Ganoderma lucidum, a well-known Chinese medicinal fungus, has been used to promote health and longevity in East Asian countries. Its medical effects on cancer, hypertension, hepatitis, and hypercholesterolemia have been demonstrated by pharmacological studies in the past two decades. [1][2][3][4] This fungus has attracted considerable attention partly because the polysaccharides isolated from the fruit bodies and the mycelium have shown antitumor and hyperglycemic activity. 3,5,6) However, the active components in the spores of G. lucidum have rarely been studied owing to the difficulty in collection and sporoderm-breaking of the spores. Recently, with the successful cultivation of G. lucidum indoors on a large scale and a breakthrough in sporodermbreaking technology, much attention has been paid to chemical components of G. lucidum spores 7,8) and their versatile biological activities.9,10) More recently, we have reported an immunomodulating polysaccharide from the spores of G. lucidum. 11) In the present study, we deal with the purification and characterization of the T lymphocyte-stimulating polysaccharide, based on the activity-guided principle, from the hot-water extract of the sporoderm-broken spores of G. lucidum. A series of sulfated or carboxymethylated derivatives of the polysaccharide were successively prepared and their structures were elucidated by chemical and spectral methods. The solution conformation and T lymphocyte proliferation effect of the polysaccharide and its derivatives are compared and discussed.
ExperimentalMaterials Spores of G. lucidum were collected in Shanxi Province, PR China. It was identified by Dr. Xiu-Lan Huang and stored as a voucher specimen (no. 99064) in the Herbarium of Phytochemistry Department of Shanghai Institute of Materia Medica, Shanghai, PR China. Before use, the sporoderm of the spores was ultrasonographically broken and the broken ratio was estimated about 60-80% by electronic microscopy. T-Dextran in a series of different molecular weights was obtained from Pharmacia. Concanavalin (Con A) was from Sigma and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Fluka. Medium RPMI-1640 was purchased from Gibco Laboratories. All other reagents were of the highest quality available and were used without further purification.Isolation and Purification of the T Lymphocyte-Stimulating Polysaccharide The sporoderm-broken spores of G. lucidum (1.0 kg), previously defatted with 95% alcohol, were decocted for 6 h with 10 l of water to half the original volume, and the residue was again decocted with 8 l of water. The combined aqueous extract was deproteinated with trichloroacetic acid, and the resulting aqueous fraction was extensively dialyzed against running water for 3 d and then against distilled water for 1 d (molecular weight cutoff 3000-5000 Da). The retentate was concentrated under reduced pressure to a small volume (1.8 l), and 4 volumes of ethanol were added stepwise with stirring at 4°C. Then the mixture was stored overnight at Ϫ10°C. T...
