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African Journal of Biotechnology Vol. 7 (17), pp. 3004-3010, 3 September, 2008 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2008 Academic Journals

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Antioxidant activity of aqueous extract of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis
Li Ping Zheng1, Li Wen Gao2, Jian Qin Zhou2, Yang Hu Sima3 and Jian Wen Wang2*
Department of Horticultural Sciences, Soochow University, Suzhou 215123, P.R. China. School of Pharmaceutical Sciences, Medical College, Soochow University, Suzhou 215123, P.R. China. 3 School of Pre-clinical Medicine and Life Sciences, Medical College, Soochow University, Suzhou 215123, P.R. China.
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Accepted 23 July, 2008

Tolypocladium sp. Ts-1 was isolated from the fruiting body of a wild Cordyceps sinensis, one of the best known traditional Chinese medicine and health foods. The antioxidant activities of hot-water extracts from cultured mycelia of Tolypocladium sp. were assessed in different in vitro systems. The extracts showed superoxide dismutase (SOD) activity of 35.6 U/mg protein and are effective in scavenging superoxide radical in a concentration dependent fashion with IC50 value of 1.3 mg/mL. As a reinforcement of the action, similar radical scavenging effects of the extracts were also discerned with both site-specific and non site-specific hydroxyl radical using the deoxyribose assay method. 1,1Diphenyl-2-picrylhydracyl (DPPH) radical scavenging activities of the extracts reached more than 75.2% at the concentrations of 3-6 mg/mL. The extracts showed moderate reducing power and ferrous ion chelating activity. Moreover, the protective effects of the extracts against hydrogen peroxide (H2O2)induced lesion to rat PC12 cell (pheochromocytoma cell line) was observed in a dose-dependent manner from 0.25 to 2.0 mg/mL. Our results suggest that the aqueous extract of Tolypocladium sp. mycelium has strong antioxidant activities and is a potential source of natural antioxidant products. This is the first report on antioxidant activity of Tolypocladium fungus isolated from wild C. sinensis, an endangered species. Key words: Aqueous extracts, antioxidant activity, free radicals, PC12 cells, Tolypocladium sp. INTRODUCTION Cordyceps sinensis (Berk.) Sac, known as Dong-ChongXia-Cao (winter worm-summer grass) in Chinese, has been considered as a tonic food and herbal medicine in China for many centuries. This precious medicinal mushroom formed on an insect larva has been used in traditional Chinese medicine to treat kidney ailments, to relieve pain, to restore general health and appetite, and to promote longevity (Zhu et al., 1998a). Nowadays, numerous pharmacological activities have been found in this medicinal fungus including antitumour, antiinflammation and antiatherosclerosis (Zhu et al., 1998b). The free radical scavenging and antioxidant activity of natural Cordyceps species and cultivated fungal mycelia, and fruiting bodies, for example, have been observed in several studies (Chen et al., 2006; Yu et al., 2006; Zhan et al., 2006). Tolypocladium sp. is one of the numerous anamorphic fungal species isolated from wild C. sinensis (Li, 1988). The aqueous extract and exopolysaccharide from Tolypocladium fungus have been found possessing antitumor and immunomodulatory activities (Yang et al., 2004; Leung et al., 2006). Some reports indicated that the tumor development has a close relationship with oxidative stress (Sun, 1990). With respect to antioxidant activity in Cordyceps, little is known about the antioxidant action of Tolypocladium fungus. As a follow-up to our previous characterization of natural antioxidants from fungal compounds (Sun et al., 2004), the antioxidant potential of hot water extract from Tolypocladium sp. Ts-1

*Corresponding author. E-mail: jwwang@suda.edu.cn. Tel: +86 512 65880025. Fax: +86 512 65880031.

Zheng et al.

3005

was evaluated by DPPH/superoxide/hydroxyl radical scavenging, reducing power and metal chelating activity in the present study. The superoxide dismutase (SOD) activity in mycelia extracts was also investigated. Finally, the oxidative injury protective effect of the extracts was examined on PC12 cells (pheochromocytoma cell line) treated with peroxide hydrogen (H2O2).
MATERIALS AND METHODS Cultivation and extraction of mycelia of Tolypocladium sp. The fungus used in this study was isolated from the fruiting body of a wild C. sinensis collected on the high plateaus (altitude over 4000–4500 m) in Sichuan, China. It was identified as one of the anamorph strains phylogenetically related to C. sinensis and belongs to Tolypocladium genus (Chen et al., 2006) and was deposited in our lab with a voucher number Ts-1. The Ts-1 mycelium was cultivated on liquid medium containing (per liter) 40 g glucose, 10 g yeast extract, 5 g peptone, 1 g KH2PO4 and 0.5 g MgSO4 in shakeflasks at 150 rpm and 28oC. After 7 days of shaking incubation, mycelium biomass was harvested from the liquid culture by filtration and dried at 50oC. The dry mycelium was pulverized and extracted with 10 volumes of distilled water at 95–100oC for 2 h. After vacuum filtration, the filtrate was collected and the residue was re-extracted under the same conditions twice again. The filtrates were combined and condensed to one-third of their total volume with a rotary evaporator under reduced pressure at 50oC, and the resultant extract was lyophilized to dryness in vacuum. The lyophilized power was stored in dark at 4oC before being used for the bioactivity tests. Determination of polysaccharide, protein, mannitol and total phenolic contents The aqueous extract (5 g) was submerged in 400 ml hot water. The supernatant was condensed to 1/3 volume and centrifuged at 3000 r/min for 10 min to discard undissolved substances, followed by adding three times of its volume of absolute ethanol, stirred vigorously and kept overnight at 4oC. The precipitate was collected by centrifugation and washed with absolute ethanol, and then was redissolved, dialyzed with a dialysis tube of 3.3 cm in diameter to remove small molecules (

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