Mycelium Culture of Ganoderma lucidum and Its Cytotoxic Principles

Transcription

1 Mycelium Culture of Ganoderma lucidum and Its Cytotoxic Principles Poungpet Poonsapayaf. Wimonmat Boonmee2 and Dusanee Thanaboripatl 'Biotechnology Section, ReseaJch and Development Institute The Government pharmaceutical Organization, Rama VI Rd., Bangkok Thailand 2Department of Applied Biology, Faculty of Science King Mongkut's Institute oftechnology Ladkrabang, Bangkok Thailand Abstract Polysaccharides obtained from Ganoderrna lucidum are reported to exhibit antitumor activity. Recentty, mycelium of this mushroom is being cultivated and the bioactive polyglycans are also found. We cultured mycelium of G. lucidum cultivated in Thailand in Potato Dextrose Broth, Yeast-Malt Extract and Glucose-Yeast Extract liquid media in combination with 20, 40, and 60 gil glucose. The effects of the media on mycelial dry weight were studied. Crude extract obtained from mycelium grown in Yeast- Malt Extract medium were higher than those obtained from other media. It consisted of significantly higher protein content with low polysaccharide compared to extracts from. other media. Whereas, crude extract of my~iium grown in Glucose- Yeast Extract medium consisted of significantly higher polysaccharide content with low protein. Cytotoxic activities of hot water extract of mycelia were tested against P-388 leukemia cens in vitro. Introduction Ga110derma lucidum, a mushroom widely used in Chinese medicine, is known for its medicinal values in Asia. The fruiting body and mycelium have been extracted for active compounds (Jong and Binningham, 1992). Its water extract polysaccharides are reported to exhibit antitumor activities (Miyazaki and Nishijirna, 1981; Sone et ai., 1985). Although this mushroom is found' growing wildly in Thailand, some strains were also introduced for cultivation. This paper reports the effects of culture medium on mycelial growth in one of the cultivated strains and the production of crude extracts with their total polysaccharides and protein contents. The cytotoxicity effects of these crude extracts were also studied. Materials Mycelium and Method culture Mycelium of Ganoderma lucidum cultivated in Thailand was kindly provided by the Research and Development Section, the Royal Chitralada Projects, Bangkok, Thailand. We cultured mycelium in Potato Dextrose Broth (PDB), Yeast-

2 malt extract and Glucose-yeast extract liquid medium (Sone et al., 1985) with varying concentrations of glucose (20, 40 and 60 gil) under static condition at 28:t2 C. Mycelium dry weights were taken for growth measurement after 28 days in culture. Extraction of mycelium Dry mycelium obtained from each medium was extracted with distilled water at 1:20 (w/v) at 80 C for 6 h. The extracts were filtered through Whatman No.1 filter paper and the filtrates were evaporated at 50 C to eliminate approximately 90% of water. The remaining filtrates were precipitated with absolute Ethanol at 1:6 (v/v) and incubated at 4 C overnight. Precipitates were collected by centrifugation at 10,000 rpm, 4 C for 20 min and dried in vacuo. Determination of total polysaccharides and protein contents Crude extracts were analysed for total polysaccharide content using Phenolsulfuric acid method (Dubois et ai., 1956) and for protein content using Lowry's method (Dunn, 1995). Biological assay MTT (3 -[4,5 -dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimeter assay used in this study was modified from Mosmann (1983) and Jiratchariyakul et af. (1996). 'BHK (Baby Hamster Kidney) cells were maintained in EMEM medium (BioWhittaker) supplemented with 10% fetal bovine serum (BioWhittaker) and 100 J.1g/mLpenicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2. Cells (2x105 cclls/ml) were inoculated in 50 J.1Laliquot to each well of microtiter plate. Various sample concentrations (50 J.1L)were added to the cultures at day I after transplantation. At day 3, the supernatant was removed by carefully inverting, flicking and blotting the plates. MTT solution (I mg/ml) was added to the cultures at 50 J.1Lper well. After 4 h incubation, untransforrned MTT was removed and 50 J.1Lisopropanol was added to each well and shake vigorously in a microplate reader. The optical density measurements were made using a microplate reader at 570 om. Three replicate wells were used to defermine each ~ata. point.. P-388 leukemia cells were maintained in RPMI-1640 medium' (BioWhittaker)supplemented with 10% fetal bovine serum (BioWhittaker) and 100 ~ml penicillin/streptomycin at 37 C in a humidified atmosphere of 5% CO2. Cells (2x I04 cells/ml) were inoculated in 100 J.LLaliquot to each web of microtiter plate. Various sample concentrations (100 J.LL)were added to the cultures at day I after

3 Results and Discussion Mycelium growth Extradion of mycelium and its total polysaccharide and protein contents

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5 Bioassay of cytotoxic activity Acknowledgement Literature Cited

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