Chemical composition of the silver fir (Abies alba) bark extract Abigenol and its antioxidant activity

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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

    Chemic baAbigen

    Eva Tavc SvSamo Krea Faculty of Phab Jozef Stefan Inc Blood Transfu

    a r t i c l

    Article history:Received 17 JuReceived in reAccepted 4 Oc

    Keywords:Silver r (AbieAbigenolAntioxidantsPhenolsLignansFlavonoids

    ifer ss. So inus e sh

    ne baromaof indomp

    tied (gallic, homovanillic, protocatehuic, p-hydroxybenzoic, vanillic and p-coumaric), three avonoids(catechin, epicatechin and catechin tetramethyl eter) and four lignans (taxiresinol, 7-(2-methyl-3,4-dihydroxytetrahydropyran-5-yloxy)-taxiresinol, secoisolariciresinol and laricinresinol).

    2013 Elsevier B.V. All rights reserved.

    1. Introdu

    The silvCentral Eurmental andin the mongated chemmonoterpetriterpenoidoleoresin, dheartwood scarce. Theactivities (Ymon mistleand anticanhave been other pheno2008). Extrspecies havmented in Pycnogenol

    CorresponE-mail add

    0926-6690/$ http://dx.doi.oction

    er r is one of the most common tree species in theope and therefore of an important economic, environ-

    social signicance (Ficko et al., 2011). It is widespreadtane vegetation zone. Previous studies, which investi-ical composition of silver r extracts, mostly identiednes and monoterpenoids in oleoresin and twig oil,s in needles and bark, sesquiterpenes in needles anditerpenoids and steroids in needles, and lignans inand knots. Bioassay tests on the silver r extracts are

    essential oil showed antioxidative and antibacterialang et al., 2009) and the extract of silver r and com-toe (Viscum album) mixture exhibited antiproliferativecerogenic features. However, many other r speciesrecognized as rich sources of lignans, avonoids andls with antioxidant activity (Li et al., 2011; Yang et al.,

    acts from different plant parts of some other conifere been extensively researched and were also imple-pharmaceutical use. The most studied among them is, a standardized extract of the maritime pine (Pinus

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

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

    protects from oxidative stress, improves immune, circulatory, andneurodegenerative disorders. It benecially inuences metabolicsyndrome diseases and plays an important role in chronic venousinsufciency and dermatology. The activity is attributed to themixture of avonoids, mainly procyanidins and phenolic acids(DAndrea, 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; Khknen 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 increasingmRNA 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

    see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.indcrop.2013.10.005al composition of the silver r (Abies alol and its antioxidant activity

    ar Benkovic a,, Tina Grohara, Dusan Zigonb, Urbanfta, Borut Strukelj a

    rmacy, University of Ljubljana, Askerceva cesta 7, Ljubljana SI-1000, Sloveniastitute, Department of Environmental Sciences, Jamova 39, Ljubljana SI-1000, Sloveniasion Centre of Slovenia, Slajmerjeva 6, Ljubljana SI-1000, Slovenia

    e i n f o

    ly 2013vised form 2 October 2013tober 2013

    s alba) extract

    a b s t r a c t

    Extracts from the bark of different coninteresting pharmacological activitietive extract of the maritime pine (Pand cosmetic products. Here we havextract is higher than of maritime piseparated with normal phase ash chmatography (HPLC). The structures UVvis absorption spectroscopy and c/ locate / indcrop

    ) bark extract

    ajgerc, Damjan Janes a,

    pecies are known to contain various polyphenols and possessfar the most extensive research was done on the antioxida-maritima) bark, which is widely used in food supplementsown, that antioxidant activity of silver r (Abies alba) barkrk extract in cultured cells. Components of the extract weretography and reversed phase high-performance liquid chro-ividual compounds were identied by mass spectrometry,arison to reference compounds. Six phenolic acids were iden-

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

    chaperone heat shock protein beta-1 (Poussard et al., 2012). Theblack spruce (Picea mariana) bark extract showed an adequatechemical reactivity toward different radicals, and antiproliferativeproperties (Garca-Prez et al., 2010). Polyphenol and avonoid-containing neurodegen2010).

