RESEARCH LETTER

Alpha-terpineol promotes triterpenoid production of Antrodia

cinnamomea in submerged culture
Zhen-Ming Lu1, Yan Geng1, Hua-Xiang Li1, Qing Sun1, Jin-Song Shi1 & Zheng-Hong Xu1,2
1
School of Pharmaceutical Science, Jiangnan University, Wuxi, China; and 2Tianjin Key Laboratory for Industrial Biological Systems and
Bioprocessing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China

Correspondence: Zheng-Hong Xu, School of Abstract

Pharmaceutical Science, Jiangnan University,
No. 1800 Lihu Avenue, Wuxi 214122, China.
Tel.: +86 510 85918206;
fax: +86 510 85918206;
e-mail: zhenghxu@jiangnan.edu.cn
Received 19 March 2014; revised 11 July
2014; accepted 15 July 2014. Final version
published online 14 August 2014.
DOI: 10.1111/1574-6968.12545
Editor: Michael Sauer
Keywords
Antrodia cinnamomea; Cinnamomum
camphora; triterpenoids; volatile compounds.
MICROBIOLOGY LETTERS
Antrodia cinnamomea is a medicinal mushroom producing potent bioactive trit-
erpenoids. However, triterpenoids of A. cinnamomea in submerged culture are
much less than those in fruiting bodies. Here we evaluated effects of different
extracts from a host-related species, Cinnamomum camphora, on the mycelial
growth and triterpenoid production of A. cinnamomea in submerged culture.
The hot water extract of the stem showed the strongest promotion of the mycelial
growth. The petroleum ether extract of the stem (PES) (0.05 g L 1) showed the
greatest stimulatory effect on content and production of triterpenoids. A total of
39 compounds including terpenoids, phenolic and aromatic compounds were
identified in the PES by GC-MS analysis. Furthermore, the effects of seven
compounds contained in the PES on the mycelial growth and triterpenoid
production of A. cinnamomea were evaluated. Among them, a-terpineol
(0.5 mg L 1) showed the greatest stimulatory effect on the triterpenoid content
(23.31 mg g 1) and triterpenoid production (91.33 mg L 1) of A. cinnamomea.
Results of LC–MS analysis showed that a-terpineol (0.5 mg L 1) stimulated
the syntheses of six triterpenoids in the mycelia of A. cinnamomea. This

indicates that a-terpineol can act as an elicitor for triterpenoid biosynthesis in

A. cinnamomea.

variety of bioactive metabolites, such as triterpenoids, ant-

Introduction
Antrodia cinnamomea (synonyms Antrodia camphorata,
Taiwanofungus camphoratus) is a treasured medicinal
mushroom only grown in Taiwan. It has been used as a folk
medicine by aboriginal tribes in Taiwan for many years to
treat food intoxication, diarrhea and hypertension, and to
enhance liver function (Wu et al., 2004; Jong, 2012). Stud-
ies have focused extensively on the biological activities of
the fruiting bodies and mycelia of A. cinnamomea; these
activities include hepatoprotection, anticancer and antioxi-
dant effects, vasorelaxation, and anti-inflammation (Ao
et al., 2009; Geethangili & Tzeng, 2011; Lu et al., 2013).
However, market demand for the fruiting bodies of A. cin-
namomea have increased due to its rare natural occurrence
and the difficulty of artificial cultivation (Du et al., 2012).
Currently, submerged fermentation has been applied in
industrial production to satisfy the large consumption
demand for this medicinal mushroom (Chen et al., 2011).
The submerged culture of A. cinnamomea contains a
roquinonol, 4-acetylantroquinonol B, ergostatrien-3b-ol,
antrodins, benzenoids, proteins, and polysaccharides
(Geethangili & Tzeng, 2011).
Recent studies have investigated the effect of natural
products on the culture of A. cinnamomea. For example,
the growth of A. cinnamomea in submerged culture was
promoted in the presence of water-soluble wood extracts
from the host (Cinnamomum kanehirae) and four host-
related species (Cinnamomum micranthum, Cinnamomum
osmophloeum, Cinnamomum camphora and Cinnamomum
kotoense); C. camphora showed the highest growth-pro-
moting activity (Shen et al., 2004). Four wood essential
oils from C. kanehirae, C. camphora, Cunninghamia
konishii, and Chamaecyparis formosensis also promoted
the growth of A. cinnamomea (Chang & Wang, 2008).
However, the natural growth factors have rarely been
used in the production of A. cinnamomea due to its com-
plex structures, and difficulty in characterization of active
ingredients.

