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 28Contents lists available at ScienceDirectIndustrial Crops and Productsjourna l h om epage: www.elsev ier .comChemic baAbigenEva Tavc SvSamo Krea Faculty of Phab Jozef Stefan Inc Blood Transfua r t i c lArticle history:Received 17 JuReceived in reAccepted 4 OcKeywords:Silver r (AbieAbigenolAntioxidantsPhenolsLignansFlavonoidsifer ss. So inus e shne baromaof indomptied (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. IntroduThe silvCentral Eurmental andin the mongated chemmonoterpetriterpenoidoleoresin, dheartwood scarce. Theactivities (Ymon mistleand anticanhave been other pheno2008). Extrspecies havmented in Pycnogenol CorresponE-mail add0926-6690/$ http://dx.doi.octioner 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 (Pinusding 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, Pycnogenolprotects 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 activityar Benkovic a,, Tina Grohara, Dusan Zigonb, Urbanfta, Borut Strukelj armacy, 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 oly 2013vised form 2 October 2013tober 2013s alba) extracta b s t r a c tExtracts 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 extractajgerc, 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 28chaperone 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 silvecommercia2. Materia2.1. ChemicAll solvBarcelona),Germany). grade puritDeventer); triuoroace2.2. Bark exSilver described inof ground bof water at under vacutrated aqueThe ethyl aglycol 400 ature. Fifty m(AABE) wasFor comwas also ppared as abprecipitatedof d-AABE. (SI22882).Maritim(F0400/Pyc2.3. AntioxiAntioxidand cell-baIn the was used. 1was added standard). Fond aliquotmeasured alated from tto the standFor in vmononuclefrom healthfusion centewere cultur1% penicillifor 30 min d-AABE andwere than idiacetate (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, Becktonson).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 adash c ash 60 c). 50e/aceon oerck tolue/8/1)plied fract(10 mnaly frar LCass s chroy ult, MAtativere 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 methodsalsents 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).tractsr 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 patente pine bark dry extract was purchased from Biolandesnogenol LOT: G/1480).dant activityant 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-dichlorouoresceinDickindecreathat fo2.4. ChThetem csolvenDiode 1908LC SoluSevcompoacid, TpoundAscenttis ExpKineteC18 wchromrate 2 mI.D., 2.7A) andwas utForgraphi21 (1 m(1 min(1 min133 (1Avaand wi2.5. FlTheica gelMercktoluenSelectiF254, Morder:acid (1was apeightytubes were adiverse(2/3) fo2.6. MTheAcquitMilfordquantition 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 analysisC system (Shimadzu Prominence) consisted of a sys-ller (CBM-20A), a column oven CPO-20AC and aivery 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 fractionszed with the HPLC. Six most concentrated and the mostctions were dried and dissolved in acetonitrile/waterMS analysis.pectrometric analysismatographic separation was performed on a Watersra-performance liquid chromatograph (Waters Corp.,, USA), with a column identical to that used for thee 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-ToFE.T. Benkovic et al. / Industrial Crops and Products 52 (2014) 23 28 25751001251501752000PEGconfluorescenceFig. 1. Antioxiuorescence. Talba bark extr(AABE) and maPremier, Wunder positThe capivoltage was100 and 20tion gas waand 1000 w4.5 103 mments wereto generatetural informa scan accuThe data stAccurate mdual spraye([M+H]+ = 5Additioncomparisonof the sampsis. Comparspectra was3. Results 3.1. AntioxiThe 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 rableantiorica comundsc sysis (Fary-rom fraced watiois. Siher 1is.entiompS fratentif thePhenntityass he lident20 40 60 80 100marimepine barkextractd-AABEAABEcentration 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, withmulation 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 discussiondative properties of the AABEioxidative 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 ofFig. 