Antitumour Activity From Tolypocladium Sp.

Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Mycelium cultivation, chemical composition and antitumour

activity of a Tolypocladium sp. fungus isolated from wild
Cordyceps sinensis
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P.H. Leung, Q.X. Zhang and J.Y. Wu
Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Hong
Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Keywords Abstract
antitumour activity, chemical constituents,
Cordyceps sinensis, Cs-HK1, mycelium culture, Aims: To examine and illustrate the morphological characteristics and growth
Tolypocladium sp. kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps
sinensis in solid and liquid cultures, and the major chemical constituents and
Correspondence antitumour effects of Cs-HK1 mycelium.
J.Y. Wu, Department of Applied Biology and
Methods and Results: The Cs-HK1 fungus was isolated from the fruiting body
Chemical Technology and the State Key
Laboratory of Chinese Medicine and
of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rap-
Molecular Pharmacology, The Hong Kong idly at 22–25C on a liquid medium containing glucose, yeast extract, peptone
Polytechnic University, Hung Hom, Kowloon, and major inorganic salts, with a specific growth rate of 1Æ1 day)1, reaching a
Hong Kong. E-mail: bcjywu@polyu.edu.hk cell density of 23Æ0 g dw l)1 in 7–9 days. Exopolysaccharides accumulated in
the liquid culture to about 0Æ3 g l)1 glucose equivalent. In comparison with
2005/1019: received 8 September 2005, natural C. sinensis, the fungal mycelium had similar contents of protein
revised 14 January 2006 and accepted 15
(11Æ7–lg) and carbohydrate (654Æ6–lg) but much higher contents of polysac-
January 2006
charide (244Æ2 mg vs 129Æ5 mg), adenosine (1116Æ8–lg vs 264Æ6 lg) and cord-
doi:10.1111/j.1365-2672.2006.02930.x ycepin (65Æ7 lg vs 20Æ8 lg) (per gram dry weight). Cyclosporin A, an antibiotic
commonly produced by Tolypocladium sp., was also detected from the mycel-
ium extract. The hot water extract of mycelium showed low cytotoxic effect on
B16 melanoma cells in culture (about 25% inhibition) but significant antitu-
mour effect in animal tests, causing 50% inhibition of B16 cell-induced tumour
growth in mice.
Conclusions: The Tolypocladium sp. fungus, Cs-HK1, can be easily cultivated
by liquid fermentation. The mycelium biomass contained the major bioactive
compounds of C. sinensis, and the mycelium extract had significant antitumour
activity.
Significance and Impact of the Study: The Cs-HK1 fungus may be a new and
promising medicinal fungus and an effective and economical substitute of the
wild C. sinensis for health care.
latory activities. Because of the natural scarcity of most

Introduction
medicinal mushrooms, the cultivation of fungal mycelia,
Medicinal fungi or mushrooms have been widely used as fruiting bodies or the whole mushroom by solid and
tonic foods and herb remedies since ancient times, and liquid (submerged) fermentation has become the major
their medicinal properties have been increasingly recog- source of mushroom supply (Smith et al. 2002).
nized through more recent scientific research. In the Cordyceps sinensis (Berk.) Sacc. (Cs), known as Dong-
search of alternative medicines and natural therapeutics Chong-Xia-Cao (winter worm-summer grass) in Chinese,
for cancer therapy, medicinal fungi are among the most is a precious medicinal mushroom formed on an insect
promising targets because of their notable immunomodu- larva. Cordyceps sinensis has been used in China since
ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 275–283 275
Mycelium of a C. sinensis fungus P.H. Leung et al.
ancient times, mainly as a general tonic to strengthen and Sichuan, China. The C. sinensis organisms were stored at
improve lung and kidney functions, to restore health after 0–4C (for no more than 5 days) before the strain isola-
prolonged sickness and to boost and maintain overall tion. The wet and soil-covered organisms were washed
body health. Nowadays the medicinal value of C. sinensis thoroughly with tap water and briefly dipped in 70% eth-
species has gained worldwide attention and attracted great anol and then in 10% bleach for 20 min, and rinsed with
research effort towards the scientific rediscovery of this sterile distilled water. The sterilized fruiting body of the
traditional remedy (Zhu et al. 1998; Buenz et al. 2005). organisms was cut into 2–5 mm sections and planted on
Numerous pharmacological activities have been found in a solid medium in petri dishes, and incubated at 20C.
the medicinal fungus including antitumour, anti-inflam- The solid medium consisted (per litre) of 10 g peptone,
mation and antiatherosclerosis (Ji 1999). The antitumour 100 g glucose, 3 g yeast extract, 0Æ5 g MgSO4, 1 g
activity of natural C. sinensis species and cultivated fungal KH2PO4, 50–100 U penicillin and 20 g agar. Isolated fila-
mycelia, and fruiting bodies, for example, has been mentous fungus colonies appearing in the culture were
observed in several studies (Yamaguchi et al. 1990; Huang picked out and transferred into fresh medium. The isola-
et al. 2000), and C. sinensis has also been used in Chinese tion and subculture of fungal colonies was repeated
medicine for the treatment of various cancerous diseases several times until bacteria-free and morphologically uni-
or as an adjuvant of cancer chemotherapy (Ji 1999). form colonies were attained. The purified fungal strains
Cordyceps sinensis is a naturally rare species, which is were maintained in potato dextrose agar medium at
only found in a few isolated areas of high plateaus at 20C.
3500–5000 m above sea level in western China. Wild or
natural C. sinensis has become increasingly scarce in
Liquid culture of fungal mycelium
recent years owing to reckless harvesting and unfavour-
able weather conditions for its proliferation. As a result of Liquid culture was carried out in shake-flasks, with 125
the natural shortage and increasing demand, the price for or 250 ml Erlenmeyer flasks, each filled with 25 or 50 ml
wild C. sinensis has increased sharply, nearly doubled in liquid medium on a shaking incubator at 150 rev min)1
the last 3–5 years. Mycelial fermentation of Cordyceps and 25C. The liquid medium was composed (per litre)
fungal species is a feasible and sustainable means for proof 40 g glucose, 10 g yeast extract, 5 g peptone, 1 g
ducing the medicinal fungus and its active compounds KH2PO4 and 0Æ5 g MgSO4. Each of the flasks was inocu-
(Zhu et al. 1998). The cultivated fungal mycelia of some lated with 1 ml 10· diluted culture broth from a starter
C. sinensis fungal species have been shown to produce culture flask, which had been prepared by shaking incu-
pharmacological effects similar to those of wild C. sinensis bation of fungal mycelium from solid culture for 7 days.
species. The shake-flask culture was run for 7–9 days and the fun-
Tolypocladium sp. is one of the numerous anamorphic gal mycelium was harvested from the flasks by filtration,
fungal species, which is phylogenetically related to and then dried at 45–50C in an oven to constant dry
C. sinensis. Although some Tolypocladium fungi have pre- weight (dw).
viously been isolated from wild C. sinensis and cultivated The natural C. sinensis organisms for comparison with
for commercial C. sinensis products, little is known about the cultivated mycelium in chemical composition was col-
the culture characteristics, chemical composition and lected from the same location as the fresh C. sinensis for
pharmacological activity of the Tolypocladium sp. mycel- fungal strain isolation. All treatments were performed in
ium. This work was to examine and illustrate the mor- triplicates or more times and the experimental results
phological characteristics and growth kinetics of a were expressed in means ± standard error (SE). The sta-
Tolypocladium sp. fungus isolated from wild C. sinensis tistical significance of treatment effects was evaluated by
species in solid and liquid cultures, and the major chem- the Student’s t-test at a probability limit of P < 0Æ05.
ical constituents and antitumour effects of the cultivated
fungal mycelium.
Analysis of sugar and total nitrogen in culture medium
Liquid medium in the culture flasks was separated from
Materials and methods
the fungal mycelium by filtration. Sugar concentration in
the liquid medium was determined by the anthrone test
Isolation of fungi from wild Cordyceps sinensis
using glucose as a standard (Chaplin and Kennedy 1994),
Fresh and complete C. sinensis organisms (complexes of a and the total (organic and inorganic) nitrogen concentra-
fungus fruit body and a caterpillar corpse) were collected tion by the standard Kjeldahl method (ASTM 3590 2002)
in late June 2000 on the plateaus of 4000–4500 m above using NH4Cl as a reference, expressed as the total
sea level in the Ganzi Tibetan Autonomous Region, Kjeldahl nitrogen (TKN).
ª 2006 The Authors

276 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 275–283
P.H. Leung et al. Mycelium of a C. sinensis fungus
cell (American Type Culture Collection-ATCC, Rockville,