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Lett 358 (2014) 36–43
Published by John Wiley & Sons Ltd. All rights reserved
a-Terpineol enhances triterpenoids of A. cinnamomea
In this study, we investigated the effect of solvent
extracts from C. camphora on the mycelial growth and
triterpenoid production of A. cinnamomea in submerged
culture. We further found that a-terpineol in C. camphora
was the active compound that promoted triterpenoid pro-
duction of A. cinnamomea.
Material and methods
Chemicals
L-Linalool,1-octen-3-ol, eucalyptol, ()-camphora, camph-
ene, a-pinene, a-terpineol, a-terpinene, trans-caryophyl-
lene, and oleanolic acid were purchased from Sigma-
Aldrich (St. Louis, MO). Peptone and yeast extract were
purchased from Oxoid Limited (Basingstoke, UK). Other
chemicals were of analytical grade and purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Strain and cultural condition
Antrodia cinnamomea ATCC 200183 was obtained from
the American Type Culture Collection (ATCC), USA. The
strain was maintained on potato dextrose agar (PDA)
slants. The slant was incubated at 26 °C for 14 days, and
then stored at 4 °C. The mycelia of A. cinnamomea were
transferred from stock slants to Petri dishes containing
PDA medium and incubated at 26 °C for 21 days to obtain
arthroconidia for subsequent inocula (Lu et al., 2011). The
arthroconidia were washed with 10 mL of 0.9% NaCl solu-
tion, diluted and adjusted to adequate concentration by
hemocytometer under light microscopy (Sun & Liu, 2006),
and used as inoculum for subsequent experiments. The ini-
tial arthroconidia concentration for submerged culture was
1.5 9 105 arthroconidia per milliliter. Each shaking flask
culture was carried out in a 500-mL Erlenmeyer flask con-
taining 100 mL of medium and incubated at 26 °C for
12 days by shaking at 100 r.p.m. The control medium for
submerged culture was 33 g glucose, 2.8 g peptone, 2.0 g
soybean flour, 2.0 g wheat bran extract, 2.0 g KH2PO4,
1.0 g MgSO4 per liter at an initial pH 4.5.
The extraction of stems and leaves from
C. camphora
Stems and leaves from C. camphora were collected on the
campus of Jiangnan University in autumn (Wuxi, China),
freeze-dried and then stored at 20 °C. The stem (100 g)
of C. camphora was extracted sequentially with 1 L of
petroleum ether, ethyl acetate, and methanol for 12 h at
room temperature with mixing. Filtration and collection
of the extracts were done three times. The filtrate was
concentrated by vacuum distillation using an rotary evap-
FEMS Microbiol Lett 358 (2014) 36–43
37
orator (R-210, Buchi, Switzerland) and an vacuum con-
troller (V-850, Buchi) maintained the temperature of
35 °C and the desired pressure. The solid residue after
organic solvent extractions was extracted with hot water
twice at 90 °C (each 1 L for 1 h). The combined water-
soluble fraction was then collected for lyophilized after
filtration. The various extracts were further dissolved with
dimethylsulfoxide for further use. The extracts were
stored at 20 °C before being used for bioactivity tests.
The extracts of leaf from C. camphora were prepared as
described above. Table 1 shows the extraction rates for
the stems and leaves obtained from C. camphora.
Gas chromatography–mass spectrometry
GC–MS analysis was performed on a Finnigan TRACE
GC–MS (Thermo Quest Finnigan Co.) equipped with a
PEG 20 M capillary column (30 m 9 0.25 mm 9
0.25 lm). Helium (flow rate, 1.0 mL min 1) was used as
the carrier gas. The GC oven temperature was maintained
initially at 40 °C for 3.0 min, followed by increases to
90 °C at a rate of 5 °C min 1, and from 90 °C to 230 °C
at a rate of 10 °C min 1, with this temperature then held
constant for 7 min. The injection volume was 0.01 mL
and the flow rate was 10 mL min 1. The mass spectra
were acquired with a source temperature of 200 °C,
under a 70.4 eV ionization potential. The ionization
mode was EI+, emission current was 0.2 mA, and detec-
tor voltage was 350 V. n-Alkanes were run under the
same conditions as the samples to calculate the Kovats
index (KI) of compounds.
Quantification of triterpenoids
Triterpenoids were quantified as described by the colorimet-
ric method with the vanillin–acetic acid system (Lu et al.,
2011). Olenolic acid (0.2 g L 1) was used as standard to
prepare the standard curve, which was used as a benchmark
for determination of the yield of triterpenoids.
Liquid chromatography–mass spectrometry
Dried mycelia (600 mg) of A. cinnamomea were extracted
using methanol (40 mL) for 12 h at room temperature.
Table 1. Yields of stem and leaf extracts from Cinnamomum
camphora
Extraction rate (g 100 g 1)
Petroleum
C. camphora ether Ethyl acetate Methanol Hot water
Stem 2.00 0.73 8.01 21.03
Leaf 2.02 1.77 10.04 24.07
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
38 Z.-M. Lu et al.

