Industrial Crops and Products 52 (2014) 23 28
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Industrial Crops and Products
journa l h om epage: www.elsev ier .com
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.
The silvCentral Eurmental andin the mongated chemmonoterpetriterpenoidoleoresin, dheartwood scarce. Theactivities (Ymon mistleand anticanhave been other pheno2008). Extrspecies havmented in Pycnogenol
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: email@example.com (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
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).
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
HPLontrot delArray00 nmtioneral sitioFA) ws of Ais Exress x (2.as chatogrL/m
the fc sepin 5
5% B 5% B
min ilablth ad
ash 60 c). 50e/aceon oerck
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
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).
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).
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
Thetem csolvenDiode 1908LC Solu
Sevcompoacid, TpoundAscenttis ExpKineteC18 wchromrate 2 mI.D., 2.7A) andwas ut
Forgraphi21 (1 m(1 min(1 min133 (1
Theica gelMercktoluenSelectiF254, Morder:acid (1was apeightytubes were adiverse(2/3) fo
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.
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.
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.
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
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
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
comundsc sysis (Fary-rom
is. Siher 1is.
ompS fratentif the
he lident20 40 60 80 100
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.
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
Thethe resthe sila favoactive
13 ctheir Mand resome o
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...