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Antioxidant flavonoids from knotwood of Jack pine and European aspen

Article  in  Holz als Roh- und Werkstoff · February 2007

DOI: 10.1007/s00107-006-0121-0

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Holz Roh Werkst (2007) 65: 1–6
DOI 10.1007/s00107-006-0121-0

ORIGINALARBEITEN · ORIGINALS

Antioxidant flavonoids from knotwood of Jack pine and European

aspen
M. Neacsu · P. C. Eklund · R. E. Sjöholm · S. P. Pietarinen · M. O. Ahotupa ·
B. R. Holmbom · S. M. Willför

Published online: 10 August 2006

© Springer-Verlag 2006

Abstract Flavonoids have recently been found in large
amounts in knotwood and stemwood of several tree species.
Six flavonoids, two flavonoid glucosides, and one cinnamic
acid derivative were isolated from Jack pine and European
aspen knotwood and structurally characterised using GC-
MS, HR-MS, and NMR spectroscopic analyses. Isolated
compounds were further assessed on basis of their potency
to inhibit lipid peroxidation and scavenge peroxyl radi-
cals. All tested compounds possessed antioxidant properties
close to that of the reference compound Trolox.
Antioxidative Flavonoide aus dem Astholz
von Jack Pine und Aspe
Zusammenfassung Im Ast- und Stammholz verschiedener
Baumarten hat man in jüngerer Zeit größere Mengen von
Flavonoiden gefunden. Sechs Flavonoide, zwei Flavonoid-
glykoside und ein Zimtsäurederivat wurden aus dem Ast-
holz von Jack Pine und Aspe isoliert und mittels GC-MS,
HR-MS und NMR Spektralanalysen strukturell bestimmt.
Ausgehend von deren Potential, eine Lipidperoxidation zu
hemmen und Peroxylradikale abzufangen, wurden die iso-
lierten Verbindungen weiter untersucht. Alle untersuchten
Verbindungen verfügten über antioxidative Eigenschaften,
die denjenigen der Referenzverbindung Trolox weitgehend
entsprachen.

M. Neacsu
Department of Chemistry and Biochemistry, University of 1 Introduction
Agricultural Sciences and Veterinary Medicine Cluj-Napoca,
Str. Manastur 3–5, Flavonoids are widely occurring polyphenols in nature and
3400 Cluj-Napoca, Romania
more than 4000 different flavonoids have been isolated from
P. C. Eklund · R. E. Sjöholm plants. Flavonoids have been found in small amounts also
Laboratory of Organic Chemistry, Process Chemistry Centre, in foliage, bark, sapwood, and heartwood of trees (Obst
Åbo Akademi University, 1998). Recently, flavonoids have been found in high con-
Biskopsgatan 8,
20500 Turku/Åbo, Finland
centrations in knotwood of some tree species (Willför et al.
2003, Pietarinen 2005, Pietarinen et al. 2005a, Pietarinen
S. P. Pietarinen · B. R. Holmbom · S. M. Willför (u) et al. 2005b, Pietarinen et al. 2006). Hardwood knots con-
Laboratory of Wood and Paper Chemistry, Process Chemistry tain mainly flavonoids, while lignans and stilbenes are pre-
Centre, Åbo Akademi University,
Porthansgatan 3,
dominant in softwood knots (Holmbom et al. 2004, Willför
20500 Turku/Åbo, Finland et al. 2003). Nevertheless, flavonoids are found in large
e-mail: swillfor@abo.fi amounts in knots of some softwood species. Polyphenols
in knotwood constitute a large potential resource of nat-
M. O. Ahotupa
ural antioxidants and radical scavengers for the food and
BioCity B 4, MCA Research Laboratory,
Tykistökatu 6B, pharmaceutical industries and for use as technical antiox-
20520 Turku/Åbo, Finland idants (Willför et al. 2003, Willför et al. 2004, Holmbom

13
2 Holz Roh Werkst (2007) 65: 1–6
et al. 2004, Pietarinen et al. 2005a, Pietarinen et al. 2005b,
Pietarinen et al. 2006).
The main composition of Pinus banksiana (Jack pine)
wood extractives and the presence of pinobanksin and
pinocembrin are well known (Lindstedt 1950, Rudloff and
Sato 1965, Lindberg et al. 2004). Pinobanksin, pinobanksin
3-acetate, and pinocembrin are also the main flavonoids in
propolis (bee-glue) (Velikova et al. 2000, Bankova et al.
2002). The chemical composition of Populus tremula (Eu-
ropean aspen) has been studied by several research groups.
