J.Y. Wu, Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chinese Medicine and Molecular Pharmacology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong. E-mail: firstname.lastname@example.org
Aims: To examine and illustrate the morphological characteristics and growth kinetics of Cs-HK1, a Tolypocladium fungus, isolated from wild Cordyceps sinensis in solid and liquid cultures, and the major chemical constituents and antitumour effects of Cs-HK1 mycelium.
Methods and Results: The Cs-HK1 fungus was isolated from the fruiting body of a wild C. sinensis and identified as a Tolypocladium sp. fungus. It grew rapidly at 22–25°C on a liquid medium containing glucose, yeast extract, peptone and major inorganic salts, with a specific growth rate of 1·1 day−1, reaching a cell density of 23·0 g dw l−1 in 7–9 days. Exopolysaccharides accumulated in the liquid culture to about 0·3 g l−1 glucose equivalent. In comparison with natural C. sinensis, the fungal mycelium had similar contents of protein (11·7–μg) and carbohydrate (654·6–μg) but much higher contents of polysaccharide (244·2 mg vs 129·5 mg), adenosine (1116·8–μg vs 264·6 μg) and cordycepin (65·7 μg vs 20·8 μg) (per gram dry weight). Cyclosporin A, an antibiotic commonly produced by Tolypocladium sp., was also detected from the mycelium extract. The hot water extract of mycelium showed low cytotoxic effect on B16 melanoma cells in culture (about 25% inhibition) but significant antitumour 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.
Medicinal fungi or mushrooms have been widely used as tonic foods and herb remedies since ancient times, and their medicinal properties have been increasingly recognized through more recent scientific research. In the search of alternative medicines and natural therapeutics for cancer therapy, medicinal fungi are among the most promising targets because of their notable immunomodulatory activities. Because of the natural scarcity of most medicinal mushrooms, the cultivation of fungal mycelia, fruiting bodies or the whole mushroom by solid and liquid (submerged) fermentation has become the major source of mushroom supply (Smith et al. 2002).
Cordyceps sinensis (Berk.) Sacc. (Cs), known as Dong-Chong-Xia-Cao (winter worm-summer grass) in Chinese, is a precious medicinal mushroom formed on an insect larva. Cordyceps sinensis has been used in China since ancient times, mainly as a general tonic to strengthen and improve lung and kidney functions, to restore health after prolonged sickness and to boost and maintain overall body health. Nowadays the medicinal value of C. sinensis species has gained worldwide attention and attracted great research effort towards the scientific rediscovery of this traditional remedy (Zhu et al. 1998; Buenz et al. 2005). Numerous pharmacological activities have been found in the medicinal fungus including antitumour, anti-inflammation and antiatherosclerosis (Ji 1999). The antitumour activity of natural C. sinensis species and cultivated fungal mycelia, and fruiting bodies, for example, has been observed in several studies (Yamaguchi et al. 1990; Huang et al. 2000), and C. sinensis has also been used in Chinese medicine for the treatment of various cancerous diseases or as an adjuvant of cancer chemotherapy (Ji 1999).
Cordyceps sinensis is a naturally rare species, which is only found in a few isolated areas of high plateaus at 3500–5000 m above sea level in western China. Wild or natural C. sinensis has become increasingly scarce in recent years owing to reckless harvesting and unfavourable weather conditions for its proliferation. As a result of the natural shortage and increasing demand, the price for wild C. sinensis has increased sharply, nearly doubled in the last 3–5 years. Mycelial fermentation of Cordyceps fungal species is a feasible and sustainable means for producing the medicinal fungus and its active compounds (Zhu et al. 1998). The cultivated fungal mycelia of some C. sinensis fungal species have been shown to produce pharmacological effects similar to those of wild C. sinensis species.
Tolypocladium sp. is one of the numerous anamorphic fungal species, which is phylogenetically related to C. sinensis. Although some Tolypocladium fungi have previously been isolated from wild C. sinensis and cultivated for commercial C. sinensis products, little is known about the culture characteristics, chemical composition and pharmacological activity of the Tolypocladium sp. mycelium. This work was to examine and illustrate the morphological characteristics and growth kinetics of a Tolypocladium sp. fungus isolated from wild C. sinensis species in solid and liquid cultures, and the major chemical constituents and antitumour effects of the cultivated fungal mycelium.
