In vitro anti-inflammatory activity of larch (Larix decidua L.) sawdust

Article abstract of DOI:10.1021/jf8024002

The influence of larch (Larix decidua L.) sawdust extracts on arachidonate metabolism has been evaluated in vitro. Extracts of different polarities were tested for their ability to inhibit prostaglandin formation by COX-1 and COX-2 and LTB₄ formation by 5-LOX. The lipophilic n-heptane extract showed the highest activity (IC₅₀ values: COX-1, 5 μg/mL; COX-2, 0.1 μg/mL; LTB₄ assay, 11.1 μg/mL). A series of abietane, pimarane, and labdane type diterpenes isolated from this extract turned out to be potent inhibitors of LTB₄ product formation, whereas their COX inhibitory activity was less pronounced. From the abietane type diterpenes, compounds possessing a 7,13-abietadiene skeleton were better inhibitors of LTB₄ formation than those with an 8,11,13-abietatriene skeleton. Compounds bearing an OH group in position 4 were more active than those substituted with a COOH group, and methylation of the COOH group led to an almost complete loss of LTB₄ formation inhibitory activity.

Full text of DOI:10.1021/jf8024002

11688 J. Agric. Food Chem. 2008, 56, 11688–11693
In Vitro Anti-inflammatory Activity of Larch (Larix
decidua L.) Sawdust
EVA M. PFERSCHY-WENZIG,† OLAF KUNERT,§ ARMIN PRESSER,§ AND
RUDOLF BAUER*,†
Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz,
Universitaetsplatz 4, 8010 Graz, Austria, and Institute of Pharmaceutical Sciences, Department of
Pharmaceutical Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria
The influence of larch (Larix decidua L.) sawdust extracts on arachidonate metabolism has been
evaluated in vitro. Extracts of different polarities were tested for their ability to inhibit prostaglandin
formation by COX-1 and COX-2 and LTB4 formation by 5-LOX. The lipophilic n-heptane extract showed
the highest activity (IC50 values: COX-1, 5 µg/mL; COX-2, 0.1 µg/mL; LTB4 assay, 11.1 µg/mL). A
series of abietane, pimarane, and labdane type diterpenes isolated from this extract turned out to be
potent inhibitors of LTB4 product formation, whereas their COX inhibitory activity was less pronounced.
From the abietane type diterpenes, compounds possessing a 7,13-abietadiene skeleton were better
inhibitors of LTB4 formation than those with an 8,11,13-abietatriene skeleton. Compounds bearing
an OH group in position 4 were more active than those substituted with a COOH group, and
methylation of the COOH group led to an almost complete loss of LTB4 formation inhibitory activity.
KEYWORDS: Larix decidua L.; larch sawdust; cyclooxygenase; lipoxygenase; anti-inflammatory; diter-
pene; abietane; labdane; pimarane
INTRODUCTION
fraction is the labdane type diterpene larixyl acetate (2), together
with lower amounts of larixol (1) and 13-epimanool. Larixol
and larixyl acetate are characteristic for larch resin as they have
only been detected in L. decidua and L. gmelinii and as they
have not been observed in any other genus. Furthermore,
abietane and pimarane type diterpenes such as abietic acid,
dehydroabietic acid, pimaric acid, and isopimaric acid are
present (1, 5, 6). Larch turpentine has been traditionally used
in external preparations for the treatment of rheumatic and
neuralgic disorders and catarrhs of the respiratory tract (1).
European larch (Larix decidua L., Pinaceae) is an up to 35 m
high, deciduous tree that is mainly native to higher regions of
the Alps, the Sudetes, and the Carpathian mountains (1). Larch
wood is robust, waterproof, and durable, but also flexible in
thin strips. The heartwood is particularly weather-proof and
therefore mainly used as construction timber. Due to its high
resin content and difficult machinability, its use for furniture
production is limited. Larch sawdust is a byproduct of larch
wood production. At present, larch sawdust is mainly used for
producing pellet fuels.
