Figure 1.
Overview of experimental design.
(A) Cultivation of Lignosus rhinocerotis and extraction of low-molecular-weight compounds using aqueous methanol. Extracts were prepared from the mycelium (LR-MH, shaken cultures; LR-MT, static cultures), culture broth (LR-BH, shaken cultures; LR-BT, static cultures), and sclerotium (LR-SC). The different developmental/morphological forms of L. rhinocerotis: (B) sclerotium from solid-substrate fermentation, (C) mycelial pellet in shaken cultures, and (D) mycelial pellicle in static cultures of liquid fermentation.
Table 1.
Antioxidant capacity of L. rhinocerotis extracts.
Table 2.
Cytotoxic effect of L. rhinocerotis extracts.
Table 3.
Chemical composition of L. rhinocerotis extracts.
Figure 2.
(A) Electrophoretic analysis of proteins in the extracts of Lignosus rhinocerotis and visualisation by Coomassie Brilliant Blue (top) and silver staining (bottom). Molecular weight (MW) of the bands was estimated from the plot of log MW vs. relative migration distance (Rf) based on the values obtained from the bands of the marker (7−200 kDa). The estimated sizes of the bands were as follows: 1, 2 (4.0 kDa), 3 (38.0 kDa), 4 (14.0 kDa), 5 (9.5 kDa), 6 (8.0 kDa), and 7 (4.7 kDa). (B) Representative SELDI-TOF-MS spectra of the low-molecular-weight proteins (5−20 kDa) in the extracts. The x-axis represents the m/z values, and the y-axis represents the intensity of the signals (µA). Peaks with signal/noise ratios (S/N) >5 were automatically detected.
Table 4.
Chemical constituents in LR-MH and LR-MT based on GC-MS analysis.
Table 5.
Chemical constituents in LR-BH and LR-BT based on GC-MS analysis.
Table 6.
Chemical constituents in LR-SC based on GC-MS analysis.
Figure 3.
The UHPLC-ESI-MS TIC (negative mode) of the extracts of Lignosus rhinocerotis.
The profiles of the extracts of the mycelium (LR-MH, LR-MT), culture broth (LR-BH, LR-BT), and sclerotium (LR-SC) were different.
Figure 4.
The MS/MS fragmentation (in negative mode) of selected low-molecular-weight compounds in the extracts of Lignosus rhinocerotis.
Collision energy was set at 35 eV. The compounds were tentatively identified based on their mass fragmentation patterns.
Table 7.
Chemical constituents in LR-MH and LR-MT based on UHPLC-ESI-MS/MS.
Table 8.
Chemical constituents in LR-BH and LR-BT based on UHPLC-ESI-MS/MS.
Table 9.
Chemical constituents in LR-SC based on UHPLC-ESI-MS/MS.
Figure 5.
The UHPLC-ESI-MS (m/z 100–1000) principal component analysis of the extracts of Lignosus rhinocerotis.
Duplicate analysis of the extracts of mycelium from shaken (MH1, MH2) and static (MT1, MT2) conditions, culture broth from shaken (BH1, BH2) and static (BT1, BT2) conditions, and sclerotium (SC1, SC2) were performed. (A) Score plot revealed that mycelia from shaken and static conditions were distinct from the sclerotium. (B) Loading plot with multiple ions common to all extracts (centre) and marker ions far from the centre, e.g. m/z 161, 325, 339, and 766, were characteristic of individual extracts. The identification of the compounds is warranted for determining biomarkers for L. rhinocerotis from different morphological/developmental stages.