Materials and methods
Isolation of fungi from wild Cordyceps sinensis
Fresh and complete C. sinensis organisms (complexes of a fungus fruit body and a caterpillar corpse) were collected in late June 2000 on the plateaus of 4000–4500 m above sea level in the Ganzi Tibetan Autonomous Region, Sichuan, China. The C. sinensis organisms were stored at 0–4°C (for no more than 5 days) before the strain isolation. The wet and soil-covered organisms were washed thoroughly with tap water and briefly dipped in 70% ethanol and then in 10% bleach for 20 min, and rinsed with sterile distilled water. The sterilized fruiting body of the organisms was cut into 2–5 mm sections and planted on a solid medium in petri dishes, and incubated at 20°C. The solid medium consisted (per litre) of 10 g peptone, 100 g glucose, 3 g yeast extract, 0·5 g MgSO4, 1 g KH2PO4, 50–100 U penicillin and 20 g agar. Isolated filamentous fungus colonies appearing in the culture were picked out and transferred into fresh medium. The isolation and subculture of fungal colonies was repeated several times until bacteria-free and morphologically uniform colonies were attained. The purified fungal strains were maintained in potato dextrose agar medium at 20°C.
Liquid culture of fungal mycelium
Liquid culture was carried out in shake-flasks, with 125 or 250 ml Erlenmeyer flasks, each filled with 25 or 50 ml liquid medium on a shaking incubator at 150 rev min−1 and 25°C. The liquid medium was composed (per litre) of 40 g glucose, 10 g yeast extract, 5 g peptone, 1 g KH2PO4 and 0·5 g MgSO4. Each of the flasks was inoculated with 1 ml 10× diluted culture broth from a starter culture flask, which had been prepared by shaking incubation of fungal mycelium from solid culture for 7 days. The shake-flask culture was run for 7–9 days and the fungal mycelium was harvested from the flasks by filtration, and then dried at 45–50°C in an oven to constant dry weight (dw).
The natural C. sinensis organisms for comparison with the cultivated mycelium in chemical composition was collected from the same location as the fresh C. sinensis for fungal strain isolation. All treatments were performed in triplicates or more times and the experimental results were expressed in means ± standard error (SE). The statistical significance of treatment effects was evaluated by the Student's t-test at a probability limit of P < 0·05.
Analysis of sugar and total nitrogen in culture medium
Liquid medium in the culture flasks was separated from the fungal mycelium by filtration. Sugar concentration in the liquid medium was determined by the anthrone test using glucose as a standard (Chaplin and Kennedy 1994), and the total (organic and inorganic) nitrogen concentration by the standard Kjeldahl method (ASTM 3590 2002) using NH4Cl as a reference, expressed as the total Kjeldahl nitrogen (TKN).
Analysis of major constituents of fungal mycelium and natural Cordyceps sinensis
For determination of the total carbohydrate and protein contents, the powdered dry mass of mycelium and natural C. sinensis samples was extracted using double-distilled water (20 ml g−1) at room temperature (about 20–25°C) for 2 h in an ultrasonic bath. The extraction mixture was then filtered and the filtrate was concentrated by evaporation and freeze-dried. The total carbohydrate content of the extract was determined by the anthrone test using glucose as a standard, and the total protein content by the Bradford method (Kruger 1996) using bovine serum albumin as a standard.
Polysaccharide (PS) fraction was obtained by hot water extraction of the sample powder (1 g in 30 ml and boiling for 2 h) followed by ethanol precipitation as described by Li et al. (2001). The precipitate was separated from the liquid by centrifugation and the PS content in the precipitate was expressed in glucose equivalent measured by the anthrone test. The exopolysaccharides (EPS) produced by the C. sinensis fungus in liquid culture were isolated from the liquid medium and quantified in the same ways.