Cyclooxygenases (COX) and 5-lipoxygenase (5-LOX) are key
enzymes in arachidonate metabolism. The isoenzymes COX-1
and COX-2 catalyze the first two steps of the cyclooxygenase
pathway of the arachidonic acid cascade, which leads to the
production of prostaglandins. As COX-1 is constitutively
expressed and COX-1 derived prostanoids play a protective role
for the gastrointestinal mucosa, COX-1 is considered to possess
housekeeping functions. COX-2 expression, however, is mainly
induced by external stimuli. Therefore, inhibition of COX-1-
derived prostanoid production has been considered to cause side
effects of NSAIDs such as gastrointestinal bleeding and ulcers,
whereas inhibition of COX-2 derived prostanoids has been
considered to be responsible for their anti-inflammatory, anal-
gesic, and antipyretic effects. Thus, selective COX-2 inhibitors
have been preferred over nonselective ones to treat inflammation
(7). However, several selective COX-2 inhibitors have been
found to cause side effects as well, such as cardiovascular
problems (8), and it was found that also COX-2 is expressed
constitutively in some tissues (9). In the first step of the
Larch wood is known to contain phenolic compounds such
as lignans and flavonols (mainly dihydroquercetin and dihy-
drokaempferol) (1). These compounds have been shown to
possess antioxidant and anti-inflammatory effects (2, 3). Larch
arabinogalactan, a water-soluble polysaccharide, which is
predominantly extracted from the wood of Larix occidentalis,
is approved by the U.S. FDA as a source of dietary fiber and
also has potential therapeutic effects as an immunostimulant
and cancer therapy adjunct (4). By drilling holes into the trunk
of L. decidua trees, larch turpentine, also known as Venice
turpentine, can be obtained. This product is composed of about
15% essential oil, 50-65% resin acids, and about 15%
nonsaponifiable resin. The main constituent of the neutral resin
* Author to whom correspondence should be addressed (telephone
++43 316 380 8700; fax ++43 316 380 9860; e-mail rudolf.bauer@
uni-graz.at).
† Department of Pharmacognosy.
§ Department of Pharmaceutical Chemistry.
10.1021/jf8024002 CCC: $40.75  2008 American Chemical Society
Published on Web 12/02/2008

Anti-inflammatory Activity of Larch Sawdust
J. Agric. Food Chem., Vol. 56, No. 24, 2008 11689
Isolation of Compounds 1-6 (Figure 1) from the n-Heptane
Extract. Thirteen grams of n-heptane extract was subjected to VLC
on silica gel (130 g, 0.04-0.063 mm, Merck, Darmstadt, Germany)
with a stepwise gradient from 0 to 100% ethyl acetate in n-hexane,
yielding 13 fractions. Fraction 6 (n-hexane/ethyl acetate 90:10, 950
mg) was further separated on a Sephadex LH20 column (GE Healthcare
Bio-Sciences AB, Uppsala, Sweden; 2.8 × 60 cm, mobile phase ethyl
acetate), yielding fractions 6a-f. Thirty milligrams of fraction 6d (297
mg, elution volume ) 67-112 mL) was purified by semipreparative
HPLC using a Hypercarb column (10 × 150 mm, 7 µm; Thermo
Electron Corp.) as stationary phase and MeCN/THF 95:5 (4 mL/min)
as mobile phase. This purification step yielded compounds 3 (isopimaric
acid, 2 mg) and 4 (palustric acid, 1.5 mg). Fraction 7 (n-hexane/ethyl
acetate 85:15, 2.3 g) was separated on a Sephadex LH20 column (2.8
× 60 cm, mobile phase ethyl acetate), yielding fractions 7a-f. Seventy-
five milligrams of fraction 7c (370 mg, elution volume ) 320-340
mL) was separated by preparative HPLC using a LiChrosorb RP 18 (7
µm) LiChroCART 250-25 column (Merck), mobile phase MeCN/H2O
65:35-90:10 in 30 min; 30-31 min, 90:10-100:0; 31-45 min, 100:
0; 10 min re-equilibration; flow rate, 20 mL/min. The separation yielded
compounds 2 (larixyl acetate, 30 mg) and 5 (dehydroabietic acid, 3
mg). Fraction 7d (230 mg, elution volume ) 340-370 mL) was further
purified on silica gel (1.8 × 26.5 cm, 0.04-0.063 mm, Merck) using
a stepwise gradient of petroleum ether/acetone 95:5-80:20, yielding
fractions 7d1- 7d10. Semipreparative HPLC of fraction 7d6 (petroleum
ether/acetone 90:10, 60 mg) using a LiChrosorb RP 18 (7 µm)
LiChroCART 250-10 column (mobile phase MeCN/H2O 65:35-90:
10 in 30 min; 30-31 min, 90:10-100:0; 31-45 min, 100:0; 10 min
re-equilibration; flow rate, 2 mL/min) resulted in the isolation of
compound 6 (dehydroabietinol, 1.5 mg). Purification of 25 mg of
fraction 9 (n-hexane/ethyl acetate 70:30; 900 mg) using the same HPLC
method led to the isolation of compound 1 (larixol, 2 mg). Structures
were assigned by comparing their spectroscopic data with data from
the literature (15-18).