After removal of the mycelia by centrifugation, the super-
natants were collected and filtered through a 0.45-lm
membrane, and the filtrate was analyzed for triterpenoids
by LC–MS.
An Agilent 1260 series HPLC system (Agilent) was
used. A Waters XBridge C18 reversed-phase analysis col-
umn (4.6 mm 9 250 mm, 5 lm) was used for the sepa-
ration of compounds in extract. Gradient elution using
solvent A (water, containing 0.5% formic acid) and sol-
vent B (acetonitrile) as the mobile phase at a flow rate of
1.0 mL min 1 was applied. The gradient conditions were:
0–10 min, 5–20% B; 10–40 min, 20–50% B; 40–70 min,
50–80% B; 70–90 min, 80–90% B; 90–110 min, 90–100%
B; 110–120 min, 100% B; 120–121 min, 100–5% B; 121–
130 min, 5% B.
The MS experiment was performed with a Bruker
micrOTOF-QII (Bruker, Germany) quadrapole mass
spectrometer. The system was operated in electrospray
ionization (ESI) with positive ionization mode. Nitrogen
was used both as a drying gas at a flow rate of 8 L min 1
and as nebulizing gas at a pressure of 1 bar. The drying
gas temperature was 330 °C, and a potential of –4000 V
in negative ionization was applied across the capillary.
Quadrupole 1 filtered the calculated m/z of each com-
pound of interest, and quadrupole 2 scanned for ions pro-
duced by nitrogen collision of these ionized compounds
in the range 50–1000 m/z. The identification of triterpe-
noids was obtained by matching their molecular ions (m/
z) obtained by ES positive ionization with those references
of isolated compounds. The peak area of specific triterpe-
noid was obtained by integration of the base peak (the
highest peak) in the extract ion chromatogram (EIC).
Statistical analysis
Experiments were carried out at least three times, with at
least three replicates per experiment. The data are pre-
sented as the mean  standard deviation (SD) and the
groups were accompanied by one-way analysis of variance
(ANOVA). Dunnett’s t-test (two-sided) was used for deter-
mining the significance. Differences at P < 0.05 level were
considered statistically significant.
Results
Effects of C. camphora extracts on the biomass
and triterpenoid production of A. cinnamomea
in submerged culture
As shown in Table 2, the presence of petroleum ether,
ethyl acetate, methanol, and hot water extracts of stems
and leaves from C. camphora promoted mycelial growth
of A. cinnamomea dose dependent on submerged culture.
Among them, the higher dose of hot water extract of the
stem gave the highest biomass (4.69  0.19 g L 1). A
previous study also showed that water-soluble extract of
the wood of C. camphora promoted the hyphal growth of
A. cinnamomea (Hsu et al., 2006).
For the triterpenoid production of A. cinnamomea,
both petroleum ether extract and ethyl acetate extract of

