Investigation of the biological activities of extracts obtained from usnea subfloridana lichen by crude, crystallized and supercritical CO2 extraction

Lichens and their metabolites are promising sources with diverse pharmacological activities. This study aimed to investigate the biological activities of Usnea subfloridana (Parmeliaceae) lichen collected from Çambaşı Plateau (Ordu). The in vitro antimicrobial and antioxidant activities of U. subfloridana extracts obtained using different drying temperatures (40°C and 80°C) and various extraction methods (supercritical extraction, crystallized lichen powder, and organic solvents) were comprehensively analyzed. Additionally, the phytochemical components of these extracts were identified using gas chromatography-mass spectrometry (GC-MS). The in vitro antimicrobial analyses revealed that all lichen extracts exhibited effective activity. Compared to antibiotics, the strongest antimicrobial effects were observed against the Gram-positive bacteria Micrococcus luteus (zone diameter 38 mm, lichen crystal-40°C) and Enterococcus faecalis (zone diameter 30.75 mm, lichen crystal-80°C). The crystallized lichen extracts of U. subfloridana demonstrated greater effectiveness against the tested microorganisms than the crude extracts and supercritical CO₂ extracts. The total phenolic content of the extracts prepared by maceration with acetone was higher than that of the others, and the supercritical extract from the lichen sample dried at 40°C exhibited almost equivalent phenolic content. The flavonoid content of this extract was also found to be significantly higher than that of all other extracts (220 mg QTE/g extract). In the antioxidant analyses, which assessed the DPPH radical-scavenging ability and Fe³⁺ ion-reducing power, the sample with the highest phenolic content showed the best results. The Fe²⁺ ion-chelating capacity was observed only in the extract with the highest flavonoid content. According to the total antioxidant activity determined using the ammonium molybdate method, the second-best result (104.59 mg AAE/g extract) was obtained from the flavonoid-rich extract prepared via supercritical extraction from the lichen sample dried at 80°C. The inhibition of lipid V peroxidation was approximately 4% for only the 0.02 mg/mL portion of this extract. While all extracts exhibited superoxide dismutase and catalase activities, none displayed peroxidase activity. Extracts with such rich phenolic and flavonoid content proportionally exhibited antioxidant activity and demonstrated promising cholinesterase and tyrosinase inhibition, which are beneficial for neurological diseases. The GC-MS analysis of the different U. subfloridana extracts showed that the highest phytochemical components were detected in the supercritical CO₂ extracts (SC-CO₂) from samples dried at 40°C and 80°C (35 components). The analysis of raw lichen crystals identified 11 components (40°C) and 12 components (80°C), with more than 90% of the lichen crystals composed of usnic acid. The acetone extracts of the lichen contained 29 and 27 components, respectively. The results suggest that the extracts and/or isolated compounds from U. subfloridana could be a potential source of various biological activities. U. subfloridana can be considered a valuable resource for future drug candidates.