultured cells. Components of the extract were

ilver fir (Abies alba) extractbigenolntioxidantshenolsignanslavonoids

separated with normal phase flash chromatography and reversed phase high-performance liquid chro-matography (HPLC). The structures of individual compounds were identified by mass spectrometry,UV–vis absorption spectroscopy and comparison to reference compounds. Six phenolic acids were iden-tified (gallic, homovanillic, protocatehuic, p-hydroxybenzoic, vanillic and p-coumaric), three flavonoids(catechin, epicatechin and catechin tetramethyl eter) and four lignans (taxiresinol, 7-(2-methyl-3,4-dihydroxytetrahydropyran-5-yloxy)-taxiresinol, secoisolariciresinol and laricinresinol).

. Introduction

The silver fir is one of the most common tree species in theentral Europe and therefore of an important economic, environ-ental and social significance (Ficko et al., 2011). It is widespread

n the montane vegetation zone. Previous studies, which investi-ated chemical composition of silver fir extracts, mostly identifiedonoterpenes and monoterpenoids in oleoresin and twig oil,

riterpenoids in needles and bark, sesquiterpenes in needles andleoresin, diterpenoids and steroids in needles, and lignans ineartwood and knots. Bioassay tests on the silver fir extracts arecarce. The essential oil showed antioxidative and antibacterialctivities (Yang et al., 2009) and the extract of silver fir and com-on mistletoe (Viscum album) mixture exhibited antiproliferative

nd anticancerogenic features. However, many other fir speciesave been recognized as rich sources of lignans, flavonoids andther phenols with antioxidant activity (Li et al., 2011; Yang et al.,008). Extracts from different plant parts of some other conifer

pecies have been extensively researched and were also imple-ented in pharmaceutical use. The most studied among them is

ycnogenol®, a standardized extract of the maritime pine (Pinus

∗ Corresponding author. Tel.: +386 1 476 97 09.E-mail address: eva.tavcar.benkovic@ffa.uni-lj.si (E.T. Benkovic).

926-6690/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2013.10.005

© 2013 Elsevier B.V. All rights reserved.

maritima) bark, widely used in food supplements and cosmeticproducts. Pycnogenol® has been reported to have a strong freeradical–scavenging activity against the reactive oxygen and nitro-gen species. Exhibiting an anti-inflammatory activity, Pycnogenol®

protects from oxidative stress, improves immune, circulatory, andneurodegenerative disorders. It beneficially influences metabolicsyndrome diseases and plays an important role in chronic venousinsufficiency and dermatology. The activity is attributed to themixture of flavonoids, mainly procyanidins and phenolic acids(D’Andrea, 2010). Apart from the maritime pine bark, many authorshave reported on high phenolic content of the bark extracts of someother pine species (Fradinho et al., 2002; Kähkönen et al., 1999;Kofujita et al., 1999). The Scots pine (Pinus sylvestris) bark was foundto contain procyanidins ranging from monomers through decamersand higher polymers (Karonen et al., 2004). Phenolic acids, cate-chin, epicatechin, procyanidin B2, taxifolin, quercetin, syringic andhomovanillic acids were found in the Monterey Pine (Pinus radi-ata) bark extracts, exhibiting antioxidant properties (Bocalandroet al., 2012). Flavangenol® is a maritime pine bark extract, avail-able on the market, with a protective effect against oxidative stressassociated with streptozotocin-induced diabetes and increasing

mRNA expression of fatty acid oxidative enzyme genes in the liver(Nakano et al., 2008; Shimada et al., 2012). Standardized pine barkextract Oligopin® is rich in procyanidins and catechins and wasshown to modulate stress-induced phosphorylation of the stress

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haperone heat shock protein beta-1 (Poussard et al., 2012). Thelack spruce (Picea mariana) bark extract showed an adequatehemical reactivity toward different radicals, and antiproliferativeroperties (García-Pérez et al., 2010). Polyphenol and flavonoid-ontaining foods protect against cardiovascular risk, cancer andeurodegeneration (Habauzit and Morand, 2011; Vauzour et al.,010).