Table 2. Effects of Cinnamomum camphora extracts on the biomass, triterpenoid content and triterpenoid production of Antrodia cinnamomea

Extract Concentration (g L 1) Biomass (g L 1) Triterpenoid content (mg g 1) Triterpenoid production (mg L 1)

Control – 3.00  0.11 16.22  0.64 48.68  1.93
DMSO 3.08  0.13 16.07  0.63 49.43  1.91
PEL 0.20 3.15  0.14 18.32  0.75 57.74  2.23*
0.05 3.04  0.12 21.98  0.82* 66.79  2.65*
PES 0.20 3.41  0.15* 19.45  0.79 66.42  2.61*
0.05 3.34  0.13* 23.51  0.84* 78.49  2.86*
EEL 0.18 3.19  0.12 18.70  0.76 59.62  2.36*
0.04 3.23  0.14 21.75  0.81* 70.19  2.65*
EES 0.07 3.38  0.15 19.56  0.78* 66.04  2.57*
0.02 3.00  0.12 19.49  0.76* 58.49  2.34*
MEL 1.00 3.68  0.16* 15.39  0.62 56.60  2.21*
0.25 3.19  0.13 15.50  0.63 49.43  2.03
MES 0.80 3.45  0.14* 16.07  0.65 55.47  2.22
0.20 3.08  0.12 15.33  0.61 47.17  1.89
WEL 2.41 4.13  0.18* 12.34  0.46* 50.94  2.05
0.60 3.56  0.15* 12.17  0.45* 43.40  1.82
WES 2.10 4.69  0.19* 12.47  0.47* 58.49  2.35*
0.53 3.79  0.17* 14.14  0.52* 53.58  2.24
Data represent means  SD (n = 3), *P < 0.05 vs. control.
EEL, ethyl acetate extract of leaf; EES, ethyl acetate extract of stem; MEL, methanol extract of leaf; MES, methanol extract of stem; PEL, petro-
leum ether extract of leaf; WEL, hot water extract of leaf; WES, hot water extract of stem.

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Lett 358 (2014) 36–43
Published by John Wiley & Sons Ltd. All rights reserved
a-Terpineol enhances triterpenoids of A. cinnamomea 39

25.53
100

90

80
11.11

Relative abundance
70

60

50
20.35
40

30
19.07
20 23.85 31.48
6.42 28.45
17.46
10 4.30 8.60
9.51 14.96
0
0 5 10 15 20 25 30
Time (min)

Fig. 1. GC–MS total ion chromatogram for the volatile compounds identified in petroleum ether extract of stems from Cinnamomum camphora.
2.97, 3-methyl-hexane; 3.13, triacontane; 3.23, 2-methyl-1-hexadecanol; 3.45, 11-octadecenal; 4.30, benzene; 5.98, a-pinene; 6.42, methyl
benzene; 7.15, camphene; 8.18, 2-b-pinene; 8.80, sabinene; 10.92, a-terpinene; 11.11, eucalyptol; 12.13, 3-carene; 12.42, styrene; 16.88,
a-cubebene; 17.46, copaene; 17.91, camphor; 18.67, a-santalene; 18.85, a-bergamotene; 19.07, trans-caryophyllene; 19.12, 4-terpineol; 19.22,
aromadendrene; 20.05, a-humulene; 20.27, c-muurolene; 20.35, a-terpineol; 20.55, germacrene-d; 20.63, isoledene; 20.70, eremophilene;
20.75, a-selinene; 20.86, c-elemene; 21.11, d-cadinene; 21.96, c-cadinene; 21.43, 1,2,3,4,4a,7-hexahydro-1,6-dimethyl-4-(1-methylethyl)-
naphthalene; 22.46, safrole; 23.76, caryophyllene oxide; 23.85, methyl eugenol; 25.08, spathulenol; 25.42, eugenol; 25.53, trans-methyl
isoeugenol.