    In searchfamily, we of the silvecommercia

    2. Materia

    2.1. Chemic

    All solvBarcelona),Germany). grade puritDeventer); triuoroace

    2.2. Bark ex

    Silver described inof ground bof water at under vacutrated aqueThe ethyl aglycol 400 ature. Fifty m(AABE) was

    For comwas also ppared as abprecipitatedof d-AABE. (SI22882).

    Maritim(F0400/Pyc

    2.3. Antioxi

    Antioxidand cell-ba

    In the was used. 1was added standard). Fond aliquotmeasured alated from tto the stand

    For in vmononuclefrom healthfusion centewere cultur1% penicillifor 30 min d-AABE andwere than i

    diacetate (DCFH-DA) for 15 min. Subsequently the cells were acti-vated with 1 g/mL PMA (phorbol 12-myristate 13-acetate). Themean uorescence intensity (MFI) value of the cell population wasmeasured with the use of a ow cytometer (FacsCalibur, Beckton

    son).sed rms

    rom

    HPLontrot delArray00 nmtioneral sitioFA) ws of Ais Exress x (2.as chatogrL/m

    m acetilized

    the fc sepin 5

    5% B 5% B

    min ilablth ad

    ash c

    ash 60 c). 50e/aceon oerck

    tolue/8/1)plied

    fract(10 mnaly

    frar LC

    ass s

    chroy ult, MA

    tativere aes wC. Thonalfoods protect against cardiovascular risk, cancer anderation (Habauzit and Morand, 2011; Vauzour et al.,

    for similar activities of other species of the Pinaceaeinvestigated the antioxidant activity and compositionr r (Abies alba) bark extract (AABE) which is beinglized under the trade name Abigenol.

    ls and methods

    als

    ents were of p.a. grade purity: acetone (Panreac; acetic acid (Merck; Germany); toluene (Riedel-de Han;The solvents used for HPLC analysis were of HPLC-y: water (Panreac; Barcelona); acetonitrile (JT Baker;formic acid (Fluka, Sigma-Aldrich Buchs; Steinheim),tic acid (Roth, Karlsruhe; Germany).

    tracts

    r bark was extracted by a two-step extraction as our patent SI23867 (Strukelj et al., 2013), briey: 5 kgark of silver r (A. alba) was rst extracted with 25 L70 C for 2 h. The aqueous extract was than evaporatedum to a volume of 5 L. In the second step the concen-ous extract was extracted with 3 L 3 L of ethyl acetate.cetate extracts were added to 25 mL of polyethylenend the ethyl acetate was then evaporated from a mix-illiliters of viscous liquid silver r (A. alba) bark extract

    obtained.parison, a dry extract from the silver r bark (d-AABE)repared, where 9 L of the ethylacetate extract (pre-ove) was concentrated to 300 mL and polyphenols were

    by the addition of 300 mL of heptane to yield 20 gThis procedure is also described in our other patent

    e pine bark dry extract was purchased from Biolandesnogenol LOT: G/1480).

    dant activity

    ant activity was measured with two methods: DPPHsed test.rst test 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent00 L of DPPH solution (3.9 mg/100 mL of methanol)to 100 L aliquot of a sample (solution of extract oror a control 100 L of methanol was added to the sec-

    of sample (100 L). After 60 min, the absorbance wast 515 nm in both solutions. The concentration was calcu-he differences of both measurements and by comparingard solution of pyrogallol (0.1 mg/mL).itro cell-based test, primary human peripheral bloodar cells (PBMCs) were isolated from human buffy coatsy donors, which were obtained from the Blood trans-r of Slovenia, following institutional guidelines. PBMCsed in an enriched media RPMI 1640 (0.5% l-glutamine,n/streptomycin in 10% FBS). They were pre-incubatedwith different concentrations (1100 g/mL) of AABE,

    maritime pine bark extract (Pycnogenol). The cellsncubated in 20 M solution of 2,7-dichlorouorescein

