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 ba…


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 ba Abigen Eva Tavcˇ Sˇv Samo Kre a Faculty of Pha b Jozˇef Stefan In c Blood Transfu a r t i c l Article history: Received 17 Ju Received in re Accepted 4 Oc Keywords: Silver fir (Abie Abigenol Antioxidants Phenols Lignans Flavonoids ifer s s. So inus e sh ne ba roma of ind omp 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). © 2013 Elsevier B.V. All rights reserved. 1. Introdu The silv Central Eur mental and in the mon gated chem monoterpe triterpenoid oleoresin, d heartwood scarce. The activities (Y mon mistle and antican have been other pheno 2008). Extr species hav mented in Pycnogenol ∗ Correspon E-mail add 0926-6690/$ – http://dx.doi.o ction er fir is one of the most common tree species in the ope and therefore of an important economic, environ- social significance (Ficko et al., 2011). It is widespread tane vegetation zone. Previous studies, which investi- ical composition of silver fir extracts, mostly identified nes and monoterpenoids in oleoresin and twig oil, s in needles and bark, sesquiterpenes in needles and iterpenoids and steroids in needles, and lignans in and knots. Bioassay tests on the silver fir extracts are essential oil showed antioxidative and antibacterial ang et al., 2009) and the extract of silver fir and com- toe (Viscum album) mixture exhibited antiproliferative cerogenic features. However, many other fir species recognized as rich sources of lignans, flavonoids and ls with antioxidant activity (Li et al., 2011; Yang et al., acts from different plant parts of some other conifer e 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: (E.T. Benkovic´). maritima) bark, widely used in food supplements and cosmetic products. Pycnogenol® has been reported to have a strong free radical–scavenging activity against the reactive oxygen and nitro- gen species. Exhibiting an anti-inflammatory activity, Pycnogenol® protects from oxidative stress, improves immune, circulatory, and neurodegenerative disorders. It beneficially influences metabolic syndrome diseases and plays an important role in chronic venous insufficiency and dermatology. The activity is attributed to the mixture of flavonoids, mainly procyanidins and phenolic acids (D’Andrea, 2010). Apart from the maritime pine bark, many authors have reported on high phenolic content of the bark extracts of some other pine species (Fradinho et al., 2002; Kähkönen et al., 1999; Kofujita et al., 1999). The Scots pine (Pinus sylvestris) bark was found to contain procyanidins ranging from monomers through decamers and higher polymers (Karonen et al., 2004). Phenolic acids, cate- chin, epicatechin, procyanidin B2, taxifolin, quercetin, syringic and homovanillic acids were found in the Monterey Pine (Pinus radi- ata) bark extracts, exhibiting antioxidant properties (Bocalandro et al., 2012). Flavangenol® is a maritime pine bark extract, avail- able on the market, with a protective effect against oxidative stress associated 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 bark extract Oligopin® is rich in procyanidins and catechins and was shown to modulate stress-induced phosphorylation of the stress see front matter © 2013 Elsevier B.V. All rights reserved. rg/10.1016/j.indcrop.2013.10.005 al composition of the silver fir (Abies al ol® and its antioxidant activity ar Benkovic´ a,∗, Tina Grohara, Dusˇan Zˇigonb, Urban fta, Borut Sˇtrukelj a rmacy, University of Ljubljana, Asˇkercˇeva cesta 7, Ljubljana SI-1000, Slovenia stitute, Department of Environmental Sciences, Jamova 39, Ljubljana SI-1000, Slovenia sion Centre of Slovenia, Sˇlajmerjeva 6, Ljubljana SI-1000, Slovenia e i n f o ly 2013 vised form 2 October 2013 tober 2013 s alba) extract a b s t r a c t Extracts from the bark of different con interesting pharmacological activitie tive extract of the maritime pine (P and cosmetic products. Here we hav extract is higher than of maritime pi separated with normal phase flash ch matography (HPLC). The structures UV–vis absorption spectroscopy and c / locate / indcrop ) bark extract ajgerc, Damjan Janesˇ a, pecies are known to contain various polyphenols and possess far the most extensive research was done on the antioxida- maritima) bark, which is widely used in food supplements own, that antioxidant activity of silver fir (Abies alba) bark rk