In search for similar activities of other species of the Pinaceaeamily, we investigated the antioxidant activity and compositionf the silver fir (Abies alba) bark extract (AABE) which is beingommercialized under the trade name Abigenol®.

. Materials and methods

.1. Chemicals

All solvents were of p.a. grade purity: acetone (Panreac;arcelona), acetic acid (Merck; Germany); toluene (Riedel-de Haën;ermany). The solvents used for HPLC analysis were of HPLC-rade purity: water (Panreac; Barcelona); acetonitrile (JT Baker;eventer); formic acid (Fluka, Sigma-Aldrich Buchs; Steinheim),

rifluoroacetic acid (Roth, Karlsruhe; Germany).

.2. Bark extracts

Silver fir bark was extracted by a two-step extraction asescribed in our patent SI23867 (Strukelj et al., 2013), briefly: 5 kgf ground bark of silver fir (A. alba) was first extracted with 25 Lf water at 70 ◦C for 2 h. The aqueous extract was than evaporatednder vacuum to a volume of 5 L. In the second step the concen-rated aqueous extract was extracted with 3 L × 3 L of ethyl acetate.he ethyl acetate extracts were added to 25 mL of polyethylenelycol 400 and the ethyl acetate was then evaporated from a mix-ure. Fifty milliliters of viscous liquid silver fir (A. alba) bark extractAABE) was obtained.

For comparison, a dry extract from the silver fir bark (d-AABE)as also prepared, where 9 L of the ethylacetate extract (pre-ared as above) was concentrated to 300 mL and polyphenols wererecipitated by the addition of 300 mL of heptane to yield 20 gf d-AABE. This procedure is also described in our other patentSI22882).

Maritime pine bark dry extract was purchased from BiolandesF0400/Pycnogenol® LOT: G/1480).

.3. Antioxidant activity

Antioxidant activity was measured with two methods: DPPHnd cell-based test.

In the first test 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagentas used. 100 �L of DPPH solution (3.9 mg/100 mL of methanol)as added to 100 �L aliquot of a sample (solution of extract or

tandard). For a control 100 �L of methanol was added to the sec-nd aliquot of sample (100 �L). After 60 min, the absorbance waseasured at 515 nm in both solutions. The concentration was calcu-

ated from the differences of both measurements and by comparingo the standard solution of pyrogallol (0.1 mg/mL).

For in vitro cell-based test, primary human peripheral bloodononuclear cells (PBMCs) were isolated from human buffy coats

rom healthy donors, which were obtained from the Blood trans-usion center of Slovenia, following institutional guidelines. PBMCsere cultured in an enriched media RPMI 1640 (0.5% l-glutamine,

% penicillin/streptomycin in 10% FBS). They were pre-incubatedor 30 min with different concentrations (1–100 �g/mL) of AABE,-AABE and maritime pine bark extract (Pycnogenol). The cellsere than incubated in 20 �M solution of 2′,7′-dichlorofluorescein

and Products 52 (2014) 23– 28

diacetate (DCFH-DA) for 15 min. Subsequently the cells were acti-vated with 1 �g/mL PMA (phorbol 12-myristate 13-acetate). Themean fluorescence intensity (MFI) value of the cell population wasmeasured with the use of a flow cytometer (FacsCalibur, BecktonDickinson). The antioxidative activity of the sample is revealed by adecreased fluorescence of an oxidized form of the DCFH-DA reagentthat forms under the influence of free radicals.

2.4. Chromatographic analysis

The HPLC system (Shimadzu Prominence) consisted of a sys-tem controller (CBM-20A), a column oven CPO-20AC and asolvent delivery pump with a degasser (DGU-20A5) with a PhotoDiode Array detector (SPD-M20A) that monitored the wavelengths190–800 nm. The responses of the detectors were recorded usingLC Solution software version 1.24 SP1.

