Heat Treatment Abolishes Cytotoxicity of the Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycota) Cold-Phosphate-Buffered Saline Extract

Cordyceps militaris is an entomopathogenic fungus that is traditionally used in East Asian medicine. The fungus contains various bioactive compounds that have potential therapeutic applications. Although the immunostimulatory effects of hot-water extracts from C. militaris were previously demonstrated through dectin-1-mediated pathways, the biological properties of cold-water extracts remain unexplored. Therefore, in this study, the physicochemical properties and biological effects of cold phosphate-buffered saline (PBS) extracts from C. militaris fruiting bodies (CPE-CM) were investigated. Cold PBS extraction (4°C for 24 h) resulted in considerable browning, which was effectively suppressed by phenylthiourea, which is a polyphenol oxidase inhibitor. CPE-CM exhibited potent concentration-dependent cytotoxicity in mouse splenocytes. In contrast to previous hot-water extract findings, a comparison between wild-type and dectin-1-knockout mice revealed that this cytotoxicity was independent of dectin-1 signaling. Importantly, cytotoxicity was completely abolished by heat treatment above 70°C, suggesting the presence of heat-labile toxic components. These findings provide new insights into C. militaris extract preparation methods and support the traditional preference for hot-water extraction in Eastern medicine. The study results also emphasize the importance of extraction conditions in determining the biological properties of medicinal mushroom preparations. Furthermore, the study suggests that hot-water extraction may be optimal for obtaining beneficial compounds while eliminating potentially harmful components.