Botanical Studies (2006) 47: 267-272.

*

Corresponding author: E-mail: jcchou@mail.ndhu.edu.tw;

Phone: 886-3-8633645; Fax: 886-3-8633630.

INTRODUCTION

Antrodia cinnamomea is a medicinal fungus that grows

naturally inside the Cinnamonum kanehirae trunk, a native

tree species of Taiwan (Chang and Chou, 1995; 2004; Wu

et al., 1997). The medicinal use of A. cinnamomea was

first discovered by native Taiwanese, who used it as an

antidote for alcohol intoxication. Recently, many studies

indicate that its medicinal applications go far beyond the

original usage. It has been reported that many chemical

components of A. cinnamomea carry functional properties

like anti-oxidant (Song and Yen, 2002; Hseu et al., 2002),

anti-cancer (Chen and Yang, 1995), anti-virus (Lee et al.,

2002), and antibiotic properties (Chen and Yang, 1995).

Therefore, demand for A. cinnamomea has far exceeded

the supply, and it is now considered among the most

expensive herbal medicines on the market (more than 5

US dollars per gram of the fresh fungal fruiting body).

The high demand is causing a serious conservation

issue since people aggressively harvest the wild A.

cinnamomea fruiting body by cutting off the C. kanehirae

trunk and endangering the tree species, which is unique

to Taiwan. In an effort to resolve the conservation issue

without sacrificing the medicinal benefits, scientists from

academia and the pharmaceutical industry have been

intensively working to develop A. cinnamomea products in

the laboratory. One major approach of the laboratory study

is to culture the fungus in hyphae forms and optimize the

chemical composition, especially production of secondary

metabolites, by culturing the hyphae (Song and Yen, 2002;

2003). Several studies have shown this approach might

be feasible, and, therefore, many commercial products

have been produced. However, no detailed clinical trial

has yet been reported, and all commercial products are

categorized as health food rather than medicine.

Another approach is to mimic the fungal growth

conditions in the laboratory to culture the fungal

fruiting body. This approach successfully grows the A.

cinnamomea fruiting body on the C. kanehirae trunk but

not on other plant species; hence, it does not resolve the

conservation issue. The best solution would be to culture

the fungus and grow the fruiting body on agar plates with

commercially available nutrient media. No scientific

report on this method had been published until very

recently when Chang and Wang (2005) reported an in vitro

fruiting body formation of an A. cinnamomea isolate on

malt extract agar (MEA) and potato dextrose agar (PDA)

media. Here, we present our efforts in another approach to

induce the growth of A. cinnamomea fruiting body in vitro

through a novel wounding procedure. The report should

stimulate new thinking on the study of A. cinnamomea

fruiting body formation under in vitro conditions.

MICROBIOLOGY

In vitro induction of fruiting body in Antrodia

cinnamomea – a medicinally important fungus

Jyh-Yuan LIN, Tzong-Zeng WU, and Jyh-Ching CHOU*

Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Shou-Feng, Hualien 97401,

TAIWAN

(Received October 7, 2005; Accepted December 30, 2005)

ABSTRACT.

The fruiting body of medicinal fungus, Antrodia cinnamomea, is a unique traditional medicine

originally used by native Taiwanese. Antrodia cinnamomea specifically grows inside the rotten trunk of

Cinnamonum kanehirae, an important native tree species in Taiwan. In vitro culture of A. cinnamomea on

agar plates to induce fruiting body formation has been shown difficult since many of its physiological and

developmental processes are unclear. Laboratory culture of A. cinnamomea on the C. kanehirae trunk showed

fruiting body formation occurred on the peripheral and lower sides of trunk, indicating that orientation had

played an important role. In addition, humidity and aeration also affected fruiting body formation. Physical

wounding of red hyphae was found to induce fruiting body formation on agar plate. Methanol extracts of

white, red hyphae, wildly grown and in vitro grown fruiting bodies analyzed by HPLC showed a distinct

pattern between hyphae and fruiting bodies.

Keywords: Abiotic stress; Antrodia cinnamomea; HPLC; Secondary metabolite; Wound induction.

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Botanical Studies, Vol. 47, 2006

MATERIALS AND METHODS

Fungal strains and chemicals

The strain ACI3 of A. cinnamomea was isolated from

a rotten C. kanehirae trunk containing A. cinnamomea

fruiting body and identified as A. cinnamomea based on

comparison of its morphological features with a standard

A. cinnamomea strain purchased from the Food Industry

Research and Development Institute (Catalog number

BCRC35716), Hsinchu, Taiwan. In addition, a molecular

comparison of strain ACI3 was performed to confirm the

taxonomic identification as mentioned in the following

section. All the chemicals used were of analytical grade or

higher.