stem and leaf from C. camphora significantly elevated trit-
erpenoid production. The maximum of triterpenoid con-
tent (23.51  0.84 mg g 1) was obtained in the presence
of a lower dose of petroleum ether extract of stem (PES)
of C. camphora, which is 1.45-fold that obtained from the
control group.
Identification of volatile compounds for
petroleum ether extract of stem from
C. camphora
The GC–MS total ion chromatogram for the volatile
compounds in the PES is shown in Fig. 1. We identified
a total of 39 (81.83% of the total) compounds (Table 3).
Among them, terpenoids (50.86%) predominated, includ-
ing nine monoterpenoids (30.57%) and 19 sesquiterpe-
noids (20.29%). There were also phenolic and aromatic
compounds in the PES.
Effects of compounds from PES on the biomass
and triterpenoid production of A. cinnamomea
in submerged culture
The effect of seven commercially available compounds
from PES on the biomass and triterpenoid production of
A. cinnamomea in shaking flasks was studied. As shown
in Table 4, except for the eucalyptol and trans-caryophyl-
lene, higher doses (1.0 mg mL 1) of the other com-
pounds significantly inhibited the mycelial growth of
A. cinnamomea. At lower doses, eucalyptol (0.5 mg L 1,
0.1 mg L 1), camphora (0.1 mg L 1) and camphene
(0.5 mg L 1) elevated the biomass of A. cinnamomea.
The medium containing camphora (0.1 mg L 1) gave the
highest yield of biomass (4.24  0.17 g L 1).
We also determined the triterpenoid production of
A. cinnamomea. The results (Table 4) showed that a-ter-
pineol (0.5 mg L 1, 0.1 mg L 1) and camphora (0.5 mg
L 1) promoted the triterpenoid content of A. cinnamo-
mea. a-Terpineol (0.5 mg L 1), camphora (0.5 mg L 1,
0.1 mg L 1), camphene (0.5 mg L 1), and trans-caryo-
phyllene (0.1 mg L 1) promoted the triterpenoid produc-
tion of A. cinnamomea. Among the seven compounds,
a-terpineol showed the most efficient promotion of trit-
erpenoid production, which was 1.36-fold of that
obtained from the control group. Further studies are
needed to elucidate the effects of other compounds in the
PES on the triterpenoid production of A. cinnamomea.
Effects of a-terpineol on triterpenoid
biosynthesis of A. cinnamomea in submerged
culture
LC–MS analyses were performed on methanol extracts of
A. cinnamomea mycelia cultured with and without a-ter-
pineol (0.5 mg L 1). Seven triterpenoids – antcin h, antcin
k, antcin e, ergosterol peroxide, 3b,15 a-dihydroxylanosta-
7,9(11),24-trien-21-oic acid, ergosta-4,6,8(14),22-tetraen-
3-one, and sulfurenic acid – were identified in the
mycelia of A. cinnamomea (Table 5). Extract ion chroma-
tograms of these seven specific triterpenoids are shown

FEMS Microbiol Lett 358 (2014) 36–43 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
40 Z.-M. Lu et al.

