Antioxidant and Cytotoxic Activities of Usnea barbata (L.) F.H. Wigg. Dry Extracts in Different Solvents

Abstract

Lichens represent a significant source of antioxidants due to numerous metabolites that can reduce free radicals. Usnea barbata (L.) F.H. Wigg. has been recognized and used since ancient times for its therapeutic effects, some of which are based on its antioxidant properties. The present study aims to analyze the phytochemical profile and to evaluate the antioxidant and cytotoxic potential of this lichen species. Five dry extracts of U. barbata (UBDE) in different solvents (acetone, ethyl acetate, ethanol, methanol, water) were prepared by refluxing at Soxhlet to achieve these proposed objectives and to identify which solvent is the most effective for the extraction. The usnic acid content (UAC) was quantified by ultra-high performance liquid chromatography (UHPLC). The total polyphenols content (TPC) and tannins content (TC) were evaluated by spectrophotometry, and the total polysaccharides (PSC) were extracted by a gravimetric method. The 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical method was used to assess the antioxidant activity (AA) and the Brine Shrimp Lethality (BSL) assay was the biotest for cytotoxic activity evaluation. The ethyl acetate extract had the highest usnic acid content, and acetone extract had the highest content of total polyphenols and tannins. The most significant antioxidant effect was reported to methanol extract, and all the extracts proved high cytotoxicity. The water extract has the lowest cytotoxicity because usnic acid is slightly soluble in this solvent, and it was not found at UHPLC analysis. All extracts recorded a moderate correlation between the content of usnic acid, polyphenols, tannins, and AA; furthermore, it has been observed that the cytotoxicity varies inversely with the antioxidant effect.

Usnic Acid and Usnea barbata (L.) F.H. Wigg. Dry Extracts Promote Apoptosis and DNA Damage in Human Blood Cells through Enhancing ROS Levels

“…U. barbata was harvested from the Călimani Mountains (900 m altitude, Suceava County, Romania). Three dry extracts were obtained in different solvents: ethyl acetate (Chemical Company S.A., Iasi, Romania), acetone and ethanol (Chimreactiv SRL Bucharest, Romania) using a method described in detail in our previous study [ 13 ]. The dry extract in ethyl acetate was used only for usnic acid isolation.…”

“…This process consists of usnic acid extraction by ultra-high performance liquid chromatography (UHPLC) with photodiode array detector (PDA), followed by collecting the separated fraction. A semi-preparative technique was adapted by our UHPLC analytical method previously validated [ 13 ]. The PerkinElmer ® Flexar ® FX-15 UHPLC system was equipped with a Flexar FX PDA-Plus photodiode array detector (PerkinElmer® Waltham, MA, USA) and a Cosmosil 5-C18-AR-2 chromatographic column with a length of 150 mm and an inner diameter of 20 mm (producer: Nacalai Tesque, Kyoto, Japan); in addition, a Gilson FC 203B fraction collector (Gilson Co, Middleton, WI, USA) was used.…”

“…The usnic acid peak was collected manually between 12.5 and 13.5 min (on approximately 15% of its height, preserving the peak purity) after four successive injections (4 × 400 µL 8 mg/mL UBEA in DMSO) at 254 nm. Its identity was confirmed by comparing the retention time of the most significant peak of the sample solution with the reference one [ 13 ]. Furthermore, the solution was collected in a previously weighed vial ( supplementary material, video sequence S1 ).…”

“…The purity of previously isolated UA was determined using the UHPLC-PDA analytical method [ 13 ]. First, the sample resulting from drying was weighed and brought to a final concentration of 160 µg/mL.…”

“…Wigg ( U. barbata ) is a fruticose thalli lichen with interesting therapeutic properties [ 11 ]; this species has been used for thousands of years in traditional medicine worldwide to treat various diseases [ 12 ]. The wide range of bio-activities (antioxidant [ 13 ], antimicrobial [ 14 ], anti-inflammatory [ 15 ], anticancer [ 16 ], cytotoxic [ 17 ], pro-oxidant [ 18 ]) is due to the content of active secondary metabolites [ 19 ] synthesized by the mycobiont (lichen-forming fungus) [ 20 ]. The phytochemical profile of U. barbata is already known.…”