Chemical composition and antifungal activity of essential oil isolated from Chamaecyparis formosensis Matsum. Wood

Abstract

The chemical composition of the essential oil of Cha- maecyparis formosensis wood has been examined. GC- MS data and retention indices for reference samples were used to identify 32 constituents. a-Eudesmol (18.06%), b-guaiene (8.0%), (-)-b-cadinene (7.89%), g-costal (7.03%), a-muurolol (6.49%), 4a-hydroxy-4b- methyldihydrocostol (5.52%), s-selinene (4.78%), san- tolina triene (4.60%), eremophilene (4.32%), humulene (4.11%), myrtenol (4.11%), and t-cadinene (3.25%) were the most abundant components. Tests with the typical wood decay fungi, Laetiporus sulphureus and Trametes versicolor, proved the antifungal activity of the oil, as the growth of L. sulphureus and T. versicolor was inhibited at concentrations of 50 and 100 m gm l y 1 , respectively. The following characteristic volatile compounds were isolated and purified from ethyl acetate fractions: epi- cubenol, chamaecynone, myrtenol, cis-myrtanol, 12- hydroxyisointermedenol and 4a-hydroxy-4b-methyldi- hydrocostol. Chamaecynone possessed the strongest antifungal activity, with an antifungal index of 88.2% and 67.3% for L. sulphureus and T. versicolor at a dose of 50 m gm l y1, respectively.

Full text

Holzforschung, Vol. 59, pp. 295–299, 2005 •Copyright by Walter de Gruyter •Berlin •New York. DOI 10.1515/HF.2005.049
Chemical composition and antifungal activity of essential oil
isolated from Chamaecyparis formosensis Matsum. wood
Sheng-Yang Wang
1
, Chi-Lin Wu
2
, Fang-Hua
Chu
2
, Shih-Chang Chien
3
, Yueh-Hsiung Kuo
4
,
Lie-Fen Shyur
3,
* and Shang-Tzen Chang
2,
*
1
Department of Forestry, National Chung-Hsing
University, Taichung, Taiwan
2
School of Forestry and Resource Conservation,
National Taiwan University, Taipei, Taiwan
3
Institute of BioAgricultural Sciences, Academia Sinica,
Taipei, Taiwan
4
Department of Chemistry, National Taiwan University,
Taipei, Taiwan
*Corresponding author.
School of Forestry and Resource Conservation, National
Taiwan University, No. 1, Section 4 Roosevelt Road, Taipei
115, Taiwan
Tel.: q886-2-33664626
Fax: q886-2-23654520
E-mail: peter@ntu.edu.tw
Abstract
The chemical composition of the essential oil of Cha-
maecyparis formosensis wood has been examined. GC-
MS data and retention indices for reference samples
were used to identify 32 constituents. a-Eudesmol
(18.06%), b-guaiene (8.0%), (–)-b-cadinene (7.89%),
g-costal (7.03%), a-muurolol (6.49%), 4a-hydroxy-4b-
methyldihydrocostol (5.52%), s-selinene (4.78%), san-
tolina triene (4.60%), eremophilene (4.32%), humulene
(4.11%), myrtenol (4.11%), and t-cadinene (3.25%) were
the most abundant components. Tests with the typical
wood decay fungi, Laetiporus sulphureus and Trametes
versicolor, proved the antifungal activity of the oil, as the
growth of L. sulphureus and T. versicolor was inhibited
at concentrations of 50 and 100 mgml
y
1
, respectively.
The following characteristic volatile compounds were
isolated and purified from ethyl acetate fractions: epi-
cubenol, chamaecynone, myrtenol, cis-myrtanol, 12-
hydroxyisointermedenol and 4a-hydroxy-4b-methyldi-
hydrocostol. Chamaecynone possessed the strongest
antifungal activity, with an antifungal index of 88.2% and
67.3% for L. sulphureus and T. versicolor at a dose of
50 mgml
y
1
, respectively.
Keywords: Chamaecyparis formosensis; essential oil; a-
eudesmol; chamaecynone; antifungal activity; Laetiporus
sulphureus; Trametes versicolor.
Introduction
Wood preservatives are widely used all over the world.
Identification of the bioactive constituents of highly dura-
ble wood species and elucidation of the mechanisms of
biodeterioration can contribute to the development of
environmentally friendly wood protection systems. In our
previous studies (Chang et al. 1998, 1999, 2000, 2001)
we demonstrated that Taiwania wood (Taiwania crypto-
merioides), one of the most important plantation soft-
woods in Taiwan, is a species with excellent antifungal
and antitermitic properties.
Chamaecyparis formosensis Matsum is also a precious
wood in Taiwan because of its good wood quality and
fragrance and its outstanding durability. It is an endemic
tree that grows at elevations of 1500–2150 m in Taiwan’s
central mountains (Liu et al. 1988) and it is known as the
Taiwan red cypress. Many traditional Japanese-style
houses are constructed of C. formosensis. Its fragrance
is an indication of the presence of essential oils, which
are primarily composed of terpenes and their oxygenated
derivatives. Besides its use in perfumes, there is a long
history of its medicinal use. The oil shows antibacterial
and antifungal effects.
Kafuka and Ichikawa (1931) studied the volatile con-
stituents of the leaves of C. formosensis and found a-
pinene to be the major constituent (85%). In addition,
several terpenoids, including camphene, dipentene, cin-
eol, a-terpinene, b-terpinene, borneol, bornyl acetate,
bornyl formate, humulene, and cadinene, were identified.
Fang et al. (1986a) re-analyzed the leaf essential oils and
identified 41 terpenes. The predominant compound was
also found to be a-pinene and other major constituents
included b-pinene, 3-carene, a-terpinene, g-muurolene,
and kaurene. The root, bark, wood, cones, and leaves of
C. formosensis have also been studied (Nozoe et al.
1966; Fang et al. 1986b; Hsu et al. 1995). However, the
relationship between the essential oils of C. formosensis
wood and its antifungal properties has not been investi-
gated in detail. The aim of this study was to examine this
relationship using Trametes versicolor and Laetiporus sul-
phureus for antifungal testing.
Materials and methods
General instruments
High-performance liquid chromatography (HPLC) was carried
out using a Jasco model PU-980 pump equipped with an RI-
930 detector and a Hibar Lichrosorb Si 60 (25=1 cm i.d.) col-
umn. IR spectra were recorded on a Bio-Rad FTS-40 instrument.
Mass spectra (MS) were obtained on a Finnigan MAT-95S mass
spectrometer. NMR spectra were recorded on Bruker Avance
400- and 500-MHz Fourier transform (FT) NMR instruments.
Sample preparation and compound isolation
Logs from 80-year-old C. formosensis Matsum were collected
from the experimental forest of National Taiwan University.
Heartwood chips were prepared from a green cut tree. A sample