Fatty Acid Analysis. Free fatty acids present in the active fractions
were converted to their methyl esters as described earlier (19), and
fatty acid methyl esters were identified by GC-MS analysis on a HP
6890 GC-MS system (Agilent Technologies, Waldbronn, Germany)
equipped with a J&W Scientific DB 225 column (30 m, i.d. 0.25 mm,
film ) 0.25 µm; Agilent Technologies). Helium (0.8 mL/min) was used
as a carrier gas, injector and detector temperatures were 220 °C, and
the following temperature program was used: 0-0.5 min, 40 °C;
40-195 °C (25 °C/min); 195-202 °C (0.8 °C/min).
Preparation of Compounds 7-9. Abietic Acid (7). Compound 7
was purified by recrystallization from its dipentyl ammonium salt
according to the method given in ref 20.
Methyl Abietate (8). Compound 8 was prepared by esterification of
compound 7 with (CH3)3SiCHN2 according to the method given in ref
21.
leukotriene pathway of arachidonate metabolism, 5-LOX cata-
lyzes arachidonic acid oxygenation. Leukotrienes such as LTB4
are potent mediators of inflammatory and allergic reactions.
Therefore, 5-LOX inhibitors are considered to possess thera-
peutic potential in a range of allergic and inflammatory diseases
(7).
In the search for new anti-inflammatory drugs, the simulta-
neous blockage of both the COX and 5-LOX pathways by dual
inhibitors has been suggested as a possible alternative approach
to selective COX-2 inhibition. Drugs possessing these charac-
teristics are believed to be more effective, with lower gastric
toxicity due to a stronger anti-inflammatory effect, on the one
hand, and because, on the other hand, the enhanced conversion
of arachidonic acid to leukotrienes, which is observed for drugs
inhibiting only the COX pathway, is avoided (10).
As a part of the European research project SAFEWASTES
(www.safewastes.info) that aims to discover bioactive constitu-
ents in organic byproduct from the food, agricultural, and
pharmaceutical industries, the anti-inflammatory potential of
larch sawdust was evaluated by investigating larch sawdust
extracts and pure compounds isolated thereof for their COX-1,
COX-2, and LTB4 formation inhibitory activities in vitro.
MATERIALS AND METHODS
General Experimental Procedures. Analytical and semipreparative
HPLC separations were performed on an Agilent 1100 series HPLC
system. For preparative HPLC a Dynamax solvent delivery system and
absorbance detector were used. NMR spectra were recorded with a
Varian Unity Inova (600 MHz) spectrometer at 25 °C using the
parameters described by Seebacher et al. (11). CDCl3 was used as
solvent and TMS as internal standard.
Plant Material and Extraction. Larch sawdust was kindly provided
by Jannach La¨rchenholz GmbH, Thalheim, Austria. A voucher specimen
of the material (LD02RN090905) is deposited in the herbarium of the
Department of Pharmacognosy in Graz.
For in vitro testing, three different extracts were prepared from
powdered larch sawdust: one with n-heptane, one with 70% ethanol,
and one with water. The powdered material was mixed with a 10-fold
amount of solvent and stirred at room temperature for 2 h. After
filtration, the extracts were concentrated at 40 °C under reduced
pressure. The remaining n-heptane was removed under a stream of N2,
and the remaining water was removed by lyophilization. The following
extract yields (% w/w of starting material) were obtained: water extract,
3.6%; 70% ethanol extract, 1.5%; and n-heptane extract, 0.5%.