    Dickindecreathat fo

    2.4. Ch

    Thetem csolvenDiode 1908LC Solu

    Sevcompoacid, TpoundAscenttis ExpKineteC18 wchromrate 2 mI.D., 2.7A) andwas ut

    Forgraphi21 (1 m(1 min(1 min133 (1

    Avaand wi

    2.5. Fl

    Theica gelMercktoluenSelectiF254, Morder:acid (1was apeightytubes were adiverse(2/3) fo

    2.6. M

    TheAcquitMilfordquantition wvolumat 40

    orthog The antioxidative activity of the sample is revealed by auorescence of an oxidized form of the DCFH-DA reagentunder the inuence of free radicals.

    atographic analysis

    C system (Shimadzu Prominence) consisted of a sys-ller (CBM-20A), a column oven CPO-20AC and a

    ivery pump with a degasser (DGU-20A5) with a Photo detector (SPD-M20A) that monitored the wavelengths. The responses of the detectors were recorded using

    software version 1.24 SP1.columns with a different mobile phase gradients andns (water, acetonitrile, methanol, formic acid, aceticere tested to achieve an optimal separation of the com-ABE: Cromolith Performance Si (1004.6 mm) Merck,press C8 (10 cm 4.6 mm, 2.7 m) Supelco, Ascen-HILIC (10 cm 4.6 mm, 2.7 m) Supelco, Phenomenex6 u XB-C18 100A, 100 mm 4.6 mm). Reversed-phaseosen as an optimal HPLC stationary phase and optimalaphic conditions were: column temperature 40 C, owin, Phenomenex Kinetex C18 column (10 cm 4.6 mm

    particle size) and gradient method using water (solventonitrile (solvent B), both containing 0.1% of formic acid,: 01 min 5% B, 110 min 530% B, 1015 min 100% B.

    ractionated samples, obtained with the ash chromato-aration, six adapted HPLC gradients were developed: FR% B, 2.5 min 10% B, 10 min 10% B, 12 min 12% B), FR 8, 10 min 30% B and 1 min 5% B, 10 min 30% B,), FR 43, 2.5 min 10% B, 8 min 12% B), FR 17 (20 min 15% B), FR10% B, 4 min 50% B, 13 min 50% B).e reference compounds were analyzed both individuallydition to the samples, using the described methods.

    hromatographic separation

    chromatographic separation was performed on a sil-olumn (4 cm 12.5 cm, particle size 0.0630.200 mm,0 mg of the AABE sample, diluted in 200 L oftone/acetic acid (3/6/1) was applied on the column.f the solvents was done using TLC (Silicagel 60 plates,). A gradient elution was performed in the followingne/acetone/acetic acid (3/6/1), toluene/acetone/acetic, acetone/acetic acid (9/1). The ow rate of 10 mL/min

    with a Bchi Pump Controller C-610. One hundred andions (60 with each solvent) were collected into glassL) with a Bchi Fraction Collector C-660. All fractions

    zed with the HPLC. Six most concentrated and the mostctions were dried and dissolved in acetonitrile/waterMS analysis.

    pectrometric analysis

    matographic separation was performed on a Watersra-performance liquid chromatograph (Waters Corp.,, USA), with a column identical to that used for the

    e HPLC analysis. The methods, optimized for each frac-djusted to the ow rate of 0.5 mL/min. The injectionere 10 L. The column temperature was maintainede LC system was interfaced with a hybrid quadrupole

    acceleration time-of-ight 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

    PEG

    con

    fluor

    esce

    nce

    Fig. 1. Antioxiuorescence. Talba bark extr(AABE) and ma

    Premier, Wunder posit

    The capivoltage was100 and 20tion gas waand 1000 w4.5 103 mments wereto generatetural informa scan accuThe data stAccurate mdual spraye([M+H]+ = 5