Analysis of major constituents of fungal mycelium and
MD, USA) that was cultured on RPMI-1640 medium
natural Cordyceps sinensis
supplemented with 10% foetal bovine serum, 100 U ml)1
For determination of the total carbohydrate and protein penicillin and 100 lg ml)1 streptomycin in 25 cm2 cul-
contents, the powdered dry mass of mycelium and nat- ture flasks at 37C in humidified atmosphere with 5%
ural C. sinensis samples was extracted using double-dis- CO2. The cells were harvested from the culture flasks at
tilled water (20 ml g)1) at room temperature (about the exponential growth phase and resuspended in fresh
20–25C) for 2 h in an ultrasonic bath. The extraction medium at a cell density of 1 · 105 cells ml)1. The cell
mixture was then filtered and the filtrate was concentra- suspension was dispensed into a 96-well microplate at
ted by evaporation and freeze-dried. The total carbohy- 100 ll per well and incubated in humidified atmosphere
drate content of the extract was determined by the with 5% CO2 at 37C for 24 h, and then treated with the
anthrone test using glucose as a standard, and the total drugs (mycelium extract) at selected doses. Cell prolifer-
protein content by the Bradford method (Kruger 1996) ation in the microplate was determined at various treat-
using bovine serum albumin as a standard. ment intervals with the MTT assay (Mosmann 1983).
Polysaccharide (PS) fraction was obtained by hot water Animal test of antitumour activity was performed on
extraction of the sample powder (1 g in 30 ml and boil- C57BL/6 male mice (18–22 g), provided by the Animal
ing for 2 h) followed by ethanol precipitation as described Unit at The First Military University in Guangzhou. The
by Li et al. (2001). The precipitate was separated from animals were quarantined in the animal room for 3 days,
the liquid by centrifugation and the PS content in the and then each was injected with 2 · 106 mouse melan-
precipitate was expressed in glucose equivalent measured oma B16/neu cells (from the cell culture described above)
by the anthrone test. The exopolysaccharides (EPS) pro- subcutaneously into the left footpad to induce tumours.
duced by the C. sinensis fungus in liquid culture were iso- The recipient mice were randomly divided into three
lated from the liquid medium and quantified in the same groups, eight mice each: a control group treated with
ways. double-distilled water, a positive control group treated
Nucleosides were analysed using the HPLC method with a proved cancer drug, cyclophosphamide and a
reported by Shiao et al. (1994) with modifications. Dry group treated with the mycelium extract, by intraperito-
sample powder was extracted using deionized water and neal injection. Body weight and tumour size were meas-
the liquid extract was then evaporated to dryness and ured daily, and the tumour volume was estimated by
redissolved in methanol for analysis. The HPLC system (a · b2)/2, where a and b are the shorter and longer
consisted of a Hitachi L-7100 Pump, a HP L-1050 UV dimensions, respectively. After 27 days of treatment, the
Detector, and an Econosphere C18 Column (Alltech, mice were killed and the tumours were removed and
Deerfield, MI, USA). The mobile phase consisted of (A) weighed.
0Æ005 mol l)1 KH2PO4 in double-distilled water and (B)
methanol at a flow rate of 1Æ0 ml min)1 in a gradient
Results
elution scheme (0–10 min: linear gradient, 0–5% B;
10–20 min: linear, 5–10% B; 20–30 min: linear, 10–20%
Morphological characteristics of Cs-HK1 fungus isolated
B; 30–40 min: 20% B isocratic and finally, flushing the
from wild Cordyceps sinensis
column with 100% A for 10–15 min). The nucleoside
peaks were detected at UV 260 nm and identified and The strain isolation eventually resulted in a pure fungal
quantified with chemical standards (Sigma, St Louis, MO, colony that was designated as Cs-HK1 fungus. The colony
USA). of Cs-HK1 on MEA medium (malt extract 30 g, peptone
3 g and agar 15 g l)1) at 26C for 10 days was white and
floccose throughout on the surface, and whitish or pale
Antitumour activity tests in cell cultures and animals
yellow on the back, producing yellowish exudates
The hot water extract of fungal mycelium was tested for (Fig. 1a). The hyphae and conidiophores of mycelia were
antitumour activity in cancer cell cultures and an animal hyaline and smooth-walled; the conidia were hyaline,
tumour model. The mycelium dry powder (10 g) was cylindrical, smooth-walled and straight or slightly curved
extracted using boiling water (10 ml) for 3 h and then with rounded ends (Fig. 1b). When the mycelium culture
separated by filtration. The liquid extract was concentra- grew very old, e.g. over 20 days, it formed many rod-
ted to dryness by evaporation and redissolved in double- shaped spores, mostly 5–10 lm long (Fig. 1c). Based on
distilled water. these morphological characteristics, Cs-HK1 fungus was
The in vitro antitumour activity or cytotoxicity of identified as a Tolypocladium species (Agency: Institute
mycelium extract was tested on the mouse melanoma B16 of Microbiology, Chinese Academy of Science, Beijing).
ª 2006 The Authors

(a) (b) (c)
10 µm
Figure 1 Morphological characteristics of Cs-HK1 fungus in solid and liquid cultures, (a) mycelium colony on solid medium in a petri dish on day
20; (b) fungal mycelia (400· magnification) from liquid culture on day 4; (c) fungal spores (1000·) in solid culture (over 20 days, bar ¼ 10 lm).
Specimens in (b) and (c) were stained with 5% lactophenol cotton blue.
25 (a)
40 1·6
20
Biomass (g l–1)
Glucose (g l–1)
20
TKN (g l–1)
Biomass (g dw l–1)
30 1·2
15 15
20 0·8
10
10
10 5 0·4
5
0 0 0·0
0 2 4 6 8 10
Culture time (d) 0
Y (g l–1): 0 1 3 0 1 3 1 0·5 3 3
Figure 2 Time courses of mycelium biomass, and sugar and nitrogen P (g l–1): 1 1 1 3 3 3 0 0·5 3 0
concentrations in Cs-HK1 fungus liquid cultures (TKN for total Kjeldahl M (+/–): + + + + + + + + – +
nitrogen). d, Biomass; h, glucose; ·, TKN.
(b) 20
Cs-HK1 was identified as an anamorph of C. sinensis but
18
not as any specific species at CABI Bioscience (IMI39260,

Bakeham, UK).
16
Growth and nutrient consumption of Cs-HK1 fungus in 14

liquid cultures
12
Figure 2 shows the typical time courses of mycelial
growth and sugar, and nitrogen consumption of the 10
Cs-HK1 fungus in shake-flask cultures. The mycelium 2 3 4 5 6
biomass showed a lag-phase in the first 1–2 days and Yeast extract (g l–1)
exponential growth in the next 2–3 days (specific growth
Figure 3 Effects of major medium components on mycelium growth
rate estimated at 1Æ1 d)1). The mycelium biomass reached
of Cs-HK1 fungus in liquid cultures (7 days), (a) biomass yields at various
a maximum concentration of 23Æ2 g dw l)1 on day 7 and combinations and concentrations of three components, yeast extract
remained little changed during days 7–9, and then started (Y), peptone (P) and inorganic salts (M); +/), presence or absence; (b)
to drop on day 10. The time courses of glucose and total effects of yeast extract concentration on biomass yield at two fixed
nitrogen (TKN) appeared to be the mirror images of the peptone concentrations. ¤, Peptone (1 g l)1); h, peptone (2 g l)1).
biomass growth curve, with both nutrient concentrations
dropping slowly in the first 2 days, and rapidly from day
2 to day 6, and levelling off thereafter. result in a lower biomass yield. Among the three, yeast
We further examined the effects of yeast extract and extract was the most beneficial for mycelium growth, and
peptone concentrations and the supply of inorganic salts the highest biomass yield was attained with 3 g l)1 yeast
(KH2PO4 and MgSO4) on mycelium growth. The results extract and 1–3 g l)1 peptone plus the inorganic salts (Y3,
(Fig. 3a) show that all the three were needed for the P1 or P3, M+). Further experiment with various yeast
mycelium growth, and the absence of any one would extract concentrations at two fixed levels of peptone
ª 2006 The Authors

(Fig. 3b) showed that 5 g l)1 of yeast extract and 1–2 g l)1 Table 1 Contents of total protein, carbohydrate and polysaccharide
peptone were sufficient for rapid biomass growth. and major nucleosides in natural Cordyceps sinensis (Cs) and Cs-HK1
fungal mycelium (mycelium cultured for 7 days)
Components Natural Cs Mycelium

Exopolysaccharide production and the effect of sugars
Extract yield (wt%) 3Æ4 5Æ6
When glucose was used as the carbon source, the EPS
Protein (mg g)1) 9Æ54 ± 1Æ11 11Æ7 ± 0Æ07
content in the liquid medium could be detected on day 3 Sugar (mg g)1) 643Æ1 ± 18Æ0 654Æ6 ± 21.5
postinoculation, increased exponentially from day 3 to Polysaccharide (mg g)1) 129Æ5 ± 14.8 244.2 ± 11Æ6
day 7, and reached 0Æ26 g l)1 on day 10 (Fig. 4). In com- (sugar content) (37Æ1%) (41Æ3%)
parison, the EPS content in the culture growing on Nucleoside (lg g)1)
sucrose was not detectable before day 5 and was only Uracil 3Æ35 ± 0Æ30 13Æ3 ± 0Æ56
0Æ11 g l)1 at the maximum on day 7. In addition, glucose Cytosine 69Æ4 ± 7Æ15 5Æ03 ± 0Æ36
Adenosine 264Æ6 ± 6Æ66 1116Æ8 ± 8Æ25
was also more favourable than sucrose for the mycelium
Cordycepin 20Æ8 ± 2Æ56 65Æ7 ± 2Æ01
growth, giving rise to a higher growth rate and a higher
maximum biomass concentration. Contents are expressed in per gram dry weight of mycelium and nat-
ural Cordyceps sinensis; values represent means ± SE, n ¼ 2.
Major constituents of Cs-HK1 mycelium and natural

Cordyceps sinensis 60
800 4
The protein and total carbohydrate contents of the
40
Cs-HK1 mycelium were similar or slightly higher than
that in the natural species, and the PS content of mycel- 600
mAu
20
ium was much higher than that of natural C. sinensis 2 25 30
(Table 1). In general, the extract yield on fungal mycel- 400 3
ium was higher than that on natural C. sinensis, which
200 1
may partially account for the higher contents of most
constituents. 4
0
In the HPLC chromatograms, all the major peaks of
Cs-HK1 mycelium matches closely with those of natural 250
C. sinensis herb, although the peak sizes are quite differ-
ent (Fig. 5). The contents of most nucleosides detected in 200
the fungal mycelium were significantly higher than those 150 1
mAu
2
in the natural C. sinensis. In particular, the contents of 3
100
cordycepin and adenosine, the two marker constituents of 4
Cordyceps species, in the mycelium extract were about 50
three and five times, respectively, of those in the natural
0
species. The adenosine and cordycepin contents in the 0 5 10 15 20 25 30 35
Elution time (min)
Figure 5 Chromatographs of nucleosides in natural Cordyceps sinen-

0·5 25 sis and Cs-HK1 mycelium (Top: ——, Mycelium; - - - -, Natural Cs)
and mycelium culture broth (Bottom: ——, Mycelium; - - - -,
0·4 20
Standard; chemical standards: 1 – uracil, 2 – cytosine, 3 – adenosine,
EPS (g l–1)
0·3 15 4 – cordycepin).
0·2 10 mycelium were also confirmed by ES-MS (data not

shown). There were no detectable quantities of adenosine
0·1 5
and cordycepin in the culture broth.
0 0 Other known constituents of C. sinensis species detec-
1 3 5 7 9
ted in the Cs-HK1 mycelium included d-mannitol
Culture time (d)
(47Æ8 mg g)1) and ergosterol (0Æ544 mg g l)1). In addi-
Figure 4 Effects of carbon sources (sugars) on exopolysaccharide tion, cyclosporine (cyclosporin A) was detected in the
(EPS) production and mycelium growth of Cs-HK1 in liquid cultures. mycelium (ethyl acetate extract only) at about 25 lg g)1.
(¤) Gls, Cell; (n) Suc, Cell; (n) Glc, EPS; (h) Scu, EPS. Cyclosporin A is a well-known antibiotic with potent
ª 2006 The Authors

Table 2 Contents of major constituents in hot water extract of 10 000

Cs-HK1 mycelium used for antitumour activity tests (extract yield ¼
Tumor size (mm3)

9Æ35%) 8000
Components Contents* 6000

)1
Protein (mg g ) 32Æ1 ± 2Æ85 4000
Sugar (mg g)1) 370Æ2 ± 6Æ37
Nucleosides (lg g)1) 2000
Uracil 142Æ9 ± 5Æ21
Cytosine 45Æ9 ± 3Æ57 0
Adenine 48Æ0 ± 6Æ63 6 9 12 15 18 21 24 27
Adenosine 401Æ4 ± 9Æ42 Days after tumor cell injection
Cordycepin 20Æ0 ± 2Æ54
Figure 7 Effect of Cs-HK1 mycelium hot water extract on B16 cell-
*Contents are expressed in per gram of extract; values represent induced tumour growth in C57BL/6 mice (error bars ¼ SE, n ¼ 8).
mean ± SE, n ¼ 2. The treatment effect was statistically significant on the Student’s t-test
at P < 0Æ05 for the mycelium extract and P < 0Æ01 for the positive
control drug cyclophosphamide (CPA). s, Control; ·, positive control;
Cell proliferation (% of control)
125
m, extract treated.
100
75
25% lower cell proliferation than the untreated control
group (Fig. 6). However, there was no further decrease in
50 cell proliferation as the extract dose was increased from
50 to 200 lg ml)1. Therefore, the in vitro cytotoxicity of
25 the mycelium extract may be considered moderate or
0
low.
0 50 100 150 200 The animal test showed that the mycelium extract
Extract dose (µg ml–1) treatment of the tumour-bearing mice resulted in 42Æ9%
decrease in the tumour weight (Table 3) and 52Æ5%
Figure 6 Effect of Cs-HK1 mycelium hot water extract on the prolif-
decrease in the tumour volume (Fig. 7) over 27 days of
eration of B16 melanoma cells in culture (48 h treatment). Cell prolif-
eration (% of control) equals the MTT value of extract-treated
treatment compared with the untreated animals. The
culture divided by that of control (error bars ¼ SE, n ¼ 3). h, control; inhibitory effect of mycelium extract on tumour growth
¤, treated. was shown to be statistically significant (P < 0Æ05).
Moreover, the positive control drug showed excellent
immunosuppressive activity that is produced by Tolypoc- antitumour effect in the animal tests, which nearly
ladium inflatum and related species (Agathos et al. 1987). halted the tumour growth. This strong effect of the posi-
The presence of cyclosporin A in the Cs-HK1 fungus is tive control drug also serves to validate the animal test
another proof for its identity as Tolypocladium sp. for the mycelium extract. Therefore, the results indicate
that the mycelium extract had significant antitumour
activity.
Antitumour activity of mycelium water extract
Table 2 shows the contents of major constituents of
Discussion
mycelium hot water extract applied to the antitumour
activity tests. In cell cultures, the extract treatment of the A Tolypocladium sp. fungus, Cs-HK1, has been isolated
B16 cells at 50 lg ml)1 or higher doses resulted in about from wild C. sinensis herb in our experiment, which may
Table 3 Effect of mycelium extract treatment

Body wt (g) on tumor growth and spleen index in vivo
Tumour wt (g) Inhibition
Treatment Day 0 Day 27 on day 27 (%) (CPA, cyclophosphamide as a positive control
drug)
Control 21Æ1 ± 1.14 27Æ2 ± 4Æ51 7Æ67 ± 2.95 –
CPA, 0Æ05 g (kg day))1 21Æ5 ± 1.37 20Æ8 ± 1Æ87 0Æ21 ± 0Æ12 97Æ26**
Mycelium extract, 0Æ5 g (kg day))1 21Æ3 ± 2Æ37 25Æ4 ± 2Æ48 4Æ38 ± 2Æ25 42Æ89*
*Significance limit P < 0Æ05; ** significance limit P < 0Æ01.
ª 2006 The Authors

be a new anamorphic species of C. sinensis. The Cs-HK1 through a reductive mechanism (Lennon and Suhadolnik
fungal mycelium from liquid cultures contained most of 1976). Cordycepin content in other Cordyceps species
the known constituents of natural C. sinensis such as such as C. sinensis is extremely low or undetectable. Fur-
cordycepin, adenosine and d-mannitol, although the con- uya et al. (1983) analysed the nucleoside contents in a
tents (quantities) of specific components differ consider- natural C. sinensis and the mycelium cultures of 17
ably. Up to date, only one Tolypocladium sp. fungus, Cordyceps species, and found detectable cordycepin only
named Tolypocladium sinensis, has been isolated from in the culture broth of C. militaris. Similarly, the HPLC
wild C. sinensis and reported originally in Chinese litera- analysis of nucleosides by Shiao et al. (1994) showed no
ture (Li 1988; Jiang and Yao 2002). Although it was detectable cordycepin content in several natural and culti-
claimed to have chemical composition and pharmacologi- vated Cordyceps species except C. militaris. However, Hsu
cal activities similar to the wild species, no relevant experi- et al. (2002) measured a cordycepin content of
mental data have been reported so far. The T. sinensis 2Æ4–5Æ4 lg g)1 in a natural C. sinensis, and 1Æ4 lg g)1 in
fungus was isolated from C. sinensis organism harvested the mycelium and 1Æ9 lg g)1 in the culture broth of a C.
in the Diqing Tibetan Autonomous Region, Yunan Prov- sinensis fungus. These contents were significantly lower
ince, China, a few hundred kilometers away from where than those detected in our present study of the natural C.
C. sinensis organism used in our study was collected. The sinensis and the Cs-HK1 mycelium (Table 1). Li et al.
fact that Tolypocladium sp. fungi have been isolated from (2004) detected cordycepin as high as 5Æ0 mg g)1 in
wild C. sinensis organisms at different regions and years cultured C. militaris.
suggests that Tolypocladium sp. is a common fungal spe- The hot water extract of Cs-HK1 mycelium exhibited
cies living on the insect host of C. sinensis. positive and significant inhibitory effect on the B16-
The optimal nutrient composition for mycelium induced tumour growth in animals but low in vitro cyto-
growth and EPS production has been reported for other toxicity against the B16 melanoma cells. These results
Cordyceps species in liquid cultures. In Cordyceps jiangx- suggest that the antitumour effect of the mycelium extract
iensis culture (Xiao et al. 2004), the EPS yield was (in animals) was mainly caused by other modes of action
0Æ78 g l)1 on glucose and 0Æ57 g l)1 on sucrose, while than by directly killing the tumour cells. Immunomodula-
the biomass yield was 9Æ7 g l)1 on glucose and tion has been widely regarded as a major mechanism of
10Æ65 g l)1 on sucrose, and maltose was the most action for the antitumour effects of water and methanol
favourable among many of the carbohydrates examined extracts of Cordyceps species (Yoshida et al. 1989; Yamag-
for EPS production (1Æ05 g l)1). In Cordyceps militaris uchi et al. 1990; Shin et al. 2003). PSs in the Cordyceps
culture (Kim et al. 2003), the EPS yield on sucrose, species may play a major role in the immunomodulatory
0Æ65 g l)1, was much higher than that on glucose, activity as many antitumour PSs from various medicinal
0Æ37 g l)1. The optimal carbohydrate for EPS accumula- mushrooms (Wasser 2002). For example, a PS fraction of
tion may depend on the fungal species, other medium C. sinensis has been shown to stimulate the production of
components and culture conditions such as pH, tem- immune-important cytokines in blood mononuclear cells
perature and dissolved oxygen. The Cs-HK1 in our (Chen et al. 1997). As a complex medicinal fungus, how-
study grew more rapidly (about 20 g dw l)1 in 7– ever, its health effects are most likely attributed to the
10 days) than those species reported in previous studies synergistic action of different constituents through mul-
(10–15 g l)1 in 10 days). A direct and meaningful com- tiple mechanisms.
parison of the EPS contents with previous studies is not As a preliminary effort for identifying the constituents
possible owing to differences in the isolation procedure responsible for the cytotoxic and antitumour effects of
and analytical method. mycelium extract, we tested the cytotoxic activity of four
Adenosine and cordycepin (3¢-deoxyadenosine) are two pure compounds present in the mycelium hot water
chemical markers as well as the chief bioactive ingredients extract and its PS fraction (Fig. 8). Of these five compo-
of C. sinensis and other Cordyceps species (Ji 1999; Hsu nents, ergosterol and PS had moderate cytotoxicity
et al. 2002). Adenosine is relatively abundant and has against B16 cells, causing about 40% inhibition of cell
been detected in most natural and cultivated Cordyceps proliferation, while d-mannitol and cordycepin had only
species in a content range of 0Æ1–3Æ2 mg g)1 depending slight inhibition and adenosine had no inhibitory effect
on species, geographical location (for natural species) and on the tumour cell proliferation. From these results, we
culture conditions (for fungal mycelia) (Shiao et al. 1994; can only suggest that ergosterol and PS contributed to
Li et al. 2004). Cordycepin was originally isolated as a in vitro inhibition of the tumour cell proliferation by the
metabolic product from the culture broth of C. militaris mycelium water extract but cannot distinguish which
fungus, and its synthesis in the Cordyceps fungus was pro- components contributed to the antitumour activity
posed to be from adenosine or phosphorylated adenosine observed in the animal tests.
ª 2006 The Authors