Several columns with a different mobile phase gradients andcompositions (water, acetonitrile, methanol, formic acid, aceticacid, TFA) were tested to achieve an optimal separation of the com-pounds of AABE: Cromolith Performance – Si (100–4.6 mm) Merck,Ascentis Express C8 (10 cm × 4.6 mm, 2.7 �m) Supelco, Ascen-tis Express HILIC (10 cm × 4.6 mm, 2.7 �m) Supelco, PhenomenexKinetex (2.6 u XB-C18 100A, 100 mm × 4.6 mm). Reversed-phaseC18 was chosen as an optimal HPLC stationary phase and optimalchromatographic conditions were: column temperature 40 ◦C, flowrate 2 mL/min, Phenomenex Kinetex® C18 column (10 cm × 4.6 mmI.D., 2.7 �m particle size) and gradient method using water (solventA) and acetonitrile (solvent B), both containing 0.1% of formic acid,was utilized: 0–1 min 5% B, 1–10 min 5–30% B, 10–15 min 100% B.

For the fractionated samples, obtained with the flash chromato-graphic separation, six adapted HPLC gradients were developed: FR21 (1 min 5% B, 2.5 min 10% B, 10 min 10% B, 12 min 12% B), FR 8(1 min 5% B, 10 min 30% B and 1 min 5% B, 10 min 30% B,), FR 43(1 min 5% B, 2.5 min 10% B, 8 min 12% B), FR 17 (20 min 15% B), FR133 (1 min 10% B, 4 min 50% B, 13 min 50% B).

Available reference compounds were analyzed both individuallyand with addition to the samples, using the described methods.

2.5. Flash chromatographic separation

The flash chromatographic separation was performed on a sil-ica gel 60 column (4 cm × 12.5 cm, particle size 0.063–0.200 mm,Merck). 500 mg of the AABE sample, diluted in 200 �L oftoluene/acetone/acetic acid (3/6/1) was applied on the column.Selection of the solvents was done using TLC (Silicagel 60 plates,F254, Merck). A gradient elution was performed in the followingorder: toluene/acetone/acetic acid (3/6/1), toluene/acetone/aceticacid (1/8/1), acetone/acetic acid (9/1). The flow rate of 10 mL/minwas applied with a Büchi Pump Controller C-610. One hundred andeighty fractions (60 with each solvent) were collected into glasstubes (10 mL) with a Büchi Fraction Collector C-660. All fractionswere analyzed with the HPLC. Six most concentrated and the mostdiverse fractions were dried and dissolved in acetonitrile/water(2/3) for LC–MS analysis.

2.6. Mass spectrometric analysis

The chromatographic separation was performed on a WatersAcquity ultra-performance liquid chromatograph (Waters Corp.,Milford, MA, USA), with a column identical to that used for thequantitative HPLC analysis. The methods, optimized for each frac-

tion were adjusted to the flow rate of 0.5 mL/min. The injectionvolumes were 10 �L. The column temperature was maintainedat 40 ◦C. The LC system was interfaced with a hybrid quadrupoleorthogonal acceleration time-of-flight mass spectrometer (Q-ToF

E.T. Benkovic et al. / Industrial Crops and Products 52 (2014) 23– 28 25

75

100

125

150

175

200

0 20 40 60 80 100

PEG

mari�mepine barkextractd-AABE

AABE

concentration of extract in cell medium ( g/ml)μ

fluor

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Fig. 1. Antioxidative activity of the samples on PBMC cells are revealed by decreasedfla(

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uorescence. The highest antioxidative activity is achieved by precipitated dry Abieslba bark extract (d-AABE), followed by Abies alba bark extract prepared with PEGAABE) and maritime pine bark extract.

remier, Waters, Milford, MA, USA). The compounds were analyzednder positive (ESI(+)) and negative (ESI(−)) ion conditions.

