Protein-rich Isolate Extracted from Cordyceps militaris Fruiting Body and Anticancer Activities on Cancer Cells

Cancer remains a global health challenge due to the limitations of conventional therapies, including severe side effects and drug resistance. While Cordyceps militaris (L.) Fr. is well-regarded in traditional medicine for its antitumor effects, research has primarily focused on small molecules like cordycepin. The anticancer potential of its total protein-rich isolate remains less characterized. This study aimed to extract, characterize, and evaluate the in vitro cytotoxic activities of the protein-rich isolate from C. militaris fruiting bodies against various human cancer cell lines. Proteins were extracted from dried fruiting bodies using an alkaline extraction method (pH 12.0) followed by isoelectric precipitation (pH 7.0) and lyophilization. The isolate was analyzed for yield, protein content, water activity, and moisture. Bioactive markers (cordycepin and adenosine) were quantified using UHPLC. Cytotoxicity was assessed via MTT assay against MCF-7 (breast), HT-29 and Caco-2 (colorectal), and HepG2 (liver) cancer cells, with HDFn (normal fibroblasts) used to determine selectivity. Results showed that the extraction process achieved a high yield of 72.61% with a protein content of 41.90 ± 0.19%. The isolate demonstrated excellent stability with a low water activity (aw) of 0.22 ± 0.01. Notably, cordycepin was significantly enriched in the isolate, increasing tenfold to 1048 mg/100 g compared to the raw powder. The protein-rich isolate showed dose-dependent cytotoxicity, with the highest potency against MCF-7 cells (IC50 = 8.68 µg/mL). Crucially, the isolate exhibited promising selectivity in this model, being nearly 55 times less toxic to normal HDFn cells (IC50 = 475.56 µg/mL). It is concluded that C. militaris protein-rich isolate is a stable, bioactive-rich fraction with potent and selective anticancer activity. The significant enrichment of cordycepin and the wide therapeutic window suggest its potential as a safe candidate for functional foods or adjuvant cancer therapies.