Table 3. Volatile compounds identified in the petroleum ether stem been well studied (Phillips et al., 2006). Recent studies
extracts from Antrodia camphora have not only identified lots of novel triterpenoids in
Retention Relative A. cinnamomea (Cherng et al., 1995, 1996; Chen et al.,
time Compound peak area (%) 2010; Huang et al., 2012), but also confirmed some
2.97 3-Methyl-hexane 1.47 pharmacological properties of several triterpenoids (Ao
3.13 Triacontane 1.26 et al., 2009; Lee et al., 2012). However, the synthetic
3.23 2-Methyl-1-hexadecanol 1.53 pathway of triterpenoids in A. cinnamomea is unclear and
3.45 11-Octadecenal 0.86 the amount of triterpenoids arise much less in the myc-
4.30 Benzene 1.14
elia produced by submerged fermentation than those pro-
5.98 a-Pinene 0.50
duced in the fruiting bodies of A. cinnamomea (Wang
6.42 Methyl benzene 3.12
7.15 Camphene 0.17 et al., 2012).
8.18 2-b-Pinene 0.68 By optimizing the fermentation conditions using the
8.60 Sabinene 1.25 response surface methodology, Chang et al. reported that
10.92 a-Terpinene 0.26 the triterpenoid content of A. cinnamomea reached
11.11 Eucalyptol 18.46 31.8 mg g 1 in flask cultures (Chang et al., 2006). Lu
12.13 3-Carene 0.66
et al. (2011) used artificial intelligence-based techniques
12.42 Styrene 0.43
to optimize fermentation medium for triterpenoid pro-
16.88 a-Cubebene 0.65
17.46 Copaene 1.79 duction from A. cinnamomea; triterpenoid production
17.91 Camphor 0.26 was 64.79 mg L 1 (Lu et al., 2011). Triterpenoid produc-
18.67 a-Santalene 0.51 tion was significantly increased when 0.3% olive oil was
18.85 a-Bergamotene 0.25 added at the beginning of fermentation and, using the
19.07 trans-Caryophyllene 4.45 uniform design method, the triterpenoid production
19.12 4-Terpineol 0.79
experimentally obtained under the optimal conditions
19.22 Aromadendrene 0.28
was 72.3% (He et al., 2012). A two-stage submerged
20.05 a-Humulene 3.85
20.27 c-Muurolene 0.39 fermentation by means of the oxygen limitation and
20.35 a-terpineol 7.80 temperature fluctuation strategies with chitosan
20.55 Germacrene-D 1.60 (100 mg L 1) as a chemical elicitor was found to increase
20.63 Isoledene 0.37 triterpenoid production of A. cinnamomea (Liu et al.,
20.70 Eremophilene 0.47 2012).
20.75 a-Selinene 0.39
In this study, we identified a-terpineol from the host-
20.86 c-Elemene 0.64
related species C. camphora that could promote the growth
21.11 d-Cadinene 2.49
21.16 c-Cadinene 0.22 as well as triterpenoid content and triterpenoid production
21.43 1,2,3,4,4a,7-Hexahydro-1, 1.00 of A. cinnamomea in submerged culture. a-Terpineol was
6-dimethyl-4-(1-methylethyl)-naphthalene identified as one of the major volatile compounds in the
22.46 Safrole 0.25 wood of host species C. kanehirae. a-Terpineol can also be
23.76 Caryophyllene oxide 0.64 found in the fruiting bodies of A. cinnamomea but cannot
23.85 Methyl eugenol 2.81
be found in cultured broth through GC–MS analysis (Chen
25.08 Spathulenol 0.41
et al., 2007). Because the fruiting bodies only grow on
25.42 Eugenol 0.32
25.53 trans-Methyl isoeugenol 17.41 rotten trunks of C. kanehirae, it is suggested that a-terpin-
eol may act as a signaling molecule to stimulate triterpe-
noid production in A. cinnamomea. Further study showed
in Supporting Information, Figs S1–S7. Compared with that syntheses of six specific triterpenoids –antcin H,
the mycelia of the control group, the peak area of sulfure- antcin K, antcin E, ergosterol peroxide, 3b,15 a-dihydroxy-
nic acid in the mycelia cultured with a-terpineol lanosta-7,9(11),24-trien-21-oic acid and ergosta-4,6,8
(0.5 mg L 1) did not change significantly, whereas those of (14),22-tetraen-3-one – were stimulated by a-terpineol
the other six triterpenoids simultaneously increased (0.5 mg L 1) (Table 5). Production of these ergostane trit-
(Table 5). erpenoids, such as antcin H (Zhankuic acid C), antcin K
and antcin E, is associated with the basidiomatal formation
of A. cinnamomea (Chang et al., 2011). However, the
Discussion
content of sulfurenic acid in the mycelia was not affected
Triterpenoids are secondary metabolites of plants, fungi, by a-terpineol (0.5 mg L 1). The precise mechanism
and bacteria with multiple biological activities (Petronelli mediating the stimulation of triterpenoid production by
et al., 2009). Their biosynthetic pathways in plants have a-terpineol should be investigated further.