The capillary voltage was set at 3.0 kV, while the sampling coneoltage was 20 V. The source and desolvation temperatures were00 and 200 ◦C, respectively. The flow rate of nitrogen desolva-ion gas was 600 L/h. The acquisition range was between m/z 50nd 1000 with argon serving as a collision gas at a pressure of.5 × 10−3 mbar in the T-wave collision cell. The MS/MS experi-ents were performed using collision energies from 5 to 30 eV

o generate the product ion spectra that provided the best struc-ural information. The data were collected in centroid mode, with

scan accumulation time of 0.2 s and an interscan delay of 0.025 s.he data station utilized the MassLynx v4.1 operating software.ccurate mass measurements were obtained with an electrosprayual sprayer using the reference compound leucine enkephalin[M+H]+ = 556.2271) at a high mass resolution of 10,000.

Additional information on each compound was obtained by theomparison of retention times and absorption spectra of the peaksf the samples and the reference compounds in the HPLC analy-is. Comparison with the reference compounds and literature masspectra was utilized where possible.

. Results and discussion

.1. Antioxidative properties of the AABE

The antioxidative potential of the samples was evaluatedhrough their ability to scavenge free radicals in PBMC cells. Inhe cells, the amount of ROS (reactive oxygen species) increasesuring the oxidative stress, which may result in the damage ofhe cell structures. The intracellular ROS generation was investi-ated with a DCFH-DA reagent. The DCFH-DA is a nonfluorescentell-permeable compound that is cleaved to a highly fluorescent′,7′-dichlorofluorescein by endogenous esterases in the cell thusreventing the back-diffusion of the dye into the extracellularpace. The compound is generally used to detect and quantify ROSuch as H2O2, CO3 and NO2 (Wardman, 2007). A lower intracellularxidation resulted in a lower fluorescence of DCFH-DA reagent. TheABE samples exhibited significantly better antioxidative proper-

ies in the cell-based assay (Fig. 1) compared to the maritime pineark extract. Dry extract (d-AABE) exhibited even higher antioxi-ant activity than the extract prepared in PEG, which is mostly due

o the absence of PEG and consequently higher concentration ofntioxidative phytochemicals. Antioxidant activity of AABE mea-ured in the cell-free assay by the DPPH method was 91% higherompared to the maritime pine bark extract.

Fig. 2. Chromatogram of the AABE. All 13 identified compounds are labeled.

The wood of the silver fir is used for general construction, andthe resinous essential oil in fragrances and inhalants. The bark ofthe silver fir represents a residue at wood processing, making ita favorable output material for production of pharmacologicallyactive antioxidant extracts.

3.2. Purification of compounds

The complexity of the ABEE extract and similarity of itscompounds prevented us to achieve the single step chromato-graphic system with a resolution sufficient for an optimal LC–MSanalysis (Fig. 2). Therefore a pre-separation with a differentstationary-phase selectivity was needed. Normal-phase FLASH col-umn chromatography was found suitable for this step.

180 fractions were obtained with flash chromatography andanalyzed with the adapted HPLC methods. The separation withcombination of both methods proved to be efficient for the LC–MSanalysis. Six fractions from the flash chromatography containingaltogether 13 highest HPLC peaks have been selected for the LC–MSanalysis.

3.3. Identification of the compounds

13 compounds were identified in 6 samples (fractions), based ontheir MS fragmentation patterns, high-resolution mass, UV-spectraand retention time (Table 1). Quantification was performed forsome of the identified compounds.

3.3.1. Phenolic acidsIdentity of 6 phenolic acids was confirmed by a comparison of

their mass spectral data and absorption spectra to the databasefrom the literature (Rothwell et al., 2012). Five phenolic acidswere identified with the use of reference compounds (presentedin Table 1 with quantitative data).

3.3.2. FlavonoidsThree flavonoids were identified in the AABE. The presence of

catechin and epicatechin was identified via their fragmentationpatterns in the MS spectra and confirmed with the use of referencecompounds.

