Chemical composition and cellular toxicity of ethnobotanical-based hot and cold aqueous preparations of the tiger's milk mushroom (Lignosus rhinocerotis)

The sclerotium of the "tiger's milk mushroom" (Lignosus rhinocerotis) is used as tonic and folk medicine for the treatment of cancer, fever, cough and asthma by the local and indigenous communities. It is traditionally prepared by either boiling or maceration-like methods; however, there is no attempt to understand how different processing methods might affect their efficacies as anticancer agents. This investigation was undertaken to evaluate the cytotoxicity of the hot and cold aqueous extracts of Lignosus rhinocerotis and to deduce the nature of the chemical component(s) that might be responsible for differential cellular toxicity of the extracts. The hot (LR-HA) and cold (LR-CA) aqueous extracts of the sclerotium of Lignosus rhinocerotis were prepared. The levels of bioactive components in the extracts were determined and chemical profiling was performed using UPLC-ESI-MS, SDS-PAGE and SELDI-TOF MS. Cytotoxicity of LR-HA and LR-CA against a panel of human cancer and normal cell lines was assessed by the MTT and trypan blue exclusion assays. Changes in cell morphology upon treatment with the extracts were observed. The chemical composition and bioactivities data were correlated to explain the nature of the cytotoxic component(s). LR-HA and LR-CA were particularly abundant in polar components. Both extracts exhibited varying degree of cytotoxicity against the cancer cell lines with LR-CA showed significantly stronger cytotoxicity (IC50: 37-355 µg/ml) than LR-HA (IC50>500 µg/ml); however, LR-CA lacked selectivity in that it also has cytotoxic effect on the normal cell lines. Based on the results of protein profiling of heat-treated LR-CA (40-100°C) coupled to the MTT assay, the cytotoxic component(s) in LR-CA were deduced to be thermo-labile, water-soluble protein/peptide(s). Our findings have shown that the use of different preparation methods (hot and cold aqueous extraction) for Lignosus rhinocerotis has resulted in extracts with distinctively different cellular toxicity in which the cytotoxic constituents were present only in LR-CA.