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Lett 358 (2014) 36–43
Published by John Wiley & Sons Ltd. All rights reserved
a-Terpineol enhances triterpenoids of A. cinnamomea 41

Table 4. Effects of compounds from Cinnamomum camphora on the biomass, triterpenoid content and triterpenoid production of Antrodia
cinnamomea in submerged culture

Compound Concentration (mg L 1) Biomass (g L 1) Triterpenoid content (mg g 1) Triterpenoid production (mg L 1)
Control – 3.72  0.14 18.05  0.68 67.17  2.53
Eucalyptol 1.0 3.74  0.15 15.09  0.58 56.44  2.16
0.5 3.81  0.16 17.08  0.63 64.99  2.56
0.1 3.85  0.16 15.47  0.58 59.59  2.38
Camphora 1.0 2.03  0.08* 13.90  0.53 28.25  1.13
0.5 3.50  0.12 20.85  0.78* 73.01  2.77*
0.1 4.24  0.17* 17.46  0.66 74.04  2.79*
Camphene 1.0 3.37  0.11 16.70  0.67 56.28  2.15
0.5 3.88  0.14 18.09  0.69 70.18  2.75*
0.1 3.74  0.15 18.03  0.67 67.44  2.63
a-Pinene 1.0 2.17  0.09* 14.02  0.56 30.42  1.22
0.5 3.01  0.11 15.98  0.58 48.09  1.91
0.1 3.49  0.12 16.74  0.63 58.42  2.31
a-Terpineol 1.0 2.04  0.08 7.35  0.26 14.99  0.61
0.5 3.92  0.15 23.31  0.85* 91.33  3.46*
0.1 3.38  0.13 20.06  0.76* 67.80  2.67
c-Terpinene 1.0 2.77  0.11 12.97  0.46 35.93  1.44
0.5 3.37  0.12 18.39  0.71 61.96  2.47
0.1 3.60  0.11 18.96  0.73 68.25  2.71
trans-Caryophyllene 1.0 3.68  0.13 16.89  0.64 62.15  2.49
0.5 3.73  0.14 16.51  0.63 61.58  2.47
0.1 3.79  0.15 18.39  0.72 69.68  2.81*
Data represent means  SD (n = 3), *P < 0.05 vs. control.

Table 5. Effect of a-terpineol (0.5 mg L 1) on triterpenoids of Antrodia cinnamomea in submerged culture