296 S.-Y. Wang et al.
Figure 1 Compounds isolated from C. formosensis.epi-Cub-
enol (1), chamaecynone (2), myrtenol (3), cis-myrtanol (4), 12-
hydroxyisointermedenol (5), 4a-hydroxy-4b-methyldihydro-
costol (6).
of 2 kg of the air-dried wood chips was subjected to hydrodis-
tillation for 8 h using a Clevenger-type apparatus. The oil (1.6%,
v/w) was dried above anhydrous Na
2
SO
4
. In a parallel experi-
ment, 10 kg of wood chips was exhaustively extracted with
methanol (MeOH). The extracts were condensed to 420 g and
then extracted with ethyl acetate (EtOAc) and after evaporation
the EtOAc-soluble fraction was obtained. The EtOAc fraction
(80 g) mixed with silica gel (160 g) was chromatographed on a
silica gel column (1.0 kg) by elution with gradients of hexane (n-
C
6
H
14
), EtOAc and MeOH. An aliquot of 500 ml of eluate was
collected for each fraction. TLC analyses were used to monitor
the chemical composition of each fraction. Fractions with similar
compositions were pooled to 45 fractions (EA-1 to EA-45). Com-
pounds 1–6 were obtained by HPLC separation and purification.
epi-Cubenol (1) (Kuo et al. 2002) wretention time (RT) 13.2 minx
and chamaecynone (2) (Nozoe et al. 1966) (RT 14.8 min) wSi-60
column, mobile phase EtOAc/n-C
6
H
14
(23:77), flow rate 1.5 ml
min
y
1
xwere isolated from the EA-7 fraction. Myrtenol (3) (Dea-
gostino et al. 2001) (RT 23.0 min) and cis-myrtanol (4) (Kim and
Lee 1997) (RT 25.0 min) were obtained from EA-11; however, the
mobile phase was changed to EtOAc/n-C
6
H
14
s25:75. 12-
Hydroxyisointermedenol (5) (Ahmed and Mahmoud 1998) was
purified from the EA-18 fraction with the same HPLC system at
20.0 min; the mobile phase was EtOAc/n-C
6
H
14
s50:50 at a flow
rate of 2.0 ml min
y
1
.4a-Hydroxy-4b-methyldihydrocostol (6)
(Gonzalez et al. 1992) in the form of colorless needle crystals
was separated from the EA-22 fraction during condensation.
Structures of six compounds isolated from C. formosensis were
identified using NMR, FTIR, and MS spectrometry. The spectral
data are consistent with those reported in the literature. The
structures of compounds isolated from C. formosensis are pre-
sented in Figure 1.
GC-MS analysis of essential oil
A Finnigan Trace GC–Polaris Q mass instrument (Finnigan-
Spectronex, USA) was used for GC-MS analysis with a fused
silica column (30 mm=0.25 mm i.d.) coated with SPB-50 (film
thickness 0.25 mm). The temperature program was as follows:
408C for 1 min, then increased at 58C min
y
1
to 2508C and held
for 10 min. The other parameters used were: injector tempera-
ture, 2508C; ion source temperature, 2008C; EI, 70 eV; carrier
gas, He at 1 ml min
y
1
; injection volume, 1 ml; split ratio, 1:50;
and mass range, 35–650 m/z. Quantification was by percentage
peak area. Identification of individual components was carried
out using the Wiley/NBS Registry of Mass Spectral Database
and a NIST MS Search. Chromatographic results expressed as
area percentages were calculated with a response factor of 1.0.
Antifungal assays
The fungi used were Trametes versicolor (L. ex Fr.) Quel. (BCRC
35253) and Laetiporus sulphureus (B. ex Fr.) Bond. (BCRC
35305). Antifungal assays were carried out in triplicate and the
data were averaged. Different concentrations of the essential oil
(50, 100, and 200 mgml
y
1
) were added to sterilized potato dex-
trose agar (PDA). Compounds isolated from the EtOAc fraction
were tested for their antifungal activity at a dose of 50 mgml
y
1
.
The test plates were incubated at 278C. When the mycelium of
fungi reached the edge of the control plate, the antifungal index
was calculated as follows:
Antifungal index (%)s(1–Da/Db)=100
where Da is the diameter of the growth zone in the experimental
dish (cm) and Db is the diameter of the growth zone in the con-
trol dish (cm).
Results and discussion
C. formosensis wood yielded 1.6% essential oil by steam
distillation. The following volatile compounds were iso-
lated, purified, and identified in the EtOAc fraction of
MeOH extracts: epi-cubenol (1); chamaecynone (2); myr-
tenol (3); cis-myrtanol (4); 12-hydroxyisointermedenol (5);
and 4a-hydroxy-4b-methyldihydrocostol (6). The results
of GC and GC/MS analyses of the C. formosensis essen-
tial oil are compiled in Table 1: a-eudesmol (18.06%),
b-guaiene (8.0%), (–)-b-cadinene (7.89%), g-costal
(7.03%), a-muurolol (6.49%), 4-a-hydroxy-4b-methyldi-
hydrocostol (5.52%), s-selinene (4.78%), santolina triene
(4.60%), eremophilene (4.32%), humulene (4.11%), myr-
tenol (4.11%), and t-cadinene (3.25%) were the main
constituents. a-Eudesmol with a yield of 18.06% is the
predominant compound. According to the results of Fang
et al. (1986a), the major compounds in leaf oil of C. for-
mosensis were: a-pinene (57.32%), b-pinene (3.25%),
3-carene (5.61%), a-terpinene (0.06%), g-muurolene
(1.54%), and kaurene (1.80%). Accordingly, there is a sig-
nificant difference between wood oil and leaf oil. Mono-
terpenes, such as a-pinene, b-pinene, camphene,
carene, limonene, etc., are predominant in the leaf oil
(Fang et al. 1986a). In the wood oil, however, we identi-
fied only the monoterpenes myrtenol, myrtenal, and
santolina triene. We found chamaecynone (a norsesqui-
terpene, C
14
) that does not seem to be present in the leaf
oil. Chamaecynone is a termiticide (Harayama and Inu-
bushi 1977). It is the first example of a natural acetylenic
compound of terpenoid origin (Nozoe et al. 1966).
In this study, 26 sesquiterpenes were identified. These
can be classified into 12 skeletal types, namely: copaene,
elemane, aromadendrane, guaiane, germacrane, humu-
lane, eremophilane, eudesmane, cadinane, longifolane,
acorane, and aristolane type (Figure 2).
Two representative fungal strains of Trametes versicol-
or (white rot fungus) and Laetiporus sulphureus (brown
rot fungus) were selected to test the antifungal activity of
the essential oils. The antifungal indices presented in
Table 2 are a clear demonstration of the excellent anti-
fungal property of the oil. The growth of L. sulphureus
and T. versicolor was completely inhibited at concentra-

