Do phenolic contents relate to their in-vitro cytotoxicity against colorectal cancer cell lines from Calvatia lilacina (puffball mushroom)?

Phenolic compounds found in mushrooms are regarded as valuable phytochemicals due to their potential health benefits, including anti-cancer potential. While antioxidant activity has traditionally served as a proxy for assessing anti-cancer potential, intrinsic variations (developmental stages) and extrinsic factors (analytical methods) complicate the establishment of clear relationships between phenolic contents and cytotoxicity against cancer cell lines. This case study thoroughly examines the correlations between phenolic contents and in-vitro cytotoxicity against colorectal cancer cell lines using fruiting bodies of Calvatia lilacina (C. lilacina) at two distinct growth stages, highlighting the potential of C. lilacina as a source of novel anti-cancer agents. Two widely used methods, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and colorimetric assays, are employed to profile phenolic compounds and estimate associated in-vitro antioxidant capacities. This comparative analysis revealed discrepancies both within different colorimetric assays (intra-method variation) and between colorimetric and HPLC-MS/MS analyses (inter-method variation). Notably, the total abundance of phenolic compounds of an extract measured via HPLC-MS/MS exhibited a strong correlation with its cytotoxicity against colorectal cancer cell lines, whereas the antioxidant capabilities determined by colorimetric assays showed no significant correlation with cytotoxic effects. This study identified crude extracts containing 2-hydroxycinnamic acid as potentially crucial cytotoxic agents against the cancer cell lines. These findings underscore the importance of temporal analysis in monitoring the dynamic changes in the composition and content of phenolic compounds in mushrooms, coupled with balanced extraction methods across a broad range of solvent polarity. Furthermore, the results indicate that antioxidant capabilities determined using colorimetric assays are not suitable for estimating anti-cancer potential, even for preliminary screenings, as interferences (such as pigments) and non-specific reactions can lead to misestimation of antioxidant capacities. In contrast, owing to the superb capability to eliminate interference in target analysis, HPLC-MS/MS emerges as a valuable tool to unambiguously identify phenolic compounds for screening nutraceuticals with anti-cancer potential.