Nucleosides were analysed using the HPLC method reported by Shiao et al. (1994) with modifications. Dry sample powder was extracted using deionized water and the liquid extract was then evaporated to dryness and redissolved in methanol for analysis. The HPLC system consisted of a Hitachi L-7100 Pump, a HP L-1050 UV Detector, and an Econosphere C18 Column (Alltech, Deerfield, MI, USA). The mobile phase consisted of (A) 0·005 mol l−1 KH2PO4 in double-distilled water and (B) methanol at a flow rate of 1·0 ml min−1 in a gradient elution scheme (0–10 min: linear gradient, 0–5% B; 10–20 min: linear, 5–10% B; 20–30 min: linear, 10–20% B; 30–40 min: 20% B isocratic and finally, flushing the column with 100% A for 10–15 min). The nucleoside peaks were detected at UV 260 nm and identified and quantified with chemical standards (Sigma, St Louis, MO, USA).
Antitumour activity tests in cell cultures and animals
The hot water extract of fungal mycelium was tested for antitumour activity in cancer cell cultures and an animal tumour model. The mycelium dry powder (10 g) was extracted using boiling water (10 ml) for 3 h and then separated by filtration. The liquid extract was concentrated to dryness by evaporation and redissolved in double-distilled water.
The in vitro antitumour activity or cytotoxicity of mycelium extract was tested on the mouse melanoma B16 cell (American Type Culture Collection-ATCC, Rockville, MD, USA) that was cultured on RPMI-1640 medium supplemented with 10% foetal bovine serum, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin in 25 cm2 culture flasks at 37°C in humidified atmosphere with 5% CO2. The cells were harvested from the culture flasks at the exponential growth phase and resuspended in fresh medium at a cell density of 1 × 105 cells ml−1. The cell suspension was dispensed into a 96-well microplate at 100 μl per well and incubated in humidified atmosphere with 5% CO2 at 37°C for 24 h, and then treated with the drugs (mycelium extract) at selected doses. Cell proliferation in the microplate was determined at various treatment intervals with the MTT assay (Mosmann 1983).
Animal test of antitumour activity was performed on C57BL/6 male mice (18–22 g), provided by the Animal Unit at The First Military University in Guangzhou. The animals were quarantined in the animal room for 3 days, and then each was injected with 2 × 106 mouse melanoma B16/neu cells (from the cell culture described above) subcutaneously into the left footpad to induce tumours. The recipient mice were randomly divided into three groups, eight mice each: a control group treated with double-distilled water, a positive control group treated with a proved cancer drug, cyclophosphamide and a group treated with the mycelium extract, by intraperitoneal injection. Body weight and tumour size were measured daily, and the tumour volume was estimated by (a × b2)/2, where a and b are the shorter and longer dimensions, respectively. After 27 days of treatment, the mice were killed and the tumours were removed and weighed.
Morphological characteristics of Cs-HK1 fungus isolated from wild Cordyceps sinensis
The strain isolation eventually resulted in a pure fungal colony that was designated as Cs-HK1 fungus. The colony of Cs-HK1 on MEA medium (malt extract 30 g, peptone 3 g and agar 15 g l−1) at 26°C for 10 days was white and floccose throughout on the surface, and whitish or pale yellow on the back, producing yellowish exudates (Fig. 1a). The hyphae and conidiophores of mycelia were hyaline and smooth-walled; the conidia were hyaline, cylindrical, smooth-walled and straight or slightly curved with rounded ends (Fig. 1b). When the mycelium culture grew very old, e.g. over 20 days, it formed many rod-shaped spores, mostly 5–10 μm long (Fig. 1c). Based on these morphological characteristics, Cs-HK1 fungus was identified as a Tolypocladium species (Agency: Institute of Microbiology, Chinese Academy of Science, Beijing). Cs-HK1 was identified as an anamorph of C. sinensis but not as any specific species at CABI Bioscience (IMI39260, Bakeham, UK).
Growth and nutrient consumption of Cs-HK1 fungus in liquid cultures
Figure 2 shows the typical time courses of mycelial growth and sugar, and nitrogen consumption of the Cs-HK1 fungus in shake-flask cultures. The mycelium biomass showed a lag-phase in the first 1–2 days and exponential growth in the next 2–3 days (specific growth rate estimated at 1·1 d−1). The mycelium biomass reached a maximum concentration of 23·2 g dw l−1 on day 7 and remained little changed during days 7–9, and then started to drop on day 10. The time courses of glucose and total nitrogen (TKN) appeared to be the mirror images of the 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.