Antifungal essential oil from Chamaecyparis formosensis 297
Table 1 Composition of wood essential oil from C. formosensis.
Peak Compound RT Concentration
No. (min) (%)
1cis-Myrtenol 17.02 4.11
2 Myrtenal 17.90 1.90
3 Santolina triene 19.03 4.60
4 (–)-b-Elemene 20.35 2.32
5 Isoledene 20.74 0.86
6 4,5-Dehydro-isolongifolene 21.94 0.13
7 Germacrene D 22.46 0.29
8a-Gurjunene 22.95 1.53
9t-Muurolene 23.07 0.78
10 (–)-Alloaromadendrene 23.37 1.35
11 Valencene 23.52 2.45
12 a-Muurolene 23.73 1.42
13 t-Cadinene 24.23 3.25
14 (–)-b-Cadinene 24.51 7.89
15 (–)-a-Cubebene 24.70 1.01
16 (–)-Calamenene 25.20 0.40
17 Humulene 25.61 4.11
18 Cadala-1,3,8-triene 26.09 0.25
19 t-Elemene 26.30 0.28
20 (q)-Calarene 26.91 0.76
21 7-(5-Hexynyl)-tricyclow4.2.2.0(2,5)xdec-7-ene 27.43 1.39
22 epi-Cubenol 27.59 2.63
23 s-Selinene 27.89 4.78
24 a-Muurolol 28.08 6.49
25 b-Guaiene 28.46 8.00
26 a-Eudesmol 28.58 18.06
27 Eremophilene 28.86 4.32
28 Chamaecynone 29.32 1.79
29 4a-Hydroxy-4b-methyldihydrocostol 29.99 5.52
30 2a-Hydroxycostol 30.40 2.68
31 Unidentified 30.70 0.28
32 12-Hydroxy-isointermedenol 31.21 2.22
33 g-Costal 31.83 7.03
34 Unidentified 32.67 0.11
35 Unidentified 34.93 0.36
Figure 2 Main sesquiterpenes skeletons of essential oils from C. formosensis. (a) copaane type, (b) elemane type, (c) aromadendrane
type, (d) guaiane type, (e) germacrane type, (f) humulane type, (g) eremophilane type, (h) eudesmane type, (i) cadinane type, (j)
longifolane type, (k) acorane type, and (l) aristolane type.

