The lichen collection from Bukit Larut, Taiping, Malaysia in 1999 included Bulbothrix isidiza, Chrysothrix xanthina, Cladonia adspersa, C. verticillata, Coccocarpia palmicola, Heterodermia flabellata, H. japonica, H. obscurata, Hypotrachyna imbricatula, Leptogium azureum, Parmelinella wallichiana, Parmotrema tinctorum, P. clavuliferum, P. reticulatum, Pertusaria sp., Physma byrsaeum, Usnea baileyi and Usnea rubrotincta. Secondary metabolites could not be detected in three lichens, Coccocarpia palmicola, Leptogium azureum and Physma byrsaeum by HPLC and TLC analysis. The other 15 lichen species showed the presence of ten classes of compounds, depsides (10 compounds), depsidones (16), quinones (5), xanthones (2), naphthopyrones (1), pulvinic acid derivatives (1), diphenylethers (1), dibenzofurans (1), aliphatic acids (4) and terpenoids (3).
Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC-MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for "dual" bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.