extract in cultured cells. Components of the extract were tography and reversed phase high-performance liquid chro- ividual compounds were identified 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). The black spruce (Picea mariana) bark extract showed an adequate chemical reactivity toward different radicals, and antiproliferative properties (García-Pérez et al., 2010). Polyphenol and flavonoid- containing neurodegen 2010). In search family, we of the silve commercia 2. Materia 2.1. Chemic All solv Barcelona), Germany). grade purit Deventer); trifluoroace 2.2. Bark ex Silver fi described in of ground b of water at under vacu trated aque The ethyl a glycol 400 a ture. Fifty m (AABE) was For com was also p pared as ab precipitated of d-AABE. (SI22882). Maritim (F0400/Pyc 2.3. Antioxi Antioxid and cell-ba In the fi was used. 1 was added standard). F ond aliquot measured a lated from t to the stand For in v mononucle from health fusion cente were cultur 1% penicilli for 30 min d-AABE and were than i diacetate (DCFH-DA) for 15 min. Subsequently the cells were acti- vated with 1 �g/mL PMA (phorbol 12-myristate 13-acetate). The mean fluorescence intensity (MFI) value of the cell population was measured with the use of a flow cytometer (FacsCalibur, Beckton son). sed fl rms rom HPL ontro t del Array 00 nm tion eral sitio FA) w s of A is Ex ress x (2. as ch atogr L/m �m acet ilized the f c sep in 5 5% B 5% B min ilabl th ad ash c flash 60 c ). 50 e/ace on o erck tolue /8/1) plied fract (10 m naly fra r LC ass s chro y ult , MA tativ ere a es w C. Th onal foods protect against cardiovascular risk, cancer and eration (Habauzit and Morand, 2011; Vauzour et al., for similar activities of other species of the Pinaceae investigated the antioxidant activity and composition r fir (Abies alba) bark extract (AABE) which is being lized 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 Haën; 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 (Sˇtrukelj et al., 2013), briefly: 5 kg ark of silver fir (A. alba) was first extracted with 25 L 70 ◦C for 2 h. The aqueous extract was than evaporated um 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 polyethylene nd the ethyl acetate was then evaporated from a mix- illiliters of viscous liquid silver fir (A. alba) bark extract obtained. parison, a dry extract from the silver fir 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 g This procedure is also described in our other patent e pine bark dry extract was purchased from Biolandes nogenol® LOT: G/1480). dant activity ant activity was measured with two methods: DPPH sed test. rst test 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent 00 �L of DPPH solution (3.9 mg/100 mL of methanol) to 100 �L aliquot of a sample (solution of extract or or a control 100 �L of methanol was added to the sec- of sample (100 �L). After 60 min, the absorbance was t 515 nm in both solutions. The concentration was calcu- he differences of both measurements and by comparing ard solution of pyrogallol (0.1 mg/mL). itro cell-based test, primary human peripheral blood ar cells (PBMCs) were isolated from human buffy coats y donors, which were obtained from the Blood trans- r of Slovenia, following institutional guidelines. PBMCs ed in an enriched media RPMI 1640 (0.5% l-glutamine, n/streptomycin in 10% FBS). They were pre-incubated with different concentrations (1–100 �g/mL) of AABE, maritime pine bark extract (Pycnogenol). The cells ncubated in 20 �M solution of 2′,7′-dichlorofluorescein Dickin decrea that fo 2.4. Ch The tem c solven Diode 190–8 LC Solu Sev compo acid, T pound Ascent tis Exp Kinete C18 w chrom rate 2 m I.D., 2.7 A) and was ut For graphi 21 (1 m (1 min (1 min 133 (1 Ava and wi 2.5. Fl The ica gel Merck toluen Selecti F254, M order: acid (1 was ap eighty tubes were a diverse (2/3) fo 2.6. M The Acquit Milford quanti tion w volum at 40 ◦ orthog The antioxidative activity of the sample is revealed by a uorescence of an oxidized form of the DCFH-DA reagent under the influence 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 and ns (water, acetonitrile, methanol, formic acid, acetic ere tested to achieve an optimal separation of the com- ABE: Cromolith Performance – Si (100–4.6 mm) Merck, press C8 (10 cm × 4.6 mm, 2.7 �m) Supelco, Ascen- HILIC (10 cm × 4.6 mm, 2.7 �m) Supelco, Phenomenex 6 u XB-C18 100A, 100 mm × 4.6 mm). Reversed-phase osen