Identification of a novel component leading to anti-tumor activity besides the major ingredient cordycepin in Cordyceps militaris extract

•Two fractions, F-0-N and F-4, separated from Cordyceps militaris extract were noticed as showing anti-tumor activity with significant level.•A modified LC/MS method using an APCI ion source with chlorine adduct ionization uncovered presence of xylitol in the F-0-N fraction.•Besides of cordycepin in F-4 fraction, xylitol showed central anti-tumor activity in the F-0-N fraction.•Xylitol contributed to the anti-tumor activity synergistically with cordycepin.

In accordance with our previous study that was carried out to identify novel anti-tumor ingredients, chromatographic separation in combination with an anti-tumor activity assay was used for analysis of Cordyceps militaris extract in this study. Various modes of chromatography including reversed-phase, cation-exchange and anion-exchange were used to separate components of Cordyceps militaris, which showed various chemical properties. Anti-tumor activity of each fraction was assessed by a Hoechst staining-based apoptosis assay using malignant melanoma MeWo cells. By these repeated approaches through chromatographic segregation and cell biological assay, we finally succeeded in identifying the target substance from a certain fraction that included neutral hydrophilic components using a pre-column and post-column chlorine adduct ionization LC-APCI-MS method. The target substance was a mono-carbohydrate, xylitol, that induced apoptotic cell death in MeWo cells but not in normal human OUMS-24 fibroblasts. This is the first study showing that Cordyceps militaris extract contains a large amount of xylitol. Thus, our results will contribute greatly to uncovering the mysterious multifunctional herbal drug Cordyceps militaris as an anti-tumor agent.