298 S.-Y. Wang et al.
Table 2 Antifungal indices of wood essential oil from C.
formosensis.
Dosage Antifungal index
(mgml
y
1
)T. versicolor L. sulphureus
50 62.4"6.6 100"0
100 100"0 100"0
200 100"0 100"0
Figure 3 Antifungal activity of the constituents (50 mgml
y
1
)of
C. formosensis against the white rot fungus Trametes versicolor
(white bar) and the brown rot fungus Laetiporus sulphureus
(black bar). Compound 1:epi-cubenol; Compound 2: chamae-
cynone; Compound 3: myrtenol; Compound 4:cis-myrtanol;
Compound 5: 12-hydroxyisointermedenol; Compound 6:4a-
hydroxy-b- methyldihydrocostol.
tions of 50 and 100 mgml
y
1
, respectively. The literature
also contains reports on similar effects of essential oils.
Tellez et al. (2000) studied the composition and biological
activities of the essential oil obtained by steam distillation
from Callicarpa americana and demonstrated that a-
eudesmol (9.4%) and humulene (10.0%) are the active
substances. Gurjunene is one of the major antifungal
constituents of the essential oil of Calea clematidea
(Flach et al. 2002). Substances such as copaene, myr-
tenol, germacrene D and a-muurolol were also reported
to be antifungal (Saito et al. 1996; Chang et al. 2000;
Gallori et al. 2001; Krauze-Baranowska et al. 2002). The
terpenoids from the EtOAc fraction were subjected to in-
depth analysis. Figure 3 shows the results for C. formo-
sensis at a dose of 50 mgml
y
1
. All compounds were
active, except epi-cubenol. Chamaecynone revealed a
significant inhibitory effect against T. versicolor and L.
sulphureus, as its antifungal index was 67.3% and 88.2%
at a dose of 50 mgml
y
1
, respectively. The pure com-
pounds isolated were less active than the crude essential
oil. This observation may be due to a synergistic effect
of all the constituents, or there may be other antifungal
compounds in the oil not yet detected.
Conclusions
The oil of C. formosensis is a fungicide. It was demon-
strated that volatile constituents of the wood oil contrib-
ute to the high decay resistance of this wood. Several
typical terpenoids with an antifungal effect were isolated
from C. formosensis; however, their antifungal perform-
ance was not as effective as that of the crude wood oil.
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