    Additioncomparisonof the sampsis. Comparspectra was

    3. Results

    3.1. Antioxi

    The antthrough ththe cells, thduring the the cell strugated with cell-permea2,7-dichlopreventing space. The csuch as H2Ooxidation reAABE sampties in the cbark extracdant activitto the abseantioxidativsured in thcompared t

    . Chr

    wooinouver rableantio

    rica

    comundsc sysis (Fary-rom

    fraced watio

    is. Siher 1is.

    enti

    ompS fratentif the

    Phenntityass

    he lident20 40 60 80 100

    marimepine barkextractd-AABE

    AABE

    centration of extract in cell medium ( g/ml)

    dative activity of the samples on PBMC cells are revealed by decreasedhe highest antioxidative activity is achieved by precipitated dry Abiesact (d-AABE), followed by Abies alba bark extract prepared with PEGritime pine bark extract.

    aters, Milford, MA, USA). The compounds were analyzedive (ESI(+)) and negative (ESI()) ion conditions.llary voltage was set at 3.0 kV, while the sampling cone

    20 V. The source and desolvation temperatures were0 C, respectively. The ow rate of nitrogen desolva-s 600 L/h. The acquisition range was between m/z 50ith argon serving as a collision gas at a pressure ofbar in the T-wave collision cell. The MS/MS experi-

    performed using collision energies from 5 to 30 eV the product ion spectra that provided the best struc-ation. The data were collected in centroid mode, with

    mulation time of 0.2 s and an interscan delay of 0.025 s.ation utilized the MassLynx v4.1 operating software.ass measurements were obtained with an electrosprayr using the reference compound leucine enkephalin56.2271) at a high mass resolution of 10,000.al information on each compound was obtained by the

    of retention times and absorption spectra of the peaksles and the reference compounds in the HPLC analy-ison with the reference compounds and literature mass

    utilized where possible.

    and discussion

    dative properties of the AABE

    ioxidative potential of the samples was evaluatedeir ability to scavenge free radicals in PBMC cells. Ine amount of ROS (reactive oxygen species) increasesoxidative stress, which may result in the damage of

    Fig. 2

    Thethe resthe sila favoactive

    3.2. Pu

    Thecompographianalysstationumn ch

    180analyzcombinanalysaltogetanalys

    3.3. Id

    13 ctheir Mand resome o

    3.3.1. Ide

    their mfrom twere ictures. The intracellular ROS generation was investi-a DCFH-DA reagent. The DCFH-DA is a nonuorescentble compound that is cleaved to a highly uorescentrouorescein by endogenous esterases in the cell thusthe back-diffusion of the dye into the extracellularompound is generally used to detect and quantify ROS2, CO3 and NO2 (Wardman, 2007). A lower intracellularsulted in a lower uorescence of DCFH-DA reagent. Theles exhibited signicantly better antioxidative proper-ell-based assay (Fig. 1) compared to the maritime pinet. Dry extract (d-AABE) exhibited even higher antioxi-y than the extract prepared in PEG, which is mostly duence of PEG and consequently higher concentration ofe phytochemicals. Antioxidant activity of AABE mea-

    e cell-free assay by the DPPH method was 91% highero the maritime pine bark extract.

    in Table 1 w

    3.3.2. FlavoThree a

    catechin anpatterns in compounds

    Catechinble structurpattern obtions at m/z 3ions, conrloss of a neAcetic acid shown in Tomatogram of the AABE. All 13 identied compounds are labeled.

    d of the silver r is used for general construction, ands essential oil in fragrances and inhalants. The bark ofr represents a residue at wood processing, making it

    output material for production of pharmacologicallyxidant extracts.