Peak area‡

a-Terpineol
RT* (min) Compounds† Formula m/z Reference Control (0.5 mg g 1)
18.5 Antcin H C29H42O6 487.3054 Chen et al. (1995) 46 776 52 091
19.0 Antcin K C29H44O6 489.3210 Shen et al. (2003) 76 572 88 457
20.0 Antcin E C29H40O6 485.2898 Cherng et al. (1996) 50 710 53 470
53.0 Ergosterol peroxide C28H44O3 429.3363 Wang (2004) 23 4803 33 2011
69.3 3b,15 a-Dihydroxylanosta-7,9(11),24-trien-21-oic acid C30H46O4 471.3469 Shen et al. (2003) 37 847 81 818
83.0 Ergosta-4,6,8(14),22-tetraen-3-one C28H40O 393.3152 Wu & Chiang (1995) 52 650 68 605
83.3 Sulfurenic acid C30H48O5 489.3574 Shen et al. (2003) 56 4623 565 000
*Retention time.
†
Identification was compared with those of identified compounds from references.
‡
Peak area of specific triterpenoid was obtained by integration of the base peak (the highest peak) in the extract ion chromatogram (EIC).

Many natural inducers for the growth and triterpe-
noid biosynthesis of A. cinnamomea might exist in the
C. camphora, in view of the potent host-specificity of this
fungus. In this study, we identified 39 volatile compounds
from PES of C. camphora. However, only seven com-
pounds that are commercially available were tested for
their stimulatory effects on the biomass and triterpenoid
production of A. cinnamomea. It will be of interest to
identify other potent inducers from the host-related spe-
cies in further study.
In summary, our study showed that a-terpineol from
C. camphora possessed a significant stimulatory effect
on the triterpenoid production of A. cinnamomea. The
biosyntheses of six triterpenoids – antcin h, antcin k, ant-
cin e, ergosterol peroxide, 3b,15 a-dihydroxylanosta-7,9
(11),24-trien-21-oic acid and ergosta-4,6,8(14),22-tetraen-
3-one – were all stimulated, whereas the biosynthesis of
sulfurenic acid was not affected. It is suggested that a-ter-
pineol can act as an elicitor for triterpenoid biosynthesis
in A. cinnamomea. However, the mechanism of action is
not fully understood and needs further study.
Acknowledgements
This work was supported by grants from the Industry-Aca-
demia Cooperation Innovation Fund Project of Jiangsu

FEMS Microbiol Lett 358 (2014) 36–43 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
42
Province, China (Grant No. BY2012052) and the National
Natural Science Foundation of China (Grant No.
31201020). We thank Yi Cai, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, China, for
his help with the LC–MS analysis.
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FEMS Microbiol Lett 358 (2014) 36–43
a-Terpineol enhances triterpenoids of A. cinnamomea
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Extract ion chromatogram of antcin H (18.5 min)
in the methanol extract of Antrodia cinnamomea mycelia
cultured with (a) and without (b) a-terpineol (0.5 mg L 1).
Fig. S2. Extract ion chromatogram of antcin K (19.0 min)
in the methanol extract of Antrodia cinnamomea mycelia
cultured with (a) and without (b) a-terpineol (0.5 mg L 1).
Fig. S3. Extract ion chromatogram of antcin E (20.0 min) in
the methanol extract of Antrodia cinnamomea mycelia cul-
tured with (a) and without (b) a-terpineol (0.5 mg L 1).
Fig. S4. Extract ion chromatogram of ergosterol peroxide
(53.0 min) in the methanol extract of Antrodia cinnamomea
FEMS Microbiol Lett 358 (2014) 36–43
43
mycelia cultured with (a) and without (b) a-terpineol
(0.5 mg L 1).
Fig. S5. Extract ion chromatogram of 3b,15 a-dihydroxylan-
osta-7,9(11),24-trien-21-oic acid (69.3 min) in the methanol
extract of Antrodia cinnamomea mycelia cultured with (a)
and without (b) a-terpineol (0.5 mg L 1).
Fig. S6. Extract ion chromatogram of ergosta-4,6,8(14),22-
tetraen-3-one (83.0 min) in the methanol extract of Antro-
dia cinnamomea mycelia cultured with (a) and without (b)
a-terpineol (0.5 mg L 1).
Fig. S7. Extract ion chromatogram of sulfurenic acid
(83.3 min) in the methanol extract of Antrodia cinnamomea
mycelia cultured with (a) and without (b) a-terpineol
(0.5 mg L 1).
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved