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-
of the silve
2.2. Bark ex
of ground b
of water at
The ethyl a
glycol 400 a
ture. Fifty m
was also p
pared as ab
In the ﬁ
was used. 1
lated from t
to the stand
For in v
for 30 min
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
s of A
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
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).
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
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).
ant activity was measured with two methods: DPPH
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
rate 2 m
21 (1 m
at 40 ◦
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.
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.
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
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
Fig. 1. Antioxi
alba bark extr
(AABE) and ma
100 and 20
tion gas wa
and 1000 w
4.5 × 10−3 m
a scan accu
The data st
([M+H]+ = 5
of the samp
the cells, th
the cell stru
space. The c
such as H2O
ties in the c
to the abse
sured in th
20 40 60 80 100
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.
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
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
ions at m/z 3
loss of a ne
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
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
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).
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
Compounds in AABE extract and MS fragments, UV spectral characteristic and content.
Product ions in positive
mode in LC–MS spectra
Product ions in negative
mode in LC–MS spectra
Absorption spectrum Content in
1 Gallic acid (C7H6O5) 169 [M−H]− 270 239 0.25%
125 [M COOH]−
2 Homovanillic acid
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
163 [M−H]− 224, 304 247 0.37%
119 [M HCOOH]−
7 Catechin 581 [2M+H]+
8 Epicatechin Identical to catechin
9 Catechin tetramethyl
691 [2M−H]− 693 [2M+H]+
391 [M+HCOOH]− 347 [M+H]+
345 [M−H]− 317 [M OCH3]+
315 [M OCH3]−
179 [M C9H10O3]−
165 [M C10H12O3]−
345 [M C6H11O4]−
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]+
E.T. Benkovic´ et al. / Industrial Crops and Products 52 (2014) 23– 28 27
Table 1 (Continued)
Product ions in positive
mode in LC–MS spectra
Product ions in negative
mode in LC–MS spectra
Absorption spectrum Content in
12 Secoisolariciresinol 693 [2M OCH3]− 295 [M 2H2O C
407 [M+HCOOH]− 203
343 [M H2O]− 163
cases, the M
of water (H
331 [M 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]
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
Fig. 3. UV–vis spectrum of taxiresinol.
tive losses o
299 [M OC
ical for lign
and 137 (3
tra with [M
to a dimer a
from the m
of the taxir
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
Fig. 4. The
725) on the
723) on the
with the los
on the neg
lar mass of m
similar to th
source of a
side with o
Arts, I.C.W., H
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),
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.
, H., E
, S., P
B1 in h
, J., Ne
, B., Kr
, D., R
proposed structure of compound 10,7-(2-methyl-3,4-
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)
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-
rk was partly supported by the Slovenian Research
nt P4-0127) and Slovenian Technological Agency (grant
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.
Yang, S.-A., Jeo
Nutr. 44 (3
Yang, X.-W., L
Yeo, H., Chin,
61 (4), 844–854.
., Neto, C.P., Evtuguin, D., Jorge, F.C., Irle, M.A., Gil, M.H., Pedrosa
, J., 2002. Chemical characterisation of bark and of alkaline bark
rom maritime pine grown in Portugal. Ind. Crops Prod. 16 (1),
M.E., Royer, M., Duque-Fernandez, A., Diouf, P.N., Stevanovic, T.,
., 2010. Antioxidant, toxicological and antiproliferative properties
an polyphenolic extracts on normal and psoriatic keratinocytes. J.
macol. 132 (1), 251–258.
Morand, C., 2011. Evidence for a protective effect of polyphenols-
foods on cardiovascular health: an update for clinicians. Ther. Adv.
is. 3 (2), 87–106.
.P., Hopia, A.I., Vuorela, H.J., Rauha, J.-P., Pihlaja, K., Kujala, T.S.,
, M., 1999. Antioxidant activity of plant extracts containing phenolic
s. J. Agric. Food Chem. 47 (10), 3954–3962.
Loponen, J., Ossipov, V., Pihlaja, K., 2004. Analysis of procyanidins
rk with reversed-phase and normal-phase high-performance liquid
raphy–electrospray ionization mass spectrometry. Analytica Chimica
ttyu, K., Ota, M., 1999. Characterization of the major components in
ﬁve Japanese tree species for chemical utilization. Wood Sci. Technol.
L., Ouyang, D.-W., Yu, P., Xia, J.-H., Pan, Y.-X., Yang, X.W., Zeng,
ng, X.R., Jin, H.Z., Zhang, W.D., 2011. Phenolic compounds of Abies
s and their NO production inhibitory activities. Chem. Biodivers. 8
rimo, N., Katagiri, N., Tsubata, M., Takahashi, J., Van Chuyen, N., 2008.
effect of astraxanthin combined with ﬂavangenol on oxidative stress
s in streptozotocin-induced diabetic rats. Int. J. Vitam. Nutr. Res. 78
ires-Alves, A., Diallo, R., Dupuy, J.-W., Dargelos, E., 2012. A natu-
idant pine bark extract, Oligopin® regulates the stress chaperone
uman skeletal muscle cells: a proteomics approach. Phytother. Res.,
Urpi-Sarda, M., Boto-Ordon˜ez, M., Knox, C., Llorach, R., Eisner, R.,
veu, V., Wishart, D., Manach, C., Andres-Lacueva, C., Scalbert, A., 2012.
plorer 2.0: a major update of the Phenol-Explorer database integrating
lyphenol metabolism and pharmacokinetics in humans and experi-
imals. Database (Oxford), http://dx.doi.org/10.1093/database/bas031.
Tokuhara, D., Tsubata, M., Kamiya, T., Kamiya-Sameshima, M.,
, R., Takagaki, K., Sai, Y., Miyamoto, K., Aburada, M., 2012. Fla-
pine bark extract) and its major component procyanidin B1 enhance
oxidation in fat-loaded models. Eur. J. Pharmacol. 677 (1–3),
eft, S., Janesˇ, D., Kocˇevar Glavacˇ, N., Tavcˇar, E., Slokar, M., Zaloker A.
ocˇi raﬁnirani antioksidativni zvlecˇek iz skorje jelke in postopek za
ridobivanje: patent application P-201100341; Patent no: SI23867 SI
odriguez-Mateos, A., Corona, G., Oruna-Concha, M.J., Spencer, J.P.E.,
phenols and human health: prevention of disease and mechanisms
Nutrients 2 (11), 1106–1131.
2007. Fluorescent and luminescent probes for measurement of oxida-
itrosative species in cells and tissues: progress, pitfalls, and prospects.
. Biol. Med. 43 (7), 995–1022.
n, S.-K., Lee, E.-J., Im, N.-K., Jhee, K.-H., Lee, S.-P., Lee, I.S., 2009. Radical
g activity of the essential oil of silver ﬁr (Abies alba). J. Clin. Biochem.
i, S.-M., Shen, Y.-H., Zhang, W.-D., 2008. Phytochemical and biological
Abies species. Chem. Biodivers. 5 (1), 56–81.
Y.-W., Park, S.-Y., Kim, J., 2004. Lignans of Rosa multiﬂora roots. Arch.
s. 27 (3), 287–290.
Chemical composition of the silver fir (Abies alba) bark extract Abigenol® and its antioxidant activity
2 Materials and methods
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