2Thethe resthe sila favoactive 3.2. PuThecompographianalysstationumn ch180analyzcombinanalysaltogetanalys3.3. Id13 ctheir Mand resome o3.3.1. Idetheir 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 w3.3.2. FlavoThree acatechin anpatterns in compoundsCatechinble 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 compoundsplexity 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 LCMScation of the compoundsounds 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 ofd 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 molecularmed 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 28Table 1Compounds in AABE extract and MS fragments, UV spectral characteristic and content.Compound (elementalcomposition)Product ions in positivemode in LCMS spectraProduct ions in negativemode in LCMS spectraAbsorption spectrum Content inAABEMax Min1 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]+2731651391238 Epicatechin Identical to catechin9 Catechin tetramethyleter691 [2MH] 693 [2M+H]+391 [M+HCOOH] 347 [M+H]+345 [MH] 317 [M OCH3]+315 [M OCH3]179 [M C9H10O3]OCH3OCH3HO165 [M C10H12O3]OCH2OCH3OCH310 7-(2-Methyl-3,4-dihydroxytetrahydropyran-5-yloxy)-taxsiresinol(C25H32O10)983 [2MH]537 [M+HCOOH]491 [MH]345 [M C6H11O4]OCH3OHHOHO315 [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]+151137E.T. Benkovic et al. / Industrial Crops and Products 52 (2014) 23 28 27Table 1 (Continued)Compound (elementalcomposition)Product ions in positivemode in LCMS spectraProduct ions in negativemode in LCMS spectraAbsorption spectrum Content inAABEMax Min12 Secoisolariciresinol 693 [2M OCH3] 295 [M 2H2O C407 [M+HCOOH] 203361 [MH]O+OCCH3 CH CH343 [M H2O] 163OH133.3.3. LignaFour difcases, the Mof water (Hima (peak compoundsis characterCompouproduct ion331 [M OCH3]OCH3137CH2OCH3Laricinresinol 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 fragments2O) 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 negative200 250 300 nm0100 0200 0300 0mAU 215280Fig. 3. UVvis spectrum of taxiresinol.mode. Two tive losses oThe positivfragments w299 [M OCical for lignand 137 (32002; EklunCompoutra with [Mto a dimer afrom the mnal characteof the taxirmental anawe postulatmethyl-3,4probable stmethyl-3,4CompouThe [M+H]+H3OH]+H233C2OH]+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 with28 E.T. Benkovic et al. / Industrial Crops and Products 52 (2014) 23 28OHOOHOOHCH3O OCH3OHFig. 4. The dihydroxytetrthe fragmen725) on the723) on thewith the loson the negfragmentatCompoular mass of mion spectra.similar to thresinol fragThe epidhave revealattributed 2005; Chatt4. ConclusThe presource of aincreasing aThose ndiassay whertherefore rethe preventside with obelieve Abither researcAcknowledThis woAgency (graRIP09/20).ReferencesArts, I.C.W., Hstudies. AmBocalandro, C.K., RoeckePinus radia38 (1), 21Chattopadhyay, S.K., Kumar, T.R.S., Maulik, P.R., Srivastava, S., Garg, A., Sharon, A.,Negi, A.S., Khanuja, S.P., 2003. Absolute conguration and anticancer activity oftaxiresinol and related lignans of Taxus wallichiana. Bioorg. Med. Chem. 11 (23),49454948.Cuadra, P., Fajardo, V., 2002. A new lignan from the Patagonian Valeriana CarnosaSm. Boletn de la Sociedad Chilena de Qumica 47 (4), 361366.DAndrea, G., 2010. Pycnogenol: a blend of procyanidins with multifaceted thera-peutic applications? Fitoterapia 81 (7), 724736.Eklund, P.C., Backman, M.J., Kronberg, L.A., Smeds, A.I., Sjholm, R.E., 2008. Identi-cation of lignans by liquid chromatography-electrospray ionization ion-trapmass spectrometry. J. Mass Spectrom. 43 (1), 97107.Erdemoglu, N., Sahin, E., Sener, B., Ide, S., 2004. Structural and spectroscopic char-acteristics of two lignans from Taxus baccata L. J. Mol. Struct. 692 (13), 5762.Ficko, A., Poljanec, A., Boncina, A., 2011. Do changes in spatial distribution, structureand abundance of silver r (Abies alba Mill.) indicate its decline? Forest Ecol.age. 2, D.Mesusaacts f2.rez, iot, Ranadiophart, V., ainingnic Dn, Monenpound, M., ine bamatog 522 (, H., E from3), 223 Wu, ., Cherolepi, 2299 M., Obitoryarker), 175, S., PntioxB1 in h://dx.dl, J.A.,, J., Neol-Ex on potal an, T., amineenol ( acid153., B., Kr. Tekovo p RS za, D., R. Polytion. n, P., and n RadicOH OHproposed 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)ionssent 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.gementsrk was partly supported by the Slovenian Researchnt P4-0127) and Slovenian Technological Agency (grantManFradinhode Jextr233Garca-PPoulof CEthnHabauzicontChroKhkneHeincomKaronenin pchroActaKofujitabark33 (Li, Y.-L.,H.Wneph(12)Nakano,Inhibiom(45Poussardral aHSPhttpRothwelCruzPhendatamenShimadaNagvangfatty147Strukelj2013njegUradVauzour2010of acWardmative Freeollman, P.C.H., 2005. Polyphenols and disease risk in epidemiologic. J. Clin. Nutr. 81 (1 Suppl.), 317S325S., Sanhueza, V., Gmez-Caravaca, A.M., Gonzlez-lvarez, J., Fernndez,l, M., Rodrguez-Estrada, M.T., 2012. Comparison of the composition ofta bark extracts obtained at bench- and pilot-scales. Ind. Crops Prod.26.Yang, S.-A., JeoscavenginNutr. 44 (3Yang, X.-W., Lstudies of Yeo, H., Chin, Pharm. Re61 (4), 844854.., Neto, C.P., Evtuguin, D., Jorge, F.C., Irle, M.A., Gil, M.H., Pedrosa, J., 2002. 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Arch.s. 27 (3), 287290.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 analysis3 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 Lignans4 ConclusionsAcknowledgementsReferences

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