| Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 99 === Cordyceps militaris is a model species for Cordyceps research, and is phylogenetically close to an oriental traditional medicine, Cordyceps sinensis. We have purified a new protein from the fruiting body of C. militaris by a series of process such as homogenization, ultra sonication, ammonium sulfate precipitation, and cation exchange chromatography through CM-52 and Resource S columns. The molecular weight of the protein was ~18 kDa as determined by SDS-PAGE analysis and designated as Cordyceps militaris protein 18, CMP18. The results of native-PAGE and the conditions used in column chromatography suggested that the isoelectric point of CMP18 might be greater than 8.8. The N-terminal amino acid sequence determined by automated Edman degradation was GPSVVVGYRTVSAAQAK. The results of PAS staining and hemagglutination assay indicated that CMP18 was not a glycoprotein and did not possess agglutination activity toward mouse red blood cells.
According to MTT cell viability test, CMP18 inhibited the cell viability in mice primary splenocytes and peritoneal macrophages, murine hepatoma cell line BNL, murine leukemia macrophage cell line RAW264.7, and murine leukemia B cell line X63. Further tests on murine hepatoma cell lines BNL revealed that CMP18 inhibited the cell viability in a time- and dose-dependent manner. By trypan blue staining and cell LDH releasing assay, we proved that CMP18 could cause cell death. Furthermore, cell cycle analysis as well as annexin V and PI double stain indicated that CMP18 might mediate BNL cell apoptosis, but not necrosis. Cell caspase-9 was activated and the mitochondria potential was lost after co-incubation with CMP18. These results showed that CMP18 might induce cell apoptosis through a mitochondria dependent pathway. After heat treatment or alkalization, CMP18 would be degraded and lost the ability to induce cell apoptosis, which allowing safe consumtion of Cordyceps militaris.
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