We further examined the effects of yeast extract and peptone concentrations and the supply of inorganic salts (KH2PO4 and MgSO4) on mycelium growth. The results (Fig. 3a) show that all the three were needed for the mycelium growth, and the absence of any one would result in a lower biomass yield. Among the three, yeast extract was the most beneficial for mycelium growth, and the highest biomass yield was attained with 3 g l−1 yeast extract and 1–3 g l−1 peptone plus the inorganic salts (Y3, P1 or P3, M+). Further experiment with various yeast extract concentrations at two fixed levels of peptone (Fig. 3b) showed that 5 g l−1 of yeast extract and 1–2 g l−1 peptone were sufficient for rapid biomass growth.
Exopolysaccharide production and the effect of sugars
When glucose was used as the carbon source, the EPS content in the liquid medium could be detected on day 3 postinoculation, increased exponentially from day 3 to day 7, and reached 0·26 g l−1 on day 10 (Fig. 4). In comparison, the EPS content in the culture growing on sucrose was not detectable before day 5 and was only 0·11 g l−1 at the maximum on day 7. In addition, glucose was also more favourable than sucrose for the mycelium growth, giving rise to a higher growth rate and a higher maximum biomass concentration.
Major constituents of Cs-HK1 mycelium and natural Cordyceps sinensis
The protein and total carbohydrate contents of the Cs-HK1 mycelium were similar or slightly higher than that in the natural species, and the PS content of mycelium was much higher than that of natural C. sinensis (Table 1). In general, the extract yield on fungal mycelium was higher than that on natural C. sinensis, which may partially account for the higher contents of most constituents.
Table 1. Contents of total protein, carbohydrate and polysaccharide and major nucleosides in natural Cordyceps sinensis (Cs) and Cs-HK1 fungal mycelium (mycelium cultured for 7 days)
Contents are expressed in per gram dry weight of mycelium and natural Cordyceps sinensis; values represent means ± SE, n = 2.
Extract yield (wt%)
Protein (mg g−1)
9·54 ± 1·11
11·7 ± 0·07
Sugar (mg g−1)
643·1 ± 18·0
654·6 ± 21.5
Polysaccharide (mg g−1) (sugar content)
129·5 ± 14.8 (37·1%)
244.2 ± 11·6 (41·3%)
Nucleoside (μg g−1)
3·35 ± 0·30
13·3 ± 0·56
69·4 ± 7·15
5·03 ± 0·36
264·6 ± 6·66
1116·8 ± 8·25
20·8 ± 2·56
65·7 ± 2·01
In the HPLC chromatograms, all the major peaks of Cs-HK1 mycelium matches closely with those of natural C. sinensis herb, although the peak sizes are quite different (Fig. 5). The contents of most nucleosides detected in the fungal mycelium were significantly higher than those in the natural C. sinensis. In particular, the contents of cordycepin and adenosine, the two marker constituents of Cordyceps species, in the mycelium extract were about three and five times, respectively, of those in the natural species. The adenosine and cordycepin contents in the mycelium were also confirmed by ES-MS (data not shown). There were no detectable quantities of adenosine and cordycepin in the culture broth.
Other known constituents of C. sinensis species detected in the Cs-HK1 mycelium included d-mannitol (47·8 mg g−1) and ergosterol (0·544 mg g l−1). In addition, cyclosporine (cyclosporin A) was detected in the mycelium (ethyl acetate extract only) at about 25 μg g−1. Cyclosporin A is a well-known antibiotic with potent immunosuppressive activity that is produced by Tolypocladium inflatum and related species (Agathos et al. 1987). The presence of cyclosporin A in the Cs-HK1 fungus is another proof for its identity as Tolypocladium sp.
Antitumour activity of mycelium water extract
Table 2 shows the contents of major constituents of mycelium hot water extract applied to the antitumour activity tests. In cell cultures, the extract treatment of the B16 cells at 50 μg ml−1 or higher doses resulted in about 25% lower cell proliferation than the untreated control group (Fig. 6). However, there was no further decrease in cell proliferation as the extract dose was increased from 50 to 200 μg ml−1. Therefore, the in vitro cytotoxicity of the mycelium extract may be considered moderate or low.
Table 2. Contents of major constituents in hot water extract of Cs-HK1 mycelium used for antitumour activity tests (extract yield = 9·35%)
*Contents are expressed in per gram of extract; values represent mean ± SE, n = 2.
Protein (mg g−1)
32·1 ± 2·85
Sugar (mg g−1)
370·2 ± 6·37
Nucleosides (μg g−1)
142·9 ± 5·21
45·9 ± 3·57
48·0 ± 6·63
401·4 ± 9·42
20·0 ± 2·54
The animal test showed that the mycelium extract treatment of the tumour-bearing mice resulted in 42·9% decrease in the tumour weight (Table 3) and 52·5% decrease in the tumour volume (Fig. 7) over 27 days of treatment compared with the untreated animals. The inhibitory effect of mycelium extract on tumour growth was shown to be statistically significant (P < 0·05). Moreover, the positive control drug showed excellent antitumour effect in the animal tests, which nearly halted the tumour growth. This strong effect of the positive control drug also serves to validate the animal test for the mycelium extract. Therefore, the results indicate that the mycelium extract had significant antitumour activity.
Table 3. Effect of mycelium extract treatment on tumor growth and spleen index in vivo (CPA, cyclophosphamide as a positive control drug)
Body wt (g)
Tumour wt (g) on day 27
*Significance limit P < 0·05; ** significance limit P < 0·01.
21·1 ± 1.14
27·2 ± 4·51
7·67 ± 2.95
CPA, 0·05 g (kg day)−1
21·5 ± 1.37
20·8 ± 1·87
0·21 ± 0·12
Mycelium extract, 0·5 g (kg day)−1
21·3 ± 2·37
25·4 ± 2·48
4·38 ± 2·25
A Tolypocladium sp. fungus, Cs-HK1, has been isolated from wild C. sinensis herb in our experiment, which may be a new anamorphic species of C. sinensis. The Cs-HK1 fungal mycelium from liquid cultures contained most of the known constituents of natural C. sinensis such as cordycepin, adenosine and d-mannitol, although the contents (quantities) of specific components differ considerably. Up to date, only one Tolypocladium sp. fungus, named Tolypocladium sinensis, has been isolated from wild C. sinensis and reported originally in Chinese literature (Li 1988; Jiang and Yao 2002). Although it was claimed to have chemical composition and pharmacological activities similar to the wild species, no relevant experimental data have been reported so far. The T. sinensis fungus was isolated from C. sinensis organism harvested in the Diqing Tibetan Autonomous Region, Yunan Province, China, a few hundred kilometers away from where C. sinensis organism used in our study was collected. The fact that Tolypocladium sp. fungi have been isolated from wild C. sinensis organisms at different regions and years suggests that Tolypocladium sp. is a common fungal species living on the insect host of C. sinensis.
The optimal nutrient composition for mycelium growth and EPS production has been reported for other Cordyceps species in liquid cultures. In Cordyceps jiangxiensis culture (Xiao et al. 2004), the EPS yield was 0·78 g l−1 on glucose and 0·57 g l−1 on sucrose, while the biomass yield was 9·7 g l−1 on glucose and 10·65 g l−1 on sucrose, and maltose was the most favourable among many of the carbohydrates examined for EPS production (1·05 g l−1). In Cordyceps militaris culture (Kim et al. 2003), the EPS yield on sucrose, 0·65 g l−1, was much higher than that on glucose, 0·37 g l−1. The optimal carbohydrate for EPS accumulation may depend on the fungal species, other medium components and culture conditions such as pH, temperature and dissolved oxygen. The Cs-HK1 in our study grew more rapidly (about 20 g dw l−1 in 7–10 days) than those species reported in previous studies (10–15 g l−1 in 10 days). A direct and meaningful comparison of the EPS contents with previous studies is not possible owing to differences in the isolation procedure and analytical method.
Adenosine and cordycepin (3′-deoxyadenosine) are two chemical markers as well as the chief bioactive ingredients of C. sinensis and other Cordyceps species (Ji 1999; Hsu et al. 2002). Adenosine is relatively abundant and has been detected in most natural and cultivated Cordyceps species in a content range of 0·1–3·2 mg g−1 depending on species, geographical location (for natural species) and culture conditions (for fungal mycelia) (Shiao et al. 1994; Li et al. 2004). Cordycepin was originally isolated as a metabolic product from the culture broth of C. militaris fungus, and its synthesis in the Cordyceps fungus was proposed to be from adenosine or phosphorylated adenosine through a reductive mechanism (Lennon and Suhadolnik 1976). Cordycepin content in other Cordyceps species such as C. sinensis is extremely low or undetectable. Furuya et al. (1983) analysed the nucleoside contents in a natural C. sinensis and the mycelium cultures of 17 Cordyceps species, and found detectable cordycepin only in the culture broth of C. militaris. Similarly, the HPLC analysis of nucleosides by Shiao et al. (1994) showed no detectable cordycepin content in several natural and cultivated Cordyceps species except C. militaris. However, Hsu et al. (2002) measured a cordycepin content of 2·4–5·4 μg g−1 in a natural C. sinensis, and 1·4 μg g−1 in the mycelium and 1·9 μg g−1 in the culture broth of a C. sinensis fungus. These contents were significantly lower than those detected in our present study of the natural C. sinensis and the Cs-HK1 mycelium (Table 1). Li et al. (2004) detected cordycepin as high as 5·0 mg g−1 in cultured C. militaris.
The hot water extract of Cs-HK1 mycelium exhibited positive and significant inhibitory effect on the B16-induced tumour growth in animals but low in vitro cytotoxicity against the B16 melanoma cells. These results suggest that the antitumour effect of the mycelium extract (in animals) was mainly caused by other modes of action than by directly killing the tumour cells. Immunomodulation has been widely regarded as a major mechanism of action for the antitumour effects of water and methanol extracts of Cordyceps species (Yoshida et al. 1989; Yamaguchi et al. 1990; Shin et al. 2003). PSs in the Cordyceps species may play a major role in the immunomodulatory activity as many antitumour PSs from various medicinal mushrooms (Wasser 2002). For example, a PS fraction of C. sinensis has been shown to stimulate the production of immune-important cytokines in blood mononuclear cells (Chen et al. 1997). As a complex medicinal fungus, however, its health effects are most likely attributed to the synergistic action of different constituents through multiple mechanisms.
As a preliminary effort for identifying the constituents responsible for the cytotoxic and antitumour effects of mycelium extract, we tested the cytotoxic activity of four pure compounds present in the mycelium hot water extract and its PS fraction (Fig. 8). Of these five components, ergosterol and PS had moderate cytotoxicity against B16 cells, causing about 40% inhibition of cell proliferation, while d-mannitol and cordycepin had only slight inhibition and adenosine had no inhibitory effect on the tumour cell proliferation. From these results, we can only suggest that ergosterol and PS contributed to in vitro inhibition of the tumour cell proliferation by the mycelium water extract but cannot distinguish which components contributed to the antitumour activity observed in the animal tests.
In conclusion, the Cs-HK1 fungus may be a promising medicinal fungus and a useful substitute for the naturally rare and endangered wild species. Fermentation cultivation of fungal mycelium is a relatively simple and cost-effective process, providing a renewable source of naturally rare medicinal fungi. It also allows for convenient and fruitful manipulation of the culture conditions to control or enhance the accumulation of desired active ingredients. In addition to mycelium biomass, the EPS in the fermentation broth may also be valuable bioactive products. There is a need to further study the relationship between the fermentation conditions and the contents of bioactive compounds in the mycelium biomass. It is also of significance to assess the health and pharmacological effects more systematically and to identify and isolate the active constituents of the new C. sinensis fungus.
This work was supported financially by grants from the Hong Kong Polytechnic University and its State Key Laboratory of Chinese Medicine and Molecular Pharmacology in Shenzhen.