as an optimal HPLC stationary phase and optimal aphic conditions were: column temperature 40 ◦C, flow in, Phenomenex Kinetex® C18 column (10 cm × 4.6 mm particle size) and gradient method using water (solvent onitrile (solvent B), both containing 0.1% of formic acid, : 0–1 min 5% B, 1–10 min 5–30% B, 10–15 min 100% B. ractionated samples, obtained with the flash 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), FR 10% B, 4 min 50% B, 13 min 50% B). e reference compounds were analyzed both individually dition to the samples, using the described methods. hromatographic separation chromatographic separation was performed on a sil- olumn (4 cm × 12.5 cm, particle size 0.063–0.200 mm, 0 mg of the AABE sample, diluted in 200 �L of tone/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 following ne/acetone/acetic acid (3/6/1), toluene/acetone/acetic , acetone/acetic acid (9/1). The flow rate of 10 mL/min with a Büchi Pump Controller C-610. One hundred and ions (60 with each solvent) were collected into glass L) with a Büchi Fraction Collector C-660. All fractions zed with the HPLC. Six most concentrated and the most ctions were dried and dissolved in acetonitrile/water –MS analysis. pectrometric analysis matographic separation was performed on a Waters ra-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 flow rate of 0.5 mL/min. The injection ere 10 �L. The column temperature was maintained e LC system was interfaced with a hybrid quadrupole 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 PEG con flu or es ce nc e Fig. 1. Antioxi fluorescence. T alba bark extr (AABE) and ma Premier, W under posit The capi voltage was 100 and 20 tion gas wa and 1000 w 4.5 × 10−3 m ments were to generate tural inform a scan accu The data st Accurate m dual spraye ([M+H]+ = 5 Addition comparison of the samp sis. Compar spectra was 3. Results 3.1. Antioxi The ant through th the cells, th during the the cell stru gated with cell-permea 2′,7′-dichlo preventing space. The c such as H2O oxidation re AABE samp ties in the c bark extrac dant activit to the abse antioxidativ sured in th compared t . Chr woo inou ver fi rable antio rifica com unds c sys is (F ary- rom frac ed w atio is. Si her 1 is. entifi omp S fra tenti f the Phen ntity ass he li dent 20 40 60 80 100 mari�me pine bark extract d-AABE AABE centration of extract in cell medium ( g/ml)μ dative activity of the samples on PBMC cells are revealed by decreased he highest antioxidative activity is achieved by precipitated dry Abies act (d-AABE), followed by Abies alba bark extract prepared with PEG ritime pine bark extract. aters, Milford, MA, USA). The compounds were analyzed ive (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 were 0 ◦C, respectively. The flow rate of nitrogen desolva- s 600 L/h. The acquisition range was between m/z 50 ith argon serving as a collision gas at a pressure of bar 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 electrospray r using the reference compound leucine enkephalin 56.2271) 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 peaks les 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 evaluated eir ability to scavenge free radicals in PBMC cells. In e amount of ROS (reactive oxygen species) increases oxidative stress, which may result in the damage of Fig. 2 The the res the sil a favo active 3.2. Pu The compo graphi analys station umn ch 180 analyz combin analys altoget analys 3.3. Id 13 c their M and re some o 3.3.1. Ide their m from t were i ctures. The intracellular ROS generation was investi- a DCFH-DA reagent. The DCFH-DA is a nonfluorescent ble compound that is cleaved to a highly fluorescent rofluorescein by endogenous esterases in the cell thus the back-diffusion of the dye into the extracellular ompound is generally used to detect and quantify ROS 2, CO3 and NO2 (Wardman, 2007). A lower intracellular sulted in a lower fluorescence of DCFH-DA reagent. The les exhibited significantly better antioxidative proper- ell-based assay (Fig. 1) compared to the maritime pine t. Dry extract (d-AABE) exhibited even higher antioxi- y than the extract prepared in PEG, which is mostly due nce of PEG and consequently higher concentration of e phytochemicals. Antioxidant activity of AABE mea- e cell-free assay by the DPPH method was 91% higher o the maritime pine bark extract. in Table 1 w 3.3.2. Flavo Three fla catechin an patterns in compounds