Antiproliferative Fatty Acids Isolated from the Polypore Fungus Onnia tomentosa

Abstract

Onnia tomentosa is a widespread root rot pathogen frequently found in coniferous forests in North America. In this study, the potential medicinal properties of this wild polypore mushroom collected from north-central British Columbia, Canada, were investigated. The ethanol extract from O. tomentosa was found to exhibit strong antiproliferative activity. Liquid-liquid extraction and bioactivity-guided fractionation, together with HPLC-MS/MS and 1D/2D NMR analyses of the ethanol extract of O. tomentosa, led to the identification of eight known linoleic oxygenated fatty acids (1.1-1.4 and 2-5), together with linoleic (6) and oleic acids (7). The autoxidation of linoleic acid upon isolation from a natural source and compound 5 as an autoxidation product of linoleic acid are reported here for the first time. GC-FID analysis of O. tomentosa, Fomitopsis officinalis, Echinodontium tinctorium, and Albatrellus flettii revealed linoleic, oleic, palmitic, and stearic acids as the major fatty acids. This study further showed that fatty acids were the major antiproliferative constituents in the ethanol extract from O. tomentosa. Linoleic acid and oleic acid had IC₅₀ values of 50.3 and 90.4 µM against human cervical cancer cells (HeLa), respectively. The results from this study have implications regarding the future exploration of O. tomentosa as a possible edible and/or medicinal mushroom. It is also recommended that necessary caution be taken when isolating unstable fatty acids from natural sources and in interpreting the results.

Keywords: Hymenochaetaceae; Onnia tomentosa; antiproliferative; autoxidation; fatty acids; linoleic acid; oleic acid.

Figure 1

Antiproliferative and immunomodulatory activities of crude extracts from O. tomentosa. Dose- (a) and time-dependent (b) MTT cell viability assays on HeLa cells. The concentration of E1 used for the time-dependent experiment (b) was 0.5 mg/mL. (c) At 1 mg/mL, crude extracts from O. tomentosa were assessed for their ability to induce TNF-α production in RAW264.7 macrophage cells as an indicator of immunomodulation. Lipopolysaccharide (LPS) was used as a positive control, whereas medium and water were used as negative controls. The results presented are representations of two separate experiments (n = 2). Error bars are SD. One-way ANOVA (Tukey Test) was used for statistical analysis. * denotes p < 0.05 as compared to the water control.

Figure 2

Molecular structures and selected MS/MS fragments of mixture 1 (1.1–1.4) and compounds (2–7) found in O. tomentosa antiproliferative fractions.

Figure 3

Autoxidation of linoleic acid. Linoleic acid was incubated at room temperature, and on different days an aliquot was taken for quantification via HPLC. Linoleic acid on days 2, 4, 10, 15, and 23 was quantified by comparing the peak area of products relative to day 0 (32 mM). The results shown (mean ± S.D.) were averaged from three replicates (n = 3).

Figure 4

Formation of HODEs and KODEs upon autoxidation of linoleic acid at room temperature. Separation of the mixture 1 (HODEs) peak could not be fully achieved, and it was analyzed as a mixture. The peaks identified were as follows: 1: mixtures of 8-HODE, 9-HODE, 10-HODE, and 12-HODE; 2/3: 13-KODE; 4: 9-KODE. All HODEs and KODEs were quantified by comparing the peak areas of products relative to day 0. HPLC was used to monitor the autoxidation reactions and the results were shown as means ± S.D., averaged from three replicates (n = 3).

Figure 5

The linoleic acid, oleic acid, and LAAP mixture exhibited antiproliferative activity against HeLa cells. HeLa cells were treated with the linoleic acid, oleic acid, and LAAP mixture for 48 h at 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 µM. Cell viability was determined using the MTT assay. The result shown is representative data from three biological replicates (n = 3). Error bars indicate standard deviation (S.D.).