Heuser and Broetz (1925) reported on the chemical con-
stituents of aspen wood. The major polyphenols in aspen
knotwood are also known (Pietarinen 2005, Pietarinen et
al. 2005b), but the individual flavonoids have not earlier
been isolated and characterised in detail.
Jack pine and European aspen have flavonoid-rich knots
that could be suitable for industrial utilisation. Here we
present results from isolation, structural characterisation,
and biotesting of polyphenols from these two species.
2 Experimental
2.1 Materials and methods
Jack pine and European aspen stemwood discs containing
knots were sampled from several trees in Cape Breton, Nova
Scotia, Canada and Southern Finland, respectively. The
discs were stored at -18 ◦ C. Knots were separated and splin-
tered manually and then air-dried overnight before grinding
with a Fritsch mill (10-mesh screen). A second drying (4 d,
room temperature) was done to remove volatile compounds.
Large-scale Soxhlet extraction was performed in two steps:
first using hexane to remove lipophilic compounds, and then
acetone to obtain phenolic compounds.
The crude Jack pine extract was adsorbed on silica (Sil-
ica gel 60, Fluka), (1 : 4, w:w) on ZIF-SIMTM 60 Barrels,
and applied to a silica column for flash chromatography. The
crude extract was first eluted with 10% acetone in cyclohex-
ane and subsequently with a stepwise increasing (10% per
step) gradient of acetone in cyclohexane (10%–60%). The
eluates were analysed by TLC, GC-FID, and GC-MS. Three
eluates were concentrated to about 1 mL. A Shimadzu Sys-
tem HPLC equipped with LC-8A pumps, FRC-10A fraction
collector, SCL-10 Avp system controller, SIL-10 AP auto
injector, SPD -10 Avp UV detector, DGU-14A degasser,
and CLASS-VP software, was used. Separations were per-
formed using a solvent gradient mode: solvent A (0.1%
formic acid in 20% acetonitrile in water) and B (0.1%
formic acid in acetonitrile). The programs used were pro-
gram I a and I b and II a and II b; “a” stands for analytical
scale (a Nucleosil 5C18, 250 mm × 4.6 mm analytical col-
umn, Phenomenex C8 (octyl, MOS), 4 mm L × 3.0 mm i.d.
13
precolumn, flow rate 1 mL/min, injection volume 30 µL),
and “b” stands for preparative scale (Phenomenex Luna 10
u C18 100A 250 × 21.20 mm, Phenomenex Luna 10 u C18
100 A50 × 21.20 mm guard column, flow rate 21 mL/min,
injection volume 500 µL). Program I a started with a lin-
ear gradient until 16 min (24% B), and then increased until
30 min (32% B) followed by an isocratic range for 15 min-
utes with 32% B, and finished by another linear gradient
until 75% B at 60 min. Program II a is a linear gradient in
three steps: (1) 0 to 16 min (0 to 24% B), (2) 16 to 50 min
(24% B to 40% B), (3) 50 to 60 min (40% B to 75% B).
The aspen knotwood extract was dried and dissolved in
methanol before preparative HPLC (Phenomenex Luna 10
u C18 100A 250 × 21.20 mm column, Phenomenex Luna
10 u C18 100 A 50 × 21.20 mm guard column, flow rate
21 mL/min, injection volume 500 µL). Separations were
performed using a solvent gradient mode (solvent A (5%
HCOOH in water) and B (methanol)) in three steps: (1) 0 to
3 min (7 to 14% B); (2) 3 to 33 min (14% B to 19% B); (3)
33 to 65 min (19% B to 65% B).
GC-FID and GC-MS analyses of the extracts, eluates,
and pure polyphenols were performed as described by
Willför et al. (2003). TLC analysis was performed on Merck
silica plates (20 × 20 cm Silica gel 60 F254 , 20 × 2.5 cm con-
centrating zone) using dichloromethane: ethanol (95 : 5) as
eluent. The exact mass giving the elemental composition
was determined with Fison’s ZabSpec high-resolution MS
instrument. The samples were applied with direct insertion
by a solid probe and the ionisation mode was EI at 40 eV
electron energy. The resolution was 10 000.
1
H and 13 C NMR data were recorded on a Bruker Av
600 spectrometer at 600.13 and 150.91 MHz, respectively.
2D experiments (COSY, HSQC, and HMBC) were recorded
using Bruker standard pulse sequences. All measurements
were conducted at 298 K, using deuterated methanol as sol-
vent. Optical rotations were measured with a Perkin Elmer
241 digital polarimeter with a 1-dm, 1-mL cell.