    tion of compounds

    plexity of the ABEE extract and similarity of its prevented us to achieve the single step chromato-tem with a resolution sufcient for an optimal LCMSig. 2). Therefore a pre-separation with a differentphase selectivity was needed. Normal-phase FLASH col-atography was found suitable for this step.tions were obtained with ash chromatography andith the adapted HPLC methods. The separation withn of both methods proved to be efcient for the LCMSx fractions from the ash chromatography containing3 highest HPLC peaks have been selected for the LCMS

    cation of the compounds

    ounds were identied in 6 samples (fractions), based ongmentation patterns, high-resolution mass, UV-spectraon time (Table 1). Quantication was performed for

    identied compounds.

    olic acids of 6 phenolic acids was conrmed by a comparison ofspectral data and absorption spectra to the databaseterature (Rothwell et al., 2012). Five phenolic acidsied with the use of reference compounds (presentedith quantitative data).

    noidsvonoids were identied in the AABE. The presence of

    d epicatechin was identied via their fragmentationthe MS spectra and conrmed with the use of reference.

    tetramethyl ether was concluded to be the most proba-e of the third avonoid on the basis of its fragmentationained in the positive and negative ionization mode. The45 [MH] and 347 [M+H]+ represented the molecular

    med with the presence of their dimer adducts and theutral small molecule (OCH3), seen on every spectrum.adduct and two fragments with postulated structures,able 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 LCMS spectra

    Product ions in negativemode in LCMS spectra

    Absorption spectrum Content inAABE

    Max Min

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

    2 Homovanillic acid(C9H10O4)

    181 [MH] 219, 280151 [M OCH3]

    133 [M OCH3 H2O]

    123 [M C2H2O2]

    3 Protocatehuic acid (C7H6O4) 153 [MH] 259, 293 279 0.77%4 p-Hydroxibenzoic acid 137 [MH] 253 224 0.104%

    93 [M COOH]

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

    6 p-Coumaric acid(C9H9O3)

    163 [MH] 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 [2MH] 693 [2M+H]+391 [M+HCOOH] 347 [M+H]+345 [MH] 317 [M OCH3]+315 [M OCH3]

    179 [M C9H10O3]

    OCH3

    OCH3

    HO

    165 [M C10H12O3]

    O

    CH2O

    CH3

    OCH3

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

    983 [2MH]537 [M+HCOOH]

    491 [MH]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 LCMS spectra

    Product ions in negativemode in LCMS spectra

    Absorption spectrum Content inAABE

    Max Min

    12 Secoisolariciresinol 693 [2M OCH3] 295 [M 2H2O C407 [M+HCOOH] 203

    361 [MH]

    O+

    O

    C

    CH3 CH

    CH

    343 [M H2O] 163

    O

    H

    13

    3.3.3. LignaFour dif

    cases, the Mof water (Hima (peak compoundsis character

    Compouproduct ion331 [M OCH3]

    OCH3

    137

    CH2

    OCH3

    Laricinresinol 719 [2MH] 721 [2M+H]+405 [M+HCOOH] 691 [2M OCH3+359 [MH] 673 [2M OCH3341 [M H2O] 361 [M+H]+

    329 [M OCH3] 331 [M OCH3+H313 [M OCH3 H287 [M C3H6O2]

    nsferent lignan compounds were identied. In all fourS spectra showed the loss of neutral small fragments

    2O) and methoxy group (OCH3). The absorption max-or shoulder) in the UVvis spectra of all four lignin

    were approximately 230 and 280 nm (Fig. 3), whichistic of the lignan moiety (Yeo et al., 2004).nd 11 was shown to be taxiresinol (C19H22O6). The

    of [MH] at m/z 345 was observed in the negative

    200 250 300 nm0

    100 0

    200 0

    300 0

    mAU 215

    280

    Fig. 3. UVvis spectrum of taxiresinol.

    mode. Two tive losses oThe positivfragments w299 [M OCical for lignand 137 (32002; Eklun

    Compoutra with [Mto a dimer afrom the mnal characteof the taxirmental anawe postulatmethyl-3,4probable stmethyl-3,4

