Lichen metabolites have multiple functions in nature and these include several biological activities against pathogens and multi-drug resistant bacteria. This study shows the antibacterial properties of 18 Usnea species collected from five provinces in the northern part of Luzon, Philippines. A total of 41 lichen specimens were extracted using acetone to a final concentration of 10 mg/ml. The crude extracts were initially subjected to TLC. Interestingly, 17 different lichen metabolites were identified. The 41 crude lichen extracts were then tested against Staphylococcus aureus and Bacillus subtilis using paper-disk diffusion assay, most of which were found to be more active against the latter test organism. In addition, 28 of the 41 lichen extracts were also tested against Mycobacterium phlei. Interestingly, most of the samples exhibited much greater activities than that of the antibiotics used as indicated by the zones of inhibition. Moreover, 15 crude extracts showing distinct spots in their TLC profiles were tested against Nocardia asteroides using TLCbioautography. Results showed that usnic acid, norstictic acid, and salazinic acid were the bioactive lichen metabolites.

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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.

Endolichenic fungi (ELF; asymptomatic microorganisms living inside healthy lichen thalli) were isolated from three Usnea species, namely U. baileyi, U. bismolliuscula and U. pectinata, collected near the town of Sagada, Philippines. A total of 101 ELF were recovered representing 12 genera (classes Sordariomycetes and Eurotiomycetes), with the genera Nemania (50.5%, 51 isolates) and Xylaria (22.8%, 23 isolates) being the most abundant.Comparative analyses on the antimicrobial activities of lichens and ELF revealed that lichen crude extracts were effective against the Gram-positive bacterium Staphylococcus aureus and the yeast Candida albicans, while ELF crude extracts were effective against S. aureus, C. albicans and the Gram-negative bacterium Escherichia coli. The broad-spectrum nature of ELF has provided medicinal and industrial advantages over the slow-growing lichens as shown on their respective bioactivities. Extracts from ELF also had a higher total flavonoid content (TFC; 6.29-85.69 mg QE/g of extract) and stronger antioxidant activities (IC 50 : 0.57-19.63 mg/ml) than lichen-derived extracts. Although only culturable ELF were identified, this study provides the first evaluation of the diversity and bioactivities of culturable ELF from fruticose lichens of the genus Usnea in the Philippines.

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