In Vitro Assay for COX-1, COX-2, and LTB4 Formation
Inhibitory Activity. COX-1 and COX-2 inhibition assays were
performed in a 96-well plate format with purified prostaglandin H
synthase (PGHS)-1 from ram seminal vesicles for COX-1 and purified
PGHS-2 from sheep placental cotyledons for COX-2 (both Cayman
Chemical Co., Ann Arbor, MI) as previously described (12). The
concentration of PGE2, the main arachidonic acid metabolite in this
reaction, was determined by a competitive PGE2 EIA kit (Assay Designs
Inc., Ann Arbor, MI). Indomethacin (ICN, Aurora, OH; IC50 COX-1
) 0.9 µM) and NS-398 (Cayman Chemical Co., IC50 COX-2 ) 2.6
µM) were used as positive controls.
Abietinol (9). Five hundred milligrams (1.58 mmol) 8 was dissolved
in 20 mL of dry Et2O and added dropwise to a suspension of 96 mg
(2.53 mmol) LiAlH4 in 10 mL of dry Et2O. After refluxing for 60 min,
excessive hydride was destroyed by the addition of a few drops of water.
The reaction mixture was diluted with 10 mL of Et2O, washed with 2
N H2SO4 and brine, dried over Na2SO4, and evaporated. Column
chromatography of the residue with CH/EtOAc (3:2) on silica gave 9
as a colorless crystallizing oil, 387 mg (85%). The recorded data are
in accordance with those reported in ref 22.
The bioassay for inhibition of 5-LOX-mediated LTB4 formation was
carried out in a 96-well plate format with stimulated human polymor-
phonuclear leukocytes as described earlier (13) with slight modifications
(14). Zileuton (Sequoia, Oxford, U.K.; IC50 ) 5.0 µM) was used as
positive control.
Test samples were dissolved in absolute ethanol. Extracts were tested
at a final concentration of 20 µg/mL in the assay mixture and pure
compounds, at 50 µM. Samples were tested in at least three independent
experiments run in duplicate. Results are given as means ( SD.
For IC50 determination, active samples were tested in at least three
concentrations in at least three independent experiments, each time in
duplicate. Calculation of IC50 values was performed by semilogarithmic
presentation of dose versus activity and logarithmic regression analysis.
RESULTS AND DISCUSSION
Aqueous, 70% ethanolic, and n-heptane extracts prepared
from larch sawdust were investigated for their inhibitory activity
on prostaglandin formation by COX-1 and COX-2 and on LTB4
formation by 5-LOX. As shown in Figure 2, the lipophilic
n-heptane extract possessed pronounced inhibitory activity in
all three assays at the screening concentration of 20 µg/mL.
IC50 values for the n-heptane extract were found to be 5 µg/mL
for COX-1, 0.1 µg/mL for COX-2, and 11.1 µg/mL for the LTB4
assay. The 70% ethanolic and water extracts were less active.

11690 J. Agric. Food Chem., Vol. 56, No. 24, 2008
Pferschy-Wenzig et al.
Figure 1. Compounds 1-9.
Figure 2. COX-1, COX-2, and LTB4 formation inhibitory activities of larch sawdust water, 70% ethanol, and n-heptane extracts (screening concentration
) 20 µg/mL in the assay mixture).
The IC50 of the 70% ethanolic extract against COX-2 was found
to be 0.8 µg/mL; for the other assays IC50 values were not
determined due to the low activity at the screening concentration.
Due to its potent dual inhibitory effect against COX isoen-
zymes with a certain COX-2 selectivity, on the one hand, and
against LTB4 product formation, on the other hand, the
n-heptane extract was considered to be most interesting for
further phytochemical and pharmacological evaluation to iden-
tify the active principles.
Fractionation of the n-heptane extract resulted in the isolation
of a series of diterpenes (compounds 1-6, Figure 1). The
bioactivity of the compounds was evaluated in the COX-1,
COX-2, and LTB4 formation assays (Figure 3; Table 1). In
contrast to the findings for the crude n-heptane extract, for which

Anti-inflammatory Activity of Larch Sawdust
J. Agric. Food Chem., Vol. 56, No. 24, 2008 11691
Figure 3. COX-1, COX-2, and LTB4 formation inhibitory activities of compounds 1-9 at a screening concentration of 50 µM in the assay mixture.