    CompouThe [M+H]+H3OH]+

    H2

    3

    3

    C2O

    H]+

    H2O]+ H]+

    ]+

    2O+ H]++

    fragments with m/z 327 and 315 were due to the respec-f the hydroxyl and subsequent loss of methoxy groups.e spectrum did not show the product ion, but the nextith proposed losses: 329 [M H2O]+; 317 [M OCH3]+;

    H3 H2O]+; 273 [M C3H6O2]+ and two fragments, typ-an structures 151 (3-methoxy-4-hydroxybenzyliden)

    -methoxy-4-hydroxy benzyl ion) (Cuadra and Fajardo,d et al., 2008; Erdemoglu et al., 2004).nd 10 (C25H32O10) showed the product ion mass spec-H] ion at m/z 491. The m/z 983 and 537 may be duend an acetic acid adducts, respectively, the last arisingobile phase. The spectrum exhibited an additional sig-rized by a mass decrease of 146 Da, which is indicativeesinol, indeed eluting at a similar retention time. Ele-lysis proposed the loss of [M C6H11O4] ion, thereforeed the most probable interpretation as the loss of 7-(2--dihydroxytetrahidropyran-5-yloxy) moiety. The mostructure of the compound 10 was concluded to be 7-(2--dihydroxytetrahidropyran-5-yloxy) taxiresinol (Fig. 4).nd 12 was found to be secoisolariciresinol (C20H26O6).ion of the compound with m/z 363 was conrmed with

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

    OH

    O

    OH

    O

    OH

    CH3

    O O

    CH3OH

    Fig. 4. The dihydroxytetr

    the fragmen725) on the723) on thewith the loson the negfragmentat

    Compoular mass of mion spectra.similar to thresinol frag

    The epidhave revealattributed 2005; Chatt

    4. Conclus

    The presource of aincreasing aThose ndiassay whertherefore rethe preventside with obelieve Abither researc

    Acknowled

    This woAgency (graRIP09/20).

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    acid153.

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    RadicOH OH

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

    t ion corresponding to the dimeric adduct [2M+H]+ (m/z positive and the complementary ion [2MH] (m/z

    negative spectrum. The product ion with m/z 345 aroses of one, and 327 with the loss of two water moleculesative spectrum. Our explanation of further molecularion pathway is shown in Table 1.nd 13, which is lariciresinol (C20H24O6) with a molecu-/z 360, gave ion products in both, positive and negative

    The fragmentation pattern in the negative spectrum ise one of secolaricinresinol. Further proposal of laricin-

    mentation pathway is represented in Table 1.emiologic studies of the role of lignans in the dieted benecial cardiovascular and anticancer activities,to their antioxidative properties (Arts and Hollman,opadhyay et al., 2003)

    ions

    sent study provides evidence for the AABE as a richt least 13 natural antioxidants which have attractedttention in the eld of nutrition, health and medicine.ngs are consistent with the results of our cell basede AABE showed high antioxidant activity. The AABE iscognized as a powerful antioxidative agent, useful inive treatment of various conditions, placing it side byther widely researched conifer extracts. Therefore wegenol is a potent product and that AABE deserves fur-h.

    gements

    rk was partly supported by the Slovenian Researchnt P4-0127) and Slovenian Technological Agency (grant

    ManFradinho

    de Jextr233

    Garca-PPoulof CEthn

    HabauzicontChro

    KhkneHeincom

    Karonenin pchroActa

    Kofujitabark33 (

    Li, Y.-L.,H.Wneph(12)

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    Chemical composition of the silver fir (Abies alba) bark extract Abigenol and its antioxidant activity1 Introduction2 Materials and methods2.1 Chemicals2.2 Bark extracts2.3 Antioxidant activity2.4 Chromatographic analysis2.5 Flash chromatographic separation2.6 Mass spectrometric analysis

    3 Results and discussion3.1 Antioxidative properties of the AABE3.2 Purification of compounds3.3 Identification of the compounds3.3.1 Phenolic acids3.3.2 Flavonoids3.3.3 Lignans

    4 ConclusionsAcknowledgementsReferences

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