Molecular identification of A. cinnamomea

based on 18S rRNA DNA sequence

Genomic DNA of A. cinnamomea was purified based

on the protocol of QIAamp DNA Mini Kit (Catalog

number 51304, QIAGEN Inc., Valencia, CA, USA). Four

universal PCR primers were synthesized for 18S rRNA

DNA amplification based on the report of White et al.

(1990). The primers were NS1 (GTA GTC ATA TGC TTG

TCT C), NS3 (GCA AGT CTG GTG CCA GCA GCC),

NS4 (CTT CCG TCA ATT CCT TTA AG), and NS8 (TCC

GCA GGT TCA CCT ACG GA). The PCR thermo-cycling

program was 98oC for 2 min, followed by 35 cycles of

95oC for 45 s, 52oC for 45 s and 72oC for 2 min, and final

step as 72oC for 10 min. The PCR products were analyzed

on 1% TAE agarose gel and purified based on protocol of

QIAGEN MiniElute Gel Extraction Kit (Catalog number

28606, QIAGEN Inc., Valencia, CA, USA). The purified

DNA was subjected to DNA sequencing analysis. Pairwise

alignment was performed based on the software tools of

NCBI web site: http://www.ncbi.nlm.nih.gov.

Growth of A. cinnamomea

Growth of A. cinnamomea was observed on a 20

×20×40 cm C. kanehirae trunk inoculated with A.

cinnamomea hyphae. The inoculated trunk was placed

in a closed container at room temperature and in a dark

environment and with water barely touching the trunk

to maintain a high moisture environment. The MEA

plates were prepared based on Blakeslee’s composition

(one liter medium containing 20 g malt extract, 20 g

glucose, 1 g peptone and 20 g agar) and were used to

culture and maintain the A. cinnamomea mycelia. The

malt extract broth (MEB) was prepared as MEA without

addition of agar for liquid suspension culture. The MEA

plates or MEB suspension culture inoculated with A.

cinnamomea mycelia were kept in an incubator at 28oC.

It took about one month for mycelia to fully cover a 10

cm (diameter) petri dish. These plates were subjected to

further experimental treatments for induction of fruiting

body. Taking a clue from A. cinnamomea’s natural habitat,

various concentrations of camphor oil produced by C.

kanehirae were applied to the MEA plates to see whether

it would influence the growth of A. cinnamomea. Th e

experimental treatments were MEA medium alone as the

control, MEA medium containing 1% Tween 20 (v/v)

only, MEA medium containing 1% Tween 20 and 0.2%

camphor oil, MEA medium containing 1% Tween 20 and

0.4% camphor oil, MEA medium containing 1% Tween

20 and 0.6% camphor oil, and MEA medium containing

1% Tween 20 and 0.8% camphor oil. The 1% Tween 20

was applied to help dissolve the camphor oil in the MEA

medium.

Abiotic factors affecting growth phase of A.

cinnamomea

Acetic acid

. A hole was made in the MEA plate, and

different amounts of acetic acid were applied through the

hole. After the acetic acid had fully diffused into the agar

plates, they were kept upside down on a fully moistened

filter paper to have a highly moist environment. The

growth of A. cinnamomea was observed and compared

with the same cultural plates to which no acetic acid was

added.

Air exchange

. The covers of MEA plates containing

three-week-old A. cinnamomea hyphae were removed, and

the plates were kept face up in an incubator at 28oC. The

continuous growth result of this treatment was compared

with the same age hyphae remaining in the sealed MEA

plates.

Wound treatment

. The MEA plate with fully grown

A. cinnamomea hyphae was rubbed with cotton swabs to

expose the skeletal hyphae. The plates rubbed with cotton

swabs were then placed upside down on a fully moist filter

paper to maintain a highly moist environment.

HPLC profile analysis of A. cinnamomea

methanol extracts

Antrodia cinnamomea hyphae and fruiting bodies

were placed in an oven at 60oC for 24 h to measure their

dry weight. The samples were then subjected to 100%

methanol extraction (Wu and Chiang, 1995; Cherng et

al., 1996). The crude methanol extracts were filtered

using a 2.2 μm syringe filter and then subjected to HPLC

analysis. A 250 × 4.6 mm HyPURITY C18 HPLC column

(ThermoHypersil-Keystone, Bellefonte, PA, USA) with

a Hitachi L-7100 HPLC pump and 7240 UV detection

system (San Jose, CA, USA) were used for the analysis

of methanol extracts of A. cinnamomea under 254 nm UV

detection for secondary metabolite profiles. The mobile

phase program of HPLC was 30-100% acetonitrile from

0 - 20 min, 100% acetonitrile for next 20 min, then, linear

replacement of acetonitrile with 100% methanol for the

next 10 min, and finally 100% methanol for next 10 min.

RESULTS

Growth pattern of A. cinnamomea on C .

kanehirae

Identification of strain ACI3 based on 18S rRNA

gene sequencing analysis showed 98% homology when

LIN

et al. —

Antrodia cinnamomea

fruiting body induction

269

compared with the sequences of A. cinnamomea strain

BCRC 35398 (1015/1027 with NS1/NS4 as the PCR

primers, and 822/832 with NS3/NS8 as the PCR primers),

indicating that the strain ACI3 was A. cinnamomea. This

conclusion was further confirmed by morphological

observation of its fruiting body, including the resupinate

growth over the C. kanehirae trunk, bright orange

color, strong bitter taste, and porous surface (Chang and

Chou, 1995). It took two months after inoculation of A.

cinnamomea on C. kanehirae to form a fruiting body. The

fruiting bodies grown on the peripheral and bottom sides

of trunk showed a healthy orange to brown porous surface

(Figure 1A, B). However, the fruiting body structure

degraded and became white when the fruiting body’s face

was turned upward (Figure 1C).

Camphor effects on A. cinnamomea growth

To see whether A. cinnamomea growth is affected

under camphor oil environment, we performed a series

of experiments to see the effects of camphor oil on

A. cinnamomea growth. As shown in Figure 2, the A.

cinnamomea could tolerate camphor oil concentration up

to 400 μl per 100 ml (shown as 0.4% in Figure 2) MEA

medium without any noticeable decrease in growth. It was

observed that 200 μl of camphor oil in 100 ml (shown

as 0.2% in Figure 2) MEA medium increased the growth

by 20%. This result indicates that A. cinnamomea grows

better under a C. kanehirae environment.

Physical induction of A. cinnamomea fruiting

body

Acetic acid was tested since the natural growth

environment of A. cinnamomea is acidic. Application of

a few drops of acetic acid to MEA plates caused fungal

hyphae to stop extension and withdraw from the spots

of acetic acid. In addition, the red color of the hyphae

Figure 1. Fruiting body of A. cinnamomea on the C. kanehirae trunk. (A) The fruiting body growing on the peripheral side of trunk;

(B) The fruiting body growing on the lower side of trunk; (C) The fruiting body growing on the higher side of trunk.

Figure 2. Growth of A .

cinnamomea hyphae in MEA

plates with various concentrations

of camphor oil of C. kanehirae;

"control" as MEA medium alone,

" tw ee n 20 " a s M E A m ed i um

conta ining 1% Tween 20 (v/ v)

only, " 0.2%" a s ME A me dium

containing 1% Tween 20 and 0.2%

camphor oil, "0.4%" as MEA

medium containing 1% Tween 20

and 0.4% cam phor oil, " 0.6%"

as MEA medium containing 1%

Tween 20 and 0.6% camphor oil,

an d "0 .8% " a s ME A m e di um

co nt ai ni ng 1% Twee n 20 a nd

0.8% camphor oil. The 1% Tween

20 is applied to help diss olving

ca mph or oil in ME A m edi um .

Data are the average ± SD of five

experiments.

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Botanical Studies, Vol. 47, 2006

turned pale and even white. Thus, it appears that acetic

acid regulated the growth rate of hyphae and changed the

growth phase.

In the air exchange experiment we found, instead of

paling, an increase in the red color hyphae in well aerated

petri dishes compared to the sealed petri dishes. This

indicates that aeration may be an important factor in the

growth of hyphae, especially in change white hyphae into

red hyphae.

Wounding treatment with cotton swabs on MEA plates

with fully grown hyphae was found to induce fruiting

bodies in agar plates (Figure 3). Fruiting bodies from

MEA plates subjected to HPLC analysis showed patterns

identical to fruiting bodies obtained from C. kanehirae

(Figure 4).

HPLC analysis of A. cinnamomea methanol

extracts

Figure 4 shows the HPLC profiles of methanol

extracts from different growth stages of A. cinnamomea.

In general, hyphal and fruiting body tissues were

significantly different. Hyphal tissues contained a higher

proportion of polar compounds than fruiting body tissues,

which contained more non-polar compounds. In addition,

the high degree of similarity between the fruiting bodies

grown from C. kanehirae trunks and those from MEA

plates with wounding treatment indicating that fruiting

body production by wounding treatment can potentially

replace wildly grown fruiting bodies for medicinal

purposes.

DISCUSSION

Medicinally important fungi play an important

role in traditional Chinese medical practice. Antrodia

cinnamomea has been proved effective in several reports

at treating liver diseases and tumors (Chen and Yang,

1995; Hseu et al., 2002; Song and Yen, 2002). Further

characterization of this fungal species may therefore

yield medicinal benefits. However, its slow growth rate

and exclusive host requirement have made its large-scale

production for medicinal purposes difficult. This report

presents a successful effort to induce A. cinnamomea

fruiting bodies from a non-C. kanehirae environment.

Our observations of A. cinnamomea growth patterns

on the C. kanehirae trunk agree with the report of Bulter

and Wood (1988) on the fruiting body formation of

Polyporaceae species, which indicated that the growth

orientation affects fruiting body formation and may be

related to its sexual reproduction processes. Further study

of this aspect may help us understand the mechanism of A.

cinnamomea fruiting body formation.

Cinnamonum kanehirae is a unique host to A.

cinnamomea in the natural environment. One possible

reason is that the camphor oil produced by C. kanehirae

deters possible competitors of A. cinnamomea. Ou r

observation showed that camphor oil indeed accelerated

the growth of A. cinnamomea indicating a positive

influence of the oil on its growth.

Generally, fungal fruiting body formation is considered

part of the aging process, which can be caused by nutrient

exhaustion, mechanical injury, chemical stimulation, or

other environmental changes (Thomas and Stanley, 1968;

Sawao et al., 1984; Hideo et al., 1985; Yoshiiyuki et al.,

Figure 3. In vitro fruiting body formation in A. cinnamomea in

MEA plates after cotton swab rubbing. (A)-(C) indicate different

magnification images. The porous structure of fruiting body was

clearly visible on surface of the fruiting bodies.

LIN

et al. —

Antrodia cinnamomea

fruiting body induction

271

medicines, we were interested to learn whether secondary

product profiles at different stages of A. cinnamomea

growth showed any changes. We found that hyphal tissues

contain a higher proportion of polar compounds than

the fruiting body tissues, which contained more non-

polar compounds. This is important information for the

pharmaceutical companies interested in the medicinal

application of A. cinnamomea. The present study may

also aid the conservation of C. kanehirae since cutting off

the tree to collect A. cinnamomea fruiting bodies will no

longer be necessary.

Acknowledgements. This work was supported by a

grant from the ROC Council for Economic Planning and

Development (CEPD-K-1010 to TZW) and by an ROC

National Science Council grant (NSC-92-2311-B-259-002

to JCC).

LITERATURE CITED

Bulter, G.M. and A.E. Wood. 1988. Effects of environmental

fac tors on ba si diom e de vel opme nt i n the res upina te

polypore Phellinus contiguous. Transactions of the British

Mycological Society (currently Mycological Research) 90:

75-83.

Chang, T.-T. and W.-N. Chou. 1995. Antrodia cinnamomum sp.

nov. on Cinnamomum kanehirai in Taiwan. Mycol. Res. 99:

756-758.

Chang, T.-T. and W.-N. Chou. 2004. Antrodia cinnamomea

reconsidered and A. salmonea sp. nov. on Cunninghamia

konishii in Taiwan. Bot. Bull. Acad. Sin. 45: 347-352.

Chang, T.-T. and W.-R. Wang. 2005. Basidiomatal formation of

Antrodia cinnamomea on artificial agar media. Bot. Bull.

Acad. Sin. 46: 151-154.

Chen, C.-H. and S .-W. Yang. 1995. New steroid acids from

Antrodia cinnamomea, a fungal parasite of Cinnamomum

micranthum. J. Nat. Prod. 58: 1655-1661.

Cherng, I.-H., D.-P. Wu, and H.-C. Chiang. 1996. Triterpenoids

from Antrodia cinnamomea. Phytochemistry 41: 263-267.

Hide o, H., T. Naoki , and M. S awao . 1 985. Iso lat ion and

identification of cyclooctasulfur, a fruiting-body inducing

substance, produced by Streptomyces albulus TO447. Agric.

Biol. Chem. 49: 101-105.

Hseu, Y.-C., W.-C. Chang, Y.-T. Hseu, C.-Y. Lee, Y.-J. Yech,

P.-C. Chen, J.-Y. Chen, and H.-L. Yang. 2002. Protection

of oxidative damage by aqueous extract from Antrodia

camphorata mycelia in normal human erythrocytes. Life

Sci. 71: 469-482.

Lee, I.-H., R.-L. Huang, C.-T. Chen, H.-C. Chen, W.-C. Hsu,

and M.-K. Lu. 2002. Antrodia camphorata polysaccharides

exhibit anti-hepatitis B virus effects. FEMS Microbiol. Lett.