Table 1. IC50 Values of the Active Diterpenes in the COX-2 and LTB4
Isopimaric acid (3), a pimarane type diterpene acid, was found
Formation Assays
to selectively inhibit LTB4 formation with an IC50 similar to
that of compound 2. Concerning the abietane type diterpenes
IC50 COX-2
IC50 LT
compound
larixol (1)
larixyl acetate (2)
isopimaric acid (3)
palustric acid (4)
dehydroabietic acid (5)
dehydroabietinol (6)
abietic acid (7)
abietic acid methyl ester (8)
abietinol (9)
isolated from the extract, dehydroabietinol (6) was one of the
most potent LTB4 formation inhibitors with an IC50 similar to
that of compounds 2 and 3. Dehydroabietic acid (5), possessing
a COOH instead of an OH group in position 4, was much less
active. Palustric acid (4), lacking the double bond in position
11, showed intermediate LTB4 formation inhibition and, in
addition, moderately inhibited COX-2.
Diterpene acids from conifers are known to possess various
bioactivities (for reviews see refs 24 and 25): Several abietane
acids display antimicrobial activity against a variety of micro-
organisms. Dehydroabietic acid derivatives have been shown
to act as antiulcer agents (26), and some abietane acids also
display cardiovascular effects. Also, some adverse effects of
diterpene acids are known: resin acids present in pulp and paper
mill effluents are known to be toxic for aquatic organisms such
as fish (27, 28). The allergenic and antiallergic effects of resin
acids are still a controversial subject; however, especially the
oxidation products of resin acids seem to possess a dermal
allergenic potential (24, 29).
Furthermore, some evidence exists concerning the anti-
inflammatory potential of diterpene acids: A mixture of laevo-
pimaric, abietic, neoabietic, palustric, and isopimaric acid in
combination with triglycerides has been suggested for the
external treatment of chronic diseases such as rheumatism and
gout (30). Abietic acid, which is also known to be present in
larch resin, has been described as an inhibitor of soybean
5-lipoxygenase (31). The compound also has been found to
inhibit PGE2 production in lipopolysaccharide-treated macroph-
ages in vitro and to inhibit rat-paw and mouse-ear edema after
oral or topical application in vivo (32).
Therefore, abietic acid (7) as well as its partial synthetically
prepared derivatives abietic acid methyl ester (8) and abietinol
(9) were tested for COX-1, COX-2, and LTB4 formation
inhibitory activities. Compound 9 was the most active of all
tested compounds, possessing an IC50 of 5.9 µM, which is about
the same as that of the positive control used in the assay (5
µM). The IC50 of compound 7 was in the range of that of
compounds 2, 3, and 6. Compound 8 was virtually inactive (IC50
> 125 µM).
assay (µM)
assay (µM)
>125
10.4
10.2
17.8
44.6
10.9
13.5
>125
5.9
95.1
57.9
a very high COX-2 inhibitory activity had been observed, the
isolated diterpenes were mainly active in the LTB4 formation
assay. They did not inhibit COX-1, and only compound 2, a
major constituent of the n-heptane extract, and compound 4,
which is present only in minor amounts, were found to possess
moderate COX-2 inhibitory activity. Their IC50 values were
determined to be 95.1 and 57.9 µM, respectively. Therefore,
compounds other than the isolated diterpenes must be respon-
sible for the COX-1 and high COX-2 inhibitory activity of the
crude n-heptane extract. One class of compounds that might
contribute to this activity is unsaturated fatty acids. Fatty acid
analysis of the fractions showing COX inhibitory activity after
the first VLC fractionation step revealed that these fractions
contained linoleic acid together with minor amounts of R-lino-
lenic acid and oleic acid. These compounds have been shown
to inhibit the COX isoenzymes as well as 5-LOX-mediated
LTΒ4 formation (12, 19), linoleic acid possessing a particularly
high COX-2 inhibitory activity (IC50 ) 0.5 µM) (23). Therefore,
it can be concluded that not the isolated diterpenes but the
unsaturated fatty acids present in the active fractions, probably
together with other, yet unidentified constituents, account for
the COX inhibitory activity of larch sawdust n-heptane extract.