Pinobanksin (1): white crystals; GC-MS (EI) (TMSi-
ether derivatives) m/z 488 [M+ ] (2%), 473 (34), 296 (100),
192 (66), 73 (75); HRMS (EI) m/z 272.0682 (calculated
−1
D = +12.2 (c 5 mg mL
for C15 H12 O5 , 272.068474); [α]24 in
◦
methanol); H NMR (MeOD−d4), 600 MHz, 25 C) δ 4.54
1
(1 H, d, J = 11.6 Hz, H-3), 5.07 (1 H, d, J = 11.6 Hz, H-2),
5.91 (1 H, d, J = 2.1 Hz, H-8), 5.94 (1H, d, J = 2.1 Hz, H-6),
7.38 (1H, m, H-4’), 7.42 (2 H, m, H-3’/5’), 7.53 (2 H, m, H-
2’/6’); 13 H NMR (MeOD−d4 ), 150.9 MHz, 25 ◦ C) δ 73.73
(C-3), 85.06 (C-2), 96.35 (C-8), 97.45 (C-6), 101.85 (C-10),
128.95 (C-2’/6’), 129.43 (C-3’/5’), 129.92 (C-4’), 138.57
(C-1’), 164.41 (C-9), 165.37 (C-5), 168.78 (C-7), 198.25
(C-4). 1 H NMR and 13 H NMR data are in accordance with
Kuroyanagi et al. (1982).
Pinobanksin 3-acetate (2): white crystals; GC-MS (EI)
(TMSi-ether derivatives) m/z 458 [M+ ] (1%), 443 (81), 296
Holz Roh Werkst (2007) 65: 1–6
(100), 73 (69); HRMS (EI) m/z 314.079 (calculated for
−1
D = +48.11 (c 8.5 mg mL
C17 H14 O6 , 314.079038); [α]24 in
◦
methanol) H NMR (MeOD−d4), 600 MHz, 25 C) δ 1.95
1
(3H, s, OAc), 5.41 (1H, d, J = 11.7 Hz, H-2), 5.83 (1H, d,
J = 11.7 Hz, H-3), 5.95 (1H, d, J = 2.1 Hz, H-8), 5.97 (1H,
d, J = 2.1 Hz, H-6), 7.38 (1H, m, H-4’), 7.41 (3H, m, H-
3’/5’, H.4’), 7.52 (2H, m, H-2’/6’); 13 H NMR (MeOD−d4),
150.9 MHz, 25 ◦ C) δ 73.73 (C-3), 85.06 (C-2), 96.65 (C-8),
97.78 (C-6), 102.11 (C-10), 128.68 (C-2’/6’), 129.64 (C-
3’/5’), 120.42 (C-4’), 137.21 (C-1’), 164.12 (C-9), 165.48
(C-5), 169.12 (C-7), 170.75, (OAc) 193.00 (C-4).
Pinocembrin (3): white powder; GC-MS (EI) (TMSi-
ether derivatives) m/z 399 [M+ ] (1%), 385 (100), 296 (11),
103 (2), 73 (25); HRMS (EI) m/z 256.0738 (calculated for
−1
D = −52.6 (c 1.9 mg mL
C15 H12 O4 , 256.073559); [α]24 in
◦
methanol); H NMR (MeOD−d4), 600 MHz, 25 C) δ 2.76
1
(1H, dd, J = 17.0, 3.1 Hz, H-3a), 3.07 (1H, dd, J = 12.8,
17.0 Hz, H-3b), 5.44 (1H, dd, J = 3.1, 12.8 Hz, H-2), 5.90
(1H, d, J = 2.2 Hz, H-6), 5.93 (1H, d, J = 2.2 Hz, H-8),
7.36 (1H, m, H-4’), 7.41 (2 H, m, H-3’/5’), 7.48 (2H, m, H-
2’/6’); 13 H NMR (MeOD−d4 ), 150.9 MHz, 25 ◦ C) δ 41.21
(C-3), 80.46 (C-2), 96.24 (C-8), 97.19 (C-6), 103.39 (C-10),
127.37 (C-2’/6’), 129.64 (C-4’), 129.72 (C-3’/5’), 140.4 (C-
1’), 164.68 (C-9), 165.51 (C-5), 168.42 (C-7), 197.33 (C-4).
1
H NMR and 13 H NMR data are in accordance with Kuroy-
anagi et al. (1983) and Jung and McLaughlin (1990).