209: 63-67.

Sawao, M., H. Hayashi, and N. Tarui. 1984. Anthranilic acid, as

a fruiting body inducing substance in Favolus arcularius,

from a strain TA7 of Actinomycetes. Agric. Biol. Chem. 48:

Figure 4. HPLC chromatograms of methanol extracts from A.

cinnamomea white mycelia (A), red mycelia (B), fruiting body

grown on C. kanehirae (C), and fruiting body grown in vitro in

MEA plates with cotton swab rubbing treatment (D). Several

key peaks have been shaded to enhance visual comparison.

1998). For commercial mushroom production, factors like

low temperature, increased aeration, increased humidity,

suitable light radiation, and wounding treatments are

commonly used. We adapted a similar treatment for A.

cinnamomea and found three factors, i.e. addition of acetic

acid, air exchange, and wound treatments, had unique

effects on growth. The wounding treatment was found

especially important for the formation of fruiting bodies.

This report opens further avenues for induction of A.

cinnamomea fruiting body in vitro and a case for study of

this phase change. The MEA plates with A. cinnamomea

hyphae before wounding treatment were one-month-old. It

is possible that nutrient levels were depleted in the MEA

plates, and this triggered phase change.

The methanol extraction of A. cinnamomea has been

used to study its secondary metabolite composition

(Wu and Chiang, 1995; Cherng et al., 1996). Since the

composition of secondary metabolites is an important

factor to influence the medicinal activities of herbal

272

Botanical Studies, Vol. 47, 2006

1669-1671.

S ong, T.-Y. and G.-C. Yen. 2002. Antioxidant properties of

Antrodia camphorata in submerged culture. J. Agric. Food

Chem. 50: 3322-3327.

Song, T.-Y. and G.-C. Yen. 2003. Protective effects of fermented

filtrate from Antrodia camphorata in submerged culture

against CCl

4

-induced hepatic toxicity in rats. J. Agric. Food

Chem. 51: 1571-1577.

Thomas , J .L . and D. Stanley. 1968. Chem ical induction of

haploid fruiting bodies in Schizophyllum commune. Proc.

Nati. Acad. Sci. USA 59: 745-751.

White, T.J., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification

and direct sequencing of fungal ribosomal RNA genes for

phylogenetics. In M.A. Innis, D.H. Gelfand, J.J. Sninsky

and T.J. White (eds.), PCR Protocol (A Guide to Methods

and Applications). Academic Press, New York.

Wu, D.-P. and H.-C. Chiang. 1995. Cons tituents of Antrodia

cinnamomea. J. Chin. Chem. Soc. 42: 797-800.

Wu, S .-H., L . Ryvarden, and T.-T. Chang. 1997. Antrodia

camphorata ("niu-chang-chih"), new combination of a

medicinal fungus in Taiwan. Bot. Bull. Acad. S in. 38:

273-275.

Yoshiiyuki, M., L. Hanashima, T. Yamaguchi, M. Takemura,

F. Sugawara, M. Saneyoshi, A., Mats ukage, S. Yoshida,

and K. S akaguc hi. 1998. A mus hroom fruit ing body-

inducing subs tance inhibits activities of replicative DNA

polymeras es. Biochem . Biophys . Res. Comm un. 249:

17-22.

人工環境誘導藥用真菌牛樟芝之子實體

林志遠　吳宗正　周志青

國立東華大學生命科學系暨生物技術研究所

　　藥用真菌牛樟芝的子實體為起始於台灣原住民的傳統草藥，但只寄生於台灣國寶級樹種牛樟的中

空腐朽的樹幹內，由於牛樟芝的生理與發生過程尚不清楚，因此希望大量地以人工環境栽培牛樟芝子

實體仍舊非常困難。我們觀察牛樟芝在牛樟樹幹上的生長情形發現牛樟芝子實體只能發生於樹幹四周

及向下的一面，向上的一面則無法長出牛樟芝的子實體，因此牛樟芝在樹幹上的生長方位對子實體的

形成非常重要。另外，牛樟芝子實體的形成也受到溼度與空氣品½的影響。我們也發現物理性的傷害

可誘導洋菜½培養基上的牛樟芝菌絲產生子實體，此子實體的甲醇萃取物經 HPLC 圖譜分析證明，非

常類似野生的牛樟芝子實體 HPLC 圖譜，且不同於菌絲體的 HPLC 圖譜。

關鍵詞：非生物性逆境；牛樟芝；高效能液相層析法；二次代謝物；創傷誘導。