However, some of the isolated diterpenes were found to be
good inhibitors of 5-LOX-mediated LTB4 formation. Larixol
(1) and larixyl acetate (2) are neutral labdane type diterpenes,
which differ only by the acetylation of the OH group in position
6. This acetylation seems to be necessary for LTB4 formation
inhibition as for larixyl acetate (2), a good inhibitory activity
was observed, whereas larixol (1) was inactive in this assay.
In conclusion, all tested abietane type diterpenes were
selective LTB4 formation inhibitors, except for compound 4.

11692 J. Agric. Food Chem., Vol. 56, No. 24, 2008
Pferschy-Wenzig et al.
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Compounds possessing a 7,13-abietadiene skeleton were better
LTB4 formation inhibitors than those with an 8,11,13-abi-
etatriene skeleton. Compound 4, possessing an 8,13-abietadiene
skeleton, showed intermediate LTB4 formation and moderate
COX-2 inhibitory activity. Compounds bearing an OH group
in position 4 were more active than those substituted with a
COOH group, and methylation of the COOH group led to an
almost complete loss of LTB4 formation inhibitory activity. The
only pimarane type diterpene tested (3) was a selective LTB4
formation inhibitor. Concerning the two labdane diterpenes
tested, only larixyl acetate (2) was a good LTB4 formation and
a moderate COX-2 inhibitor, whereas larixol (1) was inactive
in all three assays.
To be able to estimate the relevance of the bioactivities
observed in vitro for an anti-inflammatory potential in humans,
further studies will be necessary, as the pharmacokinetics of
the investigated compounds are virtually unknown. One study
has been performed concerning the biotransformation of dehy-
droabietic acid in rabbits after oral administration (33). Dehy-
droabietic acid also has been detected in the urine of humans
after inhalation exposure to rosin-based soldering fluxes (34);
however, no studies exist investigating the pharmacokinetics
of resin acids in humans after oral administration, and the
pharmacokinetics of larixol and larixyl acetate have not yet been
studied as well. Apart from that, the toxic and allergenic
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To summarize, the phytochemical and in vitro pharmacologi-
cal investigation of larch sawdust led to several interesting
results: On the one hand, a series of diterpene acids that
selectively inhibit 5-LOX-mediated LTB4 formation was iso-
lated. These compounds represent lead structures for the
development of leukotriene formation inhibitors. On the other
hand, the n-heptane extract and (to a lesser extent) compounds
2 and 4 were found to act as dual inhibitors of COX- and of
5-LOX-mediated LTB4 formation. Therefore, on the one hand,
these compounds might be lead structures for dual COX/LOX
inhibitors. On the other hand, the potent dual COX/LOX product
formation inhibition observed for the lipophilic extract makes
the further evaluation of larch sawdust and its lipophilic
constituents as potential anti-inflammatory agents highly inter-
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as the toxic and allergenic potential of the active constituents
will be necessary.
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COX, cyclooxygenase; EtO2, diethyl ether; EtOAc, ethyl
acetate; LOX, lipoxygenase; LTB4, leukotriene B4; MeCN,
acetonitrile; NSAID, nonsteroidal anti-inflammatory drug; PGE2,
prostaglandin E2; PGHS, prostaglandin H synthase; THF,
tetrahydrofurane;VLC, vacuum liquid chromatography.
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Antimalaria-Aktivita¨t. Ph.D. Thesis, Institute of Pharmaceutical
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ACKNOWLEDGMENT
Dr. Joachim Erler (Bionorica AG, Neumarkt, Germany) is
kindly acknowledged for providing the larch n-heptane extract.
We thank Elke Prettner (University Graz, Institute of Pharma-
ceutical Sciences, Department of Pharmaceutical Chemistry) for
measurement of the optical rotation of the isolated compounds.
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Received for review August 1, 2008. Revised manuscript received
October 10, 2008. Accepted October 23, 2008. This research was funded
by the Sixth Research Framework Program of the European Union
(No. 513949).
JF8024002