Dihydrokaempferol 7-O-β-glucoside (4): yellowish pow-
der; GC-MS (EI) (TMSi-ether derivatives) m/z 450 [M+ ]
(not visible), 286 (12), 270 (100), 242 (19), 197 (4), 296
(100), 166 (16), 149 (15), 107 (56); HRMS (EI) (TMS-ether
derivatives) m/z 954.3957 (calculated for C42 H78 O11 Si7 ,
−1
954.392913); [α]24 D = −31 (c 5 mg ml in methanol); 1 H
NMR spectral data (600.13 MHz, DMSO−d6 ): δ 3.12 (1H,
dd, J = 8.9, 9.5 Hz, H-4”), 3.18 (1H, dd, J = 7.7, 8.9 Hz,
H-2”), 3.24 (1H, t, J = 8.9 Hz, H-3”), 3.36 (1H, ddd, J =
2.0, 5.7, 9.5 Hz, H-5”), 3.41 (1H, dd, J = 5.7, 12.0 Hz, H-
6b”), 3.64 (1H, dd, J = 2.0, 12.0 Hz, H-6a”), 4.65 (1H, d, J
= 11.4 Hz, H-3), 4.95 (1H, d, J = 7.7 Hz, H-1”), 5.11 (1H,
d, J = 11.4 Hz, H-2), 6.12 (1H, d, J = 2.2 Hz, H-8), 6.15
(1H, d, J = 2.2 Hz, H-6), 6.78 (2H, d, J = 8.6 Hz, H-3’, C-
5’), 7.32 (2H, d, J = 8.6 Hz, H-2’, H-6’). 13 C NMR spectral
data (151.91 MHz, MeOD−d6 ): δ 62.3 (C-6”), 71.1 (C-4”),
73.1 (C-3), 74.6 (C-2”), 77.8 (C-3”), 78.3 (C-5”), 85.2 (C-
2), 97.0 (C-8), 98.3 (C-6), 101.3 (C-1”), 103.5 (C-10), 116.2
(C-3’, C-5’), 129.1 (C-1’), 130.5 (C-2’, C-6’), 159.3 (C-4’),
164.3 (C-9), 164.8 (C-5), 167.7 (C-7), 199.4 (C-4). 1 H and
13
C NMR data are in accordance with Nørbæk et al. (1999).
p-Hydroxy-cinnamic acid (6): yellowish powder; GC-
MS (EI) (TMSi-ether derivatives) m/z 164 [M+ ] (100%),
147 (48), 270 (100), 119 (40), 107 (16), 91 (36); HRMS (EI)
m/z 164.0 (calculated for C9 H8 O3 , 164); 1 H NMR spectral
data (600.13 MHz, MeOD−d4 ): δ 6.37 (1H, d, J = 15.9 Hz,
H-8), 6.88 (2H, d, J = 8.5 Hz, H-3, H-5), 7.53 (2H, d, J =
3
8.5 Hz, H-2, H-6), 7.63 (1H, d, J = 15.9 Hz). 13 C NMR spec-
tral data (151.91 MHz, MeOD−d6 ): δ 115.9 (C-8), 116.9
(C-3, C-5), 127.2 (C-1), 131.1 (C-2, C-6), 146.1 (C-7),
161.0 (C-4), 179.8 (C-9), 13 C NMR data are in accordance
with Silva et al. (2001).
Dihydrokaempferol (8): yellowish powder; GC-MS (EI)
(TMSi-ether derivatives) m/z 288 [M+ ] (30%), 259 (41),
165 (16), 153 (100), 136 (45), 107 (75); HRMS (EI) m/z
288.0637 (calculated for C15 H12 O6 , 288.063388); [α]24 D =
+28.4 (c 13.2 mg ml−1 in methanol); 1 H NMR spectral data
(600.13 MHz, MeOD−d4 ): δ 4.57 (1H, d, J = 11.6 Hz, H-3),
5.00 (1H, d, J = 11.6 Hz, H-2), 5.91 (1H, d, J = 2.1 Hz, H-
8), 5.95 (1H, d, J = 2.1 Hz, H-6), 6.86 (2H, d, J = 8.5 Hz,
H-3’, H-5’), 7.38 (2H, d, J = 8.5 Hz, H-2’, H-6’), 1 H NMR
data in accordance with Prescott et al. (2002). 13 C NMR
spectral data (151.91 MHz, MeOD−d6): δ 73.6 (C-3), 84.9
(C-2), 96.3 (C-8), 97.3 (C-6), 101.9 (C-10), 116.1 (C-3’, C-
5’), 129.3 (C-1’), 130.4 (C-2’, C-6’), 159.2 (C-4’), 164.6
(C-9), 165.3 (C-5), 168.7 (C-7), 197.5 (C-4).
Naringenin 7-O-β-glucoside (9): white-yellow powder;
GC-MS (EI) (TMSi-ether derivatives) m/z [M+ ] 866.4
(19%), 851.3 (22), 779.3 (42), 488.2 (32), 473.2 (100),
361.2 (98), 179 (22), 73 (40); HRMS (EI) (TMS-ether
derivatives) m/z 866.3601 (calculated for C39 H70 O10 Si6 ,
−1
866.358470); [α]24 D = −69.8 (c 5 mg ml
in methanol); 1 H
NMR spectral data (600.13 MHz, DMSO−d6 ): δ 2.73 (1H,
dd, J = 3.1, 17.1 Hz, H-3b) 3.13 (1H, bt, J = 8.9 Hz, H-4”),
3.20 (1H, bt, J = 8.9 Hz, H-2”), 3.25 (1H, t, J = 8.9 Hz, H-
3”), 3.33 (1H, dd J = 12.7, 17.1 Hz, H-3a), 3.36 (1H, ddd,
J = 2.1, 5.8, 9.5 Hz, H-5”), 3.42 (1H, dd, J = 5.8, 11.3 Hz,
H-6b”), 3.66 (1H, bd, J = 11.3 Hz, H-6a”), 4.94 (1H, d, J
= 7.7 Hz, H-1”), 5.44 (1H, dd, J = 3.1, 12.7 Hz, H-2), 6.12
(1H, d, J = 2.2 Hz, H-6), 6.14 (1H, d, J = 2.2 Hz, H-8), 6.7
(2H, d, J = 8.6 Hz, H-3’, C-5’), 7.32 (2H, d, J = 8.6 Hz, H-2’,
H-6’). 13 C NMR spectral data (151.91 MHz, DMSO−d6 ): δ
42.0 (C-3), 60.5 (C-6”), 69.4 (C-4”), 72.9 (C-2”), 76.2 (C-
3”), 77.0 (C-5”), 78.6 (C-2), 95.4 (C-8), 96.4 (C-6), 99.5
(C-1”), 103.2 (C-10), 115.2 (C-3’, C-5’), 128.4 (C-2’, C-6’),
128.6 (C-1’) 157.7 (C-4’), 162.7 (C-9), 16.9 (C-5), 165.2
(C-7), 197.2 (C-4).
Naringenin (10): yellow-orange powder; GC-MS (EI)
(TMSi-ether derivatives) m/z 487 [M+ ] (14%), 473 (100),
296 (54), 179 (18), 73 (44); HRMS (EI) m/z 272.0680
(calculated for C15 H12 O5 , 272.068474); [α]24 D = −16.6 (c
5 mg ml−1 in methanol); 1 H NMR spectral data (600.13 MHz,
MeOD−d4 ): δ 2.71 (1H, dd, J = 2.9, 17.1 Hz, H-3b), 3.13
(1H, dd, J = 12.9, 17.1 Hz, H-3a), 5.35 (1H, dd, J = 2.9,
12.9 Hz, H-2), 5.91 (1H, d, J = 2.1 Hz, H-6), 5.92 (1H, d, J
= 2.1 Hz, H-8), 6.84 (2 H, d, J = 8.5 Hz, H-3’, H-5’)), 7.33
(2H, d, J = 8.5 Hz, H-2’, H-6’), 1 H NMR data are in accor-
dance with Prescott et al. (2002). 13 C NMR spectral data
(151.91 MHz, MeOD−d6 ): δ 44.0 (C-3), 80.5 (C-2), 96.2
(C-8), 97.1 (C-6), 103.4 (C-10), 116.3 (C-3’, C-5’), 129.1
13
4 Holz Roh Werkst (2007) 65: 1–6
(C-2’, C-6’), 131.1 (C-1’), 159.0 (C-4’), 164.9 (C-9), 165.5
(C-5), 168.4 (C-7), 197.8 (C-4).
2.2 Biotests
The antioxidant properties of pure compounds were esti-
mated by their potency to inhibit tert-butylhydroperoxide (t-
BuOOH) induced lipid peroxidation in rat liver microsomes
in vitro monitored by luminal-enhanced chemiluminescence
detection (Ahotupa et al. 1997). The capacity of pure com-
pounds to trap peroxyl radicals was assessed using a chemi-
luminescence based methodology, where peroxyl radicals
are generated by thermal decomposition of 2,2’-azinobis(2-
amidinopropane)-hydrochloride (ABAP).
3 Results and discussion
3.1 Polyphenols in Jack pine knotwood
From 12.65 g acetone extract of Jack pine knotwood, we
isolated 258 mg pinobanksin (1) (2% of the extract), 224 mg
pinobanksin-3-acetate (2) (1.7%), and 263 mg pinocem-
brin (3) (2.1%) (Fig. 1). The structures of the compounds
were determined by GC-MS, HRMS, optical rotation, and
NMR spectroscopic analyses. The complete NMR signals
for pinobanksin 3-acetate are here presented for the first
time.