Catechin ble structur pattern obt ions at m/z 3 ions, confir loss of a ne Acetic acid shown in T omatogram of the AABE. All 13 identified compounds are labeled. d of the silver fir is used for general construction, and s essential oil in fragrances and inhalants. The bark of r represents a residue at wood processing, making it output material for production of pharmacologically xidant 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 sufficient for an optimal LC–MS ig. 2). Therefore a pre-separation with a different phase selectivity was needed. Normal-phase FLASH col- atography was found suitable for this step. tions were obtained with flash chromatography and ith the adapted HPLC methods. The separation with n of both methods proved to be efficient for the LC–MS x fractions from the flash chromatography containing 3 highest HPLC peaks have been selected for the LC–MS cation of the compounds ounds were identified in 6 samples (fractions), based on gmentation patterns, high-resolution mass, UV-spectra on time (Table 1). Quantification was performed for identified compounds. olic acids of 6 phenolic acids was confirmed by a comparison of spectral data and absorption spectra to the database terature (Rothwell et al., 2012). Five phenolic acids ified with the use of reference compounds (presented ith quantitative data). noids vonoids were identified in the AABE. The presence of d epicatechin was identified via their fragmentation the MS spectra and confirmed with the use of reference . tetramethyl ether was concluded to be the most proba- e of the third flavonoid on the basis of its fragmentation ained in the positive and negative ionization mode. The 45 [M−H]− and 347 [M+H]+ represented the molecular med with the presence of their dimer adducts and the utral 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 1 Compounds in AABE extract and MS fragments, UV spectral characteristic and content. Compound (elemental composition) Product ions in positive mode in LC–MS spectra Product ions in negative mode in LC–MS spectra Absorption spectrum Content in AABE Max Min 1 Gallic acid (C7H6O5) 169 [M−H]− 270 239 0.25% 125 [M COOH]− 2 Homovanillic acid (C9H10O4) 181 [M−H]− 219, 280 151 [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]+ 273 165 139 123 8 Epicatechin Identical to catechin 9 Catechin tetramethyl eter 691 [2M−H]− 693 [2M+H]+ 391 [M+HCOOH]− 347 [M+H]+ 345 [M−H]− 317 [M OCH3]+ 315 [M OCH3]− 179 [M C9H10O3]− O CH3 O CH3 HO 165 [M C10H12O3]− O CH2 O CH3 O CH3 10 7-(2-Methyl-3,4- dihydroxytetrahydropyran- 5-yloxy)-taxsiresinol (C25H32O10) 983 [2M−H]− 537 [M+HCOOH]− 491 [M−H]− 345 [M C6H11O4]− O CH3 OH 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 (elemental composition) Product ions in positive mode in LC–MS spectra Product ions in negative mode in LC–MS spectra Absorption spectrum Content in AABE Max Min 12 Secoisolariciresinol 693 [2M OCH3]− 295 [M 2H2O C 407 [M+HCOOH]− 203 361 [M−H]− O+ O C CH3 CH CH 343 [M H2O]− 163 O H 13 3.3.3. Ligna Four dif cases, the M of water (H ima (peak compounds is character Compou product ion 331 [M OCH3]− O CH3 137 CH2 OCH3 Laricinresinol 719 [2M−H]− 721 [2M+H]+ 405 [M+HCOOH]− 691 [2M OCH3+ 359 [M−H]− 673 [2M OCH3 341 [M H2O]− 361 [M+H]+ 329 [M OCH3]− 331 [M OCH3+H 313 [M OCH3 H 287 [M C3H6O2] ns ferent lignan compounds were identified. In all four S spectra showed the loss of neutral small fragments 2O) and methoxy group (OCH3). The absorption max- or shoulder) in the UV–vis spectra of all four lignin were approximately 230 and 280 nm (Fig. 3), which istic of the lignan moiety (Yeo et al., 2004). nd 11 was shown to be taxiresinol (C19H22O6). The of [M−H]− at m/z 345 was observed in the negative 200 250 300 nm 0 100 0 200 0 300 0 mAU 215 280 Fig. 3. UV–vis spectrum of taxiresinol. mode. Two tive losses o The positiv fragments w 299 [M OC ical for lign and 137 (3 2002; Eklun Compou tra with [M to a dimer a from the m nal characte of the taxir mental ana we postulat methyl-3,4 probable st methyl-3,4 Compou The [M+H]+ H3OH]+ H2 3 3 C2 O 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 next ith 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 due nd an acetic acid adducts, respectively, the last arising obile phase. The spectrum exhibited an additional sig- rized by a mass decrease of 146 Da, which is indicative esinol, indeed eluting at a similar retention time. Ele- lysis