Cell proliferation (% of control) Chaplin, M.F. and Kennedy, J.F. (1994) Carbohydrate Analysis:
125
A Practical Approach, 2nd edn. Oxford, England: ILR Press
100 at Oxford University Press.
Chen, Y.J., Shiao, M.S., Lee, S.S. and Wang, S.Y. (1997) Effect of
75
Cordyceps sinensis on the proliferation and differentiation of
50 human leukemic U937 cells. Life Sci 60, 2349–2359.
Furuya, T., Masao, M. and Masuzawa, M. (1983) N6-(2-hy-
25
droxyethel)adenosine, a biologically active compound from
0 cultured mycelia of Cordyceps and Isaria species. Phyto-
chemistry 22, 2509–2512.
e
ol
PS
to
in
pi
er
ni
os
ce
Hsu, T.H., Shiao, L.H., Hsieh, C. and Chang, D.M. (2002) A
st
an
en
dy
go
-m
or
Ad
comparison of the chemical composition and bioactive

Er
D
C
ingredients of the Chinese medicinal mushroom Don-

Extract component
gChongXiaCao, its counterfeit and mimic, and fermented
Figure 8 Effects of individual extract components on B16 cell prolif- mycelium of Cordyceps sinensis. Food Chem 78, 463–469.
eration (PS for polysaccharide; pure compounds at 100 lmol l)1 and Huang, B.M., Chuang, Y.M., Chen, C.F. and Leu, S.F. (2000)
PS at 100 lg ml)1, 48 h treatment; error bars ¼ SE, n ¼ 3). Effects of extracted Cordyceps sinensis on steroidogenesis in
MA-10 mouse Leydig tumor cells. Biol Pharm Bull 23,
In conclusion, the Cs-HK1 fungus may be a promising 1532–1535.
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rare and endangered wild species. Fermentation cultiva- cancer Traditional Chinese Medicine (150. Cordyceps sinensis
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effective process, providing a renewable source of natur- Science and Technology Press (in Chinese).
ally rare medicinal fungi. It also allows for convenient Jiang, Y. and Yao, Y.J. (2002) Names related to Cordyceps
and fruitful manipulation of the culture conditions to sinensis anamorph. Mycotaxon 84, 245–254.
control or enhance the accumulation of desired active Kim, S.W., Hwang, H.J., Xu, C.P., Sung, J.M., Choi, J.W. and
Yun, J.W. (2003) Optimization of submerged culture pro-
ingredients. In addition to mycelium biomass, the EPS in
cess for the production of mycelial biomass and exo-poly-
the fermentation broth may also be valuable bioactive
saccharides by Cordyceps militaris C738. J Appl Microbiol
products. There is a need to further study the relationship
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between the fermentation conditions and the contents of
Kruger, N.J. (1996) The Bradford method for protein quantita-
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tion. In The Protein Protocols Handbook ed. Walker, J.M.
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Antitumour Activity From Tolypocladium Sp.

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Journal of Applied Microbiology ISSN 1364-5072

Mycelium cultivation, chemical composition and antitumour

latory activities. Because of the natural scarcity of most

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cell (American Type Culture Collection-ATCC, Rockville,

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(a) (b) (c)

not as any specific species at CABI Bioscience (IMI39260,

Growth and nutrient consumption of Cs-HK1 fungus in 14

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Components Natural Cs Mycelium

Major constituents of Cs-HK1 mycelium and natural

Figure 5 Chromatographs of nucleosides in natural Cordyceps sinen-

0·2 10 mycelium were also confirmed by ES-MS (data not

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Table 2 Contents of major constituents in hot water extract of 10 000

Tumor size (mm3)

Components Contents* 6000

Table 3 Effect of mycelium extract treatment

*Significance limit P < 0Æ05; ** significance limit P < 0Æ01.

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Hsu, T.H., Shiao, L.H., Hsieh, C. and Chang, D.M. (2002) A

comparison of the chemical composition and bioactive

ingredients of the Chinese medicinal mushroom Don-

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ª 2006 The Authors

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