Fig. 1 Structures of the major
identified flavonoids isolated
from Jack pine (1–3) and
European aspen (4–10)
knotwood
Abb. 1 Strukturformeln der
wichtigsten identifizierten
Flavonoide, die aus dem
Astholz von Jack Pine (1–3) und
Aspe (4–10) isoliert wurden
13
3.2 Polyphenols in European aspen knotwood
The aspen knotwood extract contained dihydrokaempferol
7 O-β-glucoside (4) (4.8% of the extract), p-hydroxy-
cinnamic acid (6) (1.7%), taxifolin (7) (0.6%), dihy-
drokaempferol (8) (18.5%), naringenin 7-O-β-glucoside (9)
(6.6%), and naringenin (10) (13.2%) (Fig. 1), which is in
accordance with Pietarinen et al. (2005b). In addition, an
unknown compound (5) eluting after (4) was detected in
the extract. To isolate the major aspen polyphenols, a gra-
dient solvent program for RP-C18 columns was developed.
The first two peaks that eluted from the RP-column had the
same retention time in GC analysis, the same fragmenta-
tion pattern by GC-MS, and the same molar mass according
to HRMS, corresponding to dihydrokaempferol 7-O-β-
glucoside. The structure was elucidated and confirmed by
NMR spectroscopy. The structures of compounds (6), (8),
and (10) were determined after GC-MS, HRMS, and NMR
spectroscopic analyses and comparison to literature data.
Taxifolin (7) was compared to an authentic sample from
Sigma-Aldrich Chemie GmbH (Steinhem, Germany). Com-
pound (6) was identified as naringenin 7-O-β-glucoside.
The retention time of this high-polarity flavonoid glycoside
was unusually late to be obtained with RP-HPLC separation.
To the best of our knowledge, complete 1 H NMR and 13 C
NMR data for naringenin 7-O-β-glucoside and 13 C NMR
data for dihydrokaempferol and naringenin have not earlier
been reported.
Holz Roh Werkst (2007) 65: 1–6 5

Table 1 a Inhibition of lipid peroxidation in vitro, expressed as IC50 pinobanksin 3-acetate, or as components of propolis, have
values (i.e., concentration of a compound that inhibits lipid peroxida- further been studied in different tests (Velikova et al. 2000,
tion by 50%) and b scavenging of peroxyl radicals in vitro, expressed
as the stoichiometric factor (trapping capacity, i.e., moles peroxyl rad- Arjun et al. 2002, Bankova et al. 2002, Kumazawa et al.
icals scavenged per mole compound), by purified polyphenols from 2003). The antioxidative properties of pinocembrin have
Jack pine and European aspen knotwood and a reference compound also been determined (Santos et al. 1998, Sala et al. 2003).
(Trolox) These results are in accordance with our results (Table 1).
Tabelle 1 a Hemmung der Lipidperoxidation in vitro, als IC50 Werte
angegeben (d.h. Konzentration einer Verbindung, die Lipidperoxi- The differences in antioxidative capacities between differ-
dation um 50% hemmt) und b Abfangen von Peroxylradikalen in ent flavonoids is associated with certain structural features
vitro, als stöchiometrischer Faktor angegeben (Fangvermögen, d.h. (Rice-Evans et al. 1996, Zheng and Wang 2001, Jung et al.
abgefangene Peroxylradikalmole pro Mol Verbindung) mittels reiner 2003): multiple phenolic groups and the presence of a 4-
Polyphenole aus dem Astholz von Jack Pine und Aspe sowie einer
Referenzverbindung (Trolox) carbonyl group in the C ring, the presence of a hydroxyl
group in position 3, the presence of hydroxyl groups in
Compound a) Inhibition b) Scavenging of
of lipid peroxyl radicals,
positions 3 and 5 are all associated with high antioxidative
peroxidation, stoichiometric capacity. The pine knotwood flavonoids (Fig. 1) incorporate
IC50 (nM) factor (mol/mol) these features. Pinocembrin, which lacks the 3-OH group
Trolox (reference) 0.35 1.9 but has a 5-OH in the A ring and a 4-carbonyl group in the
Pinobanksin 6.1 0.32 C ring, was found less effective than pinobanksin and its
Pinobanksin 3-acetate 3.5 0.46
Pinocembrin 9.1 0.24 3-acetate derivative.
Dihydrokaempferol 8.6 0.08
7-O-β-glucoside
p-Hydroxy-cinnamic acid 3.2 0.06 4 Concluding remarks
Dihydrokaempferol 5.4 0.21
Naringenin 7-O-β-glucoside 5.4 0.08
Naringenin 3.6 0.13 Jack pine and European aspen knotwood are rich sources
of flavonoids able to inhibit lipid peroxidation and trap
peroxyl radicals. Six knotwood flavonoids, two flavonoid
3.3 Antioxidant properties of Jack pine glucosides, and one cinnamic acid derivative were isolated
and European aspen polyphenols using flash chromatography and preparative HPLC. The
compounds were structurally characterised using GC-MS,
The antioxidant properties of pure compounds were as- HR-MS, and NMR spectroscopic analyses. Isolated com-
sessed on the basis of their potency to inhibit lipid per- pounds were further assessed on basis of their potency to
oxidation in rat liver microsomes in vitro (Ahotupa et al. inhibit lipid peroxidation and scavenge peroxyl radicals. All
1997). All tested compounds possessed antioxidant prop- tested compounds possessed antioxidant properties close to
erties close to that of the reference compound Trolox that of the reference compound Trolox.
(Table 1), with p-hydroxy-cinnamic acid, pinobanksin 3-
acetate, and naringenin in the high end. The antioxidant Acknowledgement Jarl Hemming and Markku Reunanen are ac-
properties were in the same range as has been reported for knowledged for their help. This research was part of the European
Commission “Marie Curie Training Site” project (HPMT-CT-2001-
some knotwood flavonoids (Willför et al. 2003, Pietarinen 00297) and also part of the activities at the Åbo Akademi University
et al. 2006). The antioxidant properties were weaker for the Process Chemistry Centre within the Finnish Centre of Excellence by
flavonoid glucosides than for their corresponding aglycones the Academy of Finland.
(Table 1), which is in accordance with Hopia and Heinonen
(1999). The capacity of pure compounds to scavenge per- References
oxyl radicals was estimated by a chemiluminescence-based-
methodology (Ahotupa et al. 1997). All tested compounds Ahotupa M, Mäntylä E, Kangas L (1997) Antioxidant properties
of the triphenylethylene antiestrogen drug toremifene. Naunyn-
possessed scavenging properties close to that of the ref- Schmiedeberg’s Arch Pharmacol 356(3):297–302
erence compound Trolox (Table 1). The pine knotwood Arjun Banskota H, Nagaoka T, Sumioka L-Y, Tezuka Y, Awale S, Mi-
flavonoids were stronger than the aspen knotwood com- dorikawa K, Matsushige K, Kadota S (2002) Antiproliferative
pounds. However, taxifolin, which is present in small activity of the Netherlands propolis and its active principles in
cancer cell lines. J Ethnopharmacol 80(1):67–73
amounts in aspen wood, has a high scavenging capacity Bankova V, Popova M, Bogdanov S, Sabatini A-M (2002) Chem-
(Willför et al. 2003). ical composition of European propolis: expected and unexpected
Ondrias et al. (1997) reported that pinobanksin has an- results. J Biosci 57(5/6):530–533
tioxidant and antiproliferative capacities, inhibits peroxida- Heuser E, Broetz A (1925) The chemical nature of deciduous trees.
Papierfabrikant 23(Festheft):69–88
tion of low density lipoprotein, and reduces α-tocopherol Holmbom B, Eckerman C, Eklund P, Hemming J, Nisula L, Reunanen
radicals. The antioxidant properties of pure pinobanksin and M, Sjöholm R, Sundberg A, Sundberg K, Willför S (2004) Knots

13
6 Holz Roh Werkst (2007) 65: 1–6
in trees – a new rich source of lignans. Phytochem Rev 2(3):
331–340
Hopia AI, Heinonen M (1999) Antioxidant activity of flavonol agly-
cones and their glycosides in methyl linoleate. J Am Oil Chem
Soc 76(1):139–144
Jung HA, Jung ME, Kim JY, Chung HY, Choi JS (2003) Inhibitory
activity of flavonoids from Prunus davidiana and other flavonoids
on total ROS and hydroxyl radical generation. Arch Pharm Res
26(10):809–815
Jung JH, McLaughlin JL (1990) 13 C-1 H NMR long-range coup-
ling and deuterium isotope effects of flavanones. Phytochemistry
29(4):1271–1275
Kumazawa S, Hamasaka T, Nakayama T (2003) Antioxidant activity of
propolis of various geographic origins. Food Chem 84(3):329–339
Kuroyanagi M, Yamamoto Y, Fukushima S, Ueno A, Noro T, Miyase
T (1982) Chemical studies on the constituents of Polygonum no-
dosum. Chem Pharm Bull 30(5):1602–1608
Kuroyanagi M, Noro T, Fukushima S, Aiyama R, Ikuta A, Itokawa
I, Morita M (1983) Studies of constituents of seeds of Alpinia
katsumadai Hayata. Chem Pharm Bull 31(5):1544–1550
Lindberg LE, Willför SM, Holmbom BR (2004) Antibacterial effects
of knotwood extractives on paper mill bacteria. J Ind Microbiol
Biotechnol 31(3):137–147
Lindstedt G (1950) Constituents of pine heartwood. XXI. Structure of
pinobanksin. Acta Chem Scand 4:772–781
Nørbæk R, Nielsen JK, Kondo T (1999) Flavonoids from flowers
of two Crocus chrysanthus-biflorus cultivars: “Eye-catcher” and
“Spring Pearl” (Iridaceae). Phytochemistry 51(8):1139–1146
Obst JR (1998) Special (secondary) metabolites from wood. In: Bruce
A, Palfreyman JW (eds) Forest Products Biotechnology. Taylor
& Francis, London, pp 151–165
Ondrias K, Stasko A, Hromadova M, Suchy V, Nagy M (1997)
Pinobanksin inhibits peroxidation of low density lipoprotein and
it has electron donor properties reducing α-tocopherol radicals.
Die Pharmazie 52(7):566–567
Pietarinen S (2005) Extractives in Stemwood and Knots of Acacia and
Aspen Trees. Doctoral Thesis, Faculty of Chemical Engineering,
Åbo Akademi University, Åbo, Finland
Pietarinen S, Willför S, Sjöholm R, Holmbom B (2005a) Bioac-
tive phenolic substances in important tree species. Part 3. Knots
and stemwood of Acacia crassicarpa and A. mangium. Holz-
forschung 59(1):94–101
13
View publication stats
Pietarinen SP, Willför SM, Vikström FA, Holmbom BR (2005b) As-
pen knots, a rich source of flavonoids. J Wood Chem Technol, in
press
Pietarinen S, Willför S, Ahotupa M, Hemming J, Holmbom B (2006)
Knotwood and bark extracts: strong antioxidants from waste ma-
terials. J Wood Sci, published online first: DOI: 10.1007/s10086-
005-0780-1
Prescott AG, Stamford PJN, Wheeler G, Firmin JL (2002) In vitro
properties of a recombinant flavonol synthase from Arabidopsis
thaliana. Phytochemistry 60(6):589–593
Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant
activity relationship of flavonoids and phenolic acids. Free Radi-
cal Biol Med 20(7):933–956
Rudloff E, Sato A (1965) Chemical composition of the heartwood ex-
tractive of Pinus banksiana and Pinus resinosa. In: Proc 1st Can
Wood Chem Symp, Toronto (1963), pp 69–73
Sala A, Recio MC, Schinella GR, Mànez S, Giner RM, Cerdà-Nicolàs
M, Rı̀os J-L (2003) Assessment of the anti-inflammatory activity
and free radical scavenger activity of tiliroside. Eur J Pharmacol
461(1):53–61
Santos AC, Uyemura SA, Lopes JC, Bazon JN, Mingatto FE, Curti C
(1998) Effect of naturally occurring flavonoids on lipid peroxida-
tion and membrane permeability transition in mitochondria. Free
Radical Biol Med 24(9):1455–1461
Silva AMS, Alkorta I, Elguero J, Silva VLM (2001) A 13 C NMR
study of the structure of four cinnamic acids and their methyl
esters. J Mol Struct 595(1-3):1–6
Velikova M, Bankova V, Sorkun K, Houcine S, Tsvetkova I, Kujumgiev
A (2000) Propolis from the Mediterranean region: chemical com-
position and antimicrobial activity. J Biosci 55(9/10):790–193
Willför SM, Ahotupa MO, Hemming JE, Reunanen MHT, Eklund
PC, Sjöholm RE, Eckerman CSE, Pohjamo SP, Holmbom BR
(2003) Antioxidative activity of knotwood extractives and phe-
nolic compounds of selected tree species. J Agric Food Chem
51(26):7600–7606
Willför SM, Nisula L, Hemming JE, Reunanen MHT, Holmbom BR
(2004) Bioactive phenolic substances in industrially important
tree species. Part 1: Knots and stemwood of different spruce
species. Holzforschung 58(4):335–344
Zheng W, Wang SY (2001) Antioxidative capacity and phenolic
compounds in selected herbs. J Agric Food Chem 49(11):
5165–5170