proposed the loss of [M C6H11O4]− ion, therefore ed the most probable interpretation as the loss of 7-(2- -dihydroxytetrahidropyran-5-yloxy) moiety. The most ructure 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 confirmed 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 fragmen 725) on the 723) on the with the los on the neg fragmentat Compou lar mass of m ion spectra. similar to th resinol frag The epid have reveal attributed 2005; Chatt 4. Conclus The pre source of a increasing a Those findi assay wher therefore re the prevent side with o believe Abi ther researc Acknowled This wo Agency (gra RIP09/20). References Arts, I.C.W., H studies. Am Bocalandro, C. K., Roecke Pinus radia 38 (1), 21– Chattopadhyay, S.K., Kumar, T.R.S., Maulik, P.R., Srivastava, S., Garg, A., Sharon, A., Negi, A.S., Khanuja, S.P., 2003. Absolute configuration and anticancer activity of taxiresinol and related lignans of Taxus wallichiana. Bioorg. Med. Chem. 11 (23), 4945–4948. Cuadra, P., Fajardo, V., 2002. A new lignan from the Patagonian Valeriana Carnosa Sm. Boletín de la Sociedad Chilena de Química 47 (4), 361–366. D’Andrea, G., 2010. Pycnogenol: a blend of procyanidins with multifaceted thera- peutic applications? Fitoterapia 81 (7), 724–736. Eklund, P.C., Backman, M.J., Kronberg, L.A., Smeds, A.I., Sjöholm, R.E., 2008. Identi- fication of lignans by liquid chromatography-electrospray ionization ion-trap mass spectrometry. J. Mass Spectrom. 43 (1), 97–107. 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 (1–3), 57–62. Ficko, A., Poljanec, A., Boncina, A., 2011. Do changes in spatial distribution, structure and abundance of silver fir (Abies alba Mill.) indicate its decline? Forest Ecol. age. 2 , D.M esusa acts f 2. érez, iot, R anadi ophar t, V., aining nic D n, M onen pound , M., ine ba matog 522 ( , H., E from 3), 223 Wu, ., Che rolepi , 2299 M., O bitory arker ), 175 , S., P ntiox B1 in h ://dx.d l, J.A., , J., Ne ol-Ex on po tal an , T., amine enol ( acid 153. , B., Kr . Tek ovo p RS za , D., R . Poly tion. n, P., and n Radic OH 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 [2M−H]− (m/z negative spectrum. The product ion with m/z 345 arose s of one, and 327 with the loss of two water molecules ative spectrum. Our explanation of further molecular ion 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 is e one of secolaricinresinol. Further proposal of laricin- mentation pathway is represented in Table 1. emiologic studies of the role of lignans in the diet ed beneficial 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 rich t least 13 natural antioxidants which have attracted ttention in the field of nutrition, health and medicine. ngs are consistent with the results of our cell based e AABE showed high antioxidant activity. The AABE is cognized as a powerful antioxidative agent, useful in ive treatment of various conditions, placing it side by ther widely researched conifer extracts. Therefore we genol® is a potent product and that AABE deserves fur- h. gements rk was partly supported by the Slovenian Research nt P4-0127) and Slovenian Technological Agency (grant Man Fradinho de J extr 23–3 García-P Poul of C Ethn Habauzi cont Chro Kähköne Hein com Karonen in p chro Acta Kofujita bark 33 ( Li, Y.-L., H.W neph (12) Nakano, Inhi biom (4–5 Poussard ral a HSP http Rothwel Cruz Phen data men Shimada Nag vang fatty 147– Sˇtrukelj 2013 njeg Urad Vauzour 2010 of ac Wardma tive Free ollman, P.C.H., 2005. Polyphenols and disease risk in epidemiologic . J. Clin. Nutr. 81 (1 Suppl.), 317S–325S. , Sanhueza, V., Gómez-Caravaca, A.M., González-Álvarez, J., Fernández, l, M., Rodríguez-Estrada, M.T., 2012. Comparison of the composition of ta bark extracts obtained at bench- and pilot-scales. Ind. Crops Prod. 26. 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Chemical composition of the silver fir (Abies alba) bark extract Abigenol® and its antioxidant activity 1 Introduction 2 Materials and methods 2.1 Chemicals 2.2 Bark extracts 2.3 Antioxidant activity 2.4 Chromatographic analysis 2.5 Flash chromatographic separation 2.6 Mass spectrometric analysis 3 Results and discussion 3.1 Antioxidative properties of the AABE 3.2 Purification of compounds 3.3 Identification of the compounds 3.3.1 Phenolic acids 3.3.2 Flavonoids 3.3.3 Lignans 4 Conclusions Acknowledgements References


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