Catechin tetramethyl ether was concluded to be the most proba-ble structure of the third flavonoid on the basis of its fragmentationpattern obtained in the positive and negative ionization mode. Theions at m/z 345 [M−H]− and 347 [M+H]+ represented the molecularions, confirmed with the presence of their dimer adducts and the

loss of a neutral small molecule (OCH3), seen on every spectrum.Acetic acid adduct and two fragments with postulated structures,shown in Table 1 arose in the negative spectrum.

26 E.T. Benkovic et al. / Industrial Crops and Products 52 (2014) 23– 28

Table 1Compounds in AABE extract and MS fragments, UV spectral characteristic and content.

Compound (elementalcomposition)

Product ions in positivemode in LC–MS spectra

Product ions in negativemode in LC–MS spectra

Absorption spectrum Content inAABE

Max Min

1 Gallic acid (C7H6O5) 169 [M−H]− 270 239 0.25%125 [M COOH]−

2 Homovanillic acid(C9H10O4)

181 [M−H]− 219, 280151 [M OCH3]−

133 [M OCH3 H2O]−

123 [M C2H2O2]−

3 Protocatehuic acid (C7H6O4) 153 [M−H]− 259, 293 279 0.77%

4 p-Hydroxibenzoic acid 137 [M−H]− 253 224 0.104%93 [M COOH]−

5 Vanillic acid (C8H8O4) 260, 291 235, 280 0.106%

6 p-Coumaric acid(C9H9O3)

163 [M−H]− 224, 304 247 0.37%119 [M HCOOH]−

7 Catechin 581 [2M+H]+

291 [M+H]+

273165139123

8 Epicatechin Identical to catechin

9 Catechin tetramethyleter

691 [2M−H]− 693 [2M+H]+391 [M+HCOOH]− 347 [M+H]+345 [M−H]− 317 [M OCH3]+315 [M OCH3]−

179 [M C9H10O3]−

OCH3

OCH3

HO

165 [M C10H12O3]−

O

CH2

OCH3

OCH3

10 7-(2-Methyl-3,4-dihydroxytetrahydropyran-5-yloxy)-taxsiresinol(C25H32O10)

983 [2M−H]−

537 [M+HCOOH]−

491 [M−H]−

345 [M C6H11O4]−

O

CH3OH

HO

HO

315 [M OCH3]−

273 [M C3H6O2]−

11 Taxiresinol 327 [M H2O]− 329 [M H2O]+

315 [M OCH3]− 317 [M OCH3]+

273 [M C3H6O2]− 299 [M OCH3 H2O]+

151

137

E.T. Benkovic et al. / Industrial Crops and Products 52 (2014) 23– 28 27

Table 1 (Continued)

Compound (elementalcomposition)

Product ions in positivemode in LC–MS spectra

Product ions in negativemode in LC–MS spectra

Absorption spectrum Content inAABE

Max Min

12 Secoisolariciresinol 693 [2M OCH3]− 295 [M 2H2O CH3OH]+

407 [M+HCOOH]− 203

361 [M−H]−

O+

O

CH2

CH3 CH 3

CH3

343 [M H2O]− 163

331 [M OCH3]−

OO

CH3

CH2

137

O

CH2

OCH3

13 Laricinresinol 719 [2M−H]− 721 [2M+H]+

405 [M+HCOOH]− 691 [2M OCH3+H]+

359 [M−H]− 673 [2M OCH3 H2O]+ H]+

341 [M H2O]− 361 [M+H]+

329 [M OCH3]− 331 [M OCH3+H]+

313 [M OCH3 H2O+ H]+

287 [M C3H6O2]+

3

coici

p

.3.3. LignansFour different lignan compounds were identified. In all four

ases, the MS spectra showed the loss of neutral small fragmentsf water (H2O) and methoxy group (OCH3). The absorption max-ma (peak or shoulder) in the UV–vis spectra of all four lignin

ompounds were approximately 230 and 280 nm (Fig. 3), whichs characteristic of the lignan moiety (Yeo et al., 2004).

Compound 11 was shown to be taxiresinol (C19H22O6). Theroduct ion of [M−H]− at m/z 345 was observed in the negative

200 250 300 nm0

100 0

200 0

300 0

mAU 215

280

Fig. 3. UV–vis spectrum of taxiresinol.

mode. Two fragments with m/z 327 and 315 were due to the respec-tive losses of the hydroxyl and subsequent loss of methoxy groups.The positive spectrum did not show the product ion, but the nextfragments with proposed losses: 329 [M H2O]+; 317 [M OCH3]+;299 [M OCH3 H2O]+; 273 [M C3H6O2]+ and two fragments, typ-ical for lignan structures 151 (3-methoxy-4-hydroxybenzyliden)and 137 (3-methoxy-4-hydroxy benzyl ion) (Cuadra and Fajardo,2002; Eklund et al., 2008; Erdemoglu et al., 2004).

Compound 10 (C25H32O10) showed the product ion mass spec-tra with [M−H]− ion at m/z 491. The m/z 983 and 537 may be dueto a dimer and an acetic acid adducts, respectively, the last arisingfrom the mobile phase. The spectrum exhibited an additional sig-nal characterized by a mass decrease of 146 Da, which is indicativeof the taxiresinol, indeed eluting at a similar retention time. Ele-mental analysis proposed the loss of [M C6H11O4]− ion, thereforewe postulated the most probable interpretation as the loss of 7-(2-methyl-3,4-dihydroxytetrahidropyran-5-yloxy) moiety. The most

probable structure of the compound 10 was concluded to be 7-(2-methyl-3,4-dihydroxytetrahidropyran-5-yloxy) taxiresinol (Fig. 4).

Compound 12 was found to be secoisolariciresinol (C20H26O6).The [M+H]+ ion of the compound with m/z 363 was confirmed with

28 E.T. Benkovic et al. / Industrial Crops

OH

OH

O

OH

O

OH

CH3

O O

CH3

OH

OH

Fd

t77wof

lisr

ha2

4

siTattsbt

A

AR

R

A

B

ig. 4. The proposed structure of compound 10,7-(2-methyl-3,4-ihydroxytetrahydropyran-5-yloxy)-taxiresinol.

he fragment ion corresponding to the dimeric adduct [2M+H]+ (m/z25) on the positive and the complementary ion [2M−H]− (m/z23) on the negative spectrum. The product ion with m/z 345 aroseith the loss of one, and 327 with the loss of two water molecules

n the negative spectrum. Our explanation of further molecularragmentation pathway is shown in Table 1.

Compound 13, which is lariciresinol (C20H24O6) with a molecu-ar mass of m/z 360, gave ion products in both, positive and negativeon spectra. The fragmentation pattern in the negative spectrum isimilar to the one of secolaricinresinol. Further proposal of laricin-esinol fragmentation pathway is represented in Table 1.

The epidemiologic studies of the role of lignans in the dietave revealed beneficial cardiovascular and anticancer activities,ttributed to their antioxidative properties (Arts and Hollman,005; Chattopadhyay et al., 2003)

. Conclusions

The present study provides evidence for the AABE as a richource of at least 13 natural antioxidants which have attractedncreasing attention in the field of nutrition, health and medicine.hose findings are consistent with the results of our cell basedssay where AABE showed high antioxidant activity. The AABE isherefore recognized as a powerful antioxidative agent, useful inhe preventive treatment of various conditions, placing it side byide with other widely researched conifer extracts. Therefore weelieve Abigenol® is a potent product and that AABE deserves fur-her research.

cknowledgements

This work was partly supported by the Slovenian Researchgency (grant P4-0127) and Slovenian Technological Agency (grantIP09/20).

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