Usnic acid (UA) is a compound with multiple biological activities that make it useful in various industries, e.g., pharmaceutical, cosmetic, dentistry, and agricultural sectors. Lichens are the primary source of UA, which is primarily extracted using acetone. This study aimed to investigate the solubility of UA in numerous natural deep eutectic solvents (NADESs) and use a mixture of thymol and camphor as a NADES in the optimization of the UA extraction process with the design of experiments method. For numerical optimization, the following parameters were employed in the experiment to confirm the model: a camphor-to-thymol ratio of 0.3, a liquid-to-solid ratio of 60, and a time of 30 min. The obtained experimental results aligned well with the predicted values, with the mean experimental value falling within the confidence interval, exhibiting deviations between 11.93 and 14.96. By employing this model, we were able to optimize the extraction procedure, facilitating the isolation of approximately 91% of the total UA content through a single extraction, whereas a single acetone extraction yielded only 78.4% of UA.

NADESs represent a modern class of extraction media that align with the principles of green chemistry. They are considered non-toxic and biodegradable, but relatively little is known about their biological activity. This study investigated the antioxidant, antibacterial, and antifungal properties of 40 NADESs. The systems were developed from primary (PRIM) based on choline chloride (ChCl), and specialized (HEVO) plant-derived metabolites, particularly based on thymol and menthol. Their antioxidant activity was evaluated using spectrophotometric tests. The antimicrobial activity was evaluated by the disk diffusion method. The data obtained were analyzed using principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). NADESs based on PRIM exhibited negligible antioxidant activity and relatively low antimicrobial activity. By contrast, NADESs containing HEVO, particularly thymol-based systems, indicated significant antioxidant activity, with stronger activity observed at higher molar proportions of thymol. In the 1,8-cineole:thymol system, ABTS activity ranged from 167.37 ± 24.17 to 861.25 ± 33.03 mg Trolox equivalents/mL NADES (molar ratios 9:1 and 1:9, respectively). The 1,8-cineole:thymol system (1:9) also showed strong antimicrobial activity, with a maximum inhibition zone of 39.33 ± 2.52 mm against Staphylococcus aureus. In summary, NADESs based on HEVO exhibit significantly stronger biological activity than those containing only PRIM.

Carlina acaulis L. (Asteraceae) is a medicinal plant with a long history of use as an herbal remedy. Recently, this species attracted the interest of agrochemical companies due to the presence of valuable biopesticides among which the most promising is carlina oxide (COx). To stimulate the biosynthesis of its specialised metabolites by elicitation, a polysaccharide – chitosan was supplemented as chitosan lactate (ChL). The aim was to evaluate the effects of ChL soil and foliar application on the C. acaulis biomass, photosynthetic efficiency, and levels of the COx, chlorogenic acids, protocatechuic acid, schaftosides, and triterpenic acids. ChL has been shown to have no effect on shoot growth while considerably increasing root biomass. It improved photosynthetic efficiency and enhanced levels of specialised metabolites. Soil application of ChL significantly increased chlorogenic and neochlorogenic acid content in leaves, by approximately 330% and 70%, respectively compared to controls. In contrast, foliar application more strongly increased COx levels in roots (55% for foliar vs. 16% for soil treatment). This study has demonstrated that the application of ChL can be used as an inexpensive and accessible way to boost the production of valuable compounds in C. acaulis.

Usnic acid (UA) is one of the most extensively studied specialized metabolites of lichens, attracting considerable interest due to its antimicrobial, anti-inflammatory, and cytotoxic properties. The efficiency of UA extraction from lichens depends on multiple interrelated biological and technological factors. This systematic review aims to synthesize and critically evaluate reported strategies for UA extraction from wild-grown lichen biomass, with particular emphasis on extraction efficiency, practicality, and application potential. This systematic literature review, based on the Scopus database was conducted by including original research articles reporting UA extraction from wild-growing lichens. The analysis covered species selection, sample pre-treatment, solvent type, and extraction methodology. A total of 117 studies were included. Due to the predominantly non-polar nature of UA, higher extraction efficiencies were generally achieved using solvents, including acetone, supercritical CO₂, vegetable oils, and lipophilic green solvent systems. Pre-treatment strategies such as grinding or flaking significantly enhanced extraction performance by improving mass transfer. Alongside conventional methods (maceration, reflux, Soxhlet), non-conventional techniques such as Supercritical Fluid Extraction (SFE), Ultrasound- (UAE), and Microwave-Assisted Extraction (MAE) enabled faster and more selective UA extraction with reduced solvent use. Notably, SFE have been reported as particularly promising in terms of selectivity, process control, and potential suitability for scale-up, with commercially available supercritical CO₂ extracts of Usnea species supporting the feasibility of this approach. This review provides a consolidated and application-oriented overview of UA extraction, highlighting strategies that balance efficiency, selectivity, sustainability, and practical implementation.

Background: Shikonins are natural naphthoquinones that exhibit a range of biological activities. They are typically extracted using nonpolar solvents; however, green extraction approaches remain underexplored. Methods: Phytochemical profiling of E. vulgare root extracts was performed using HPLC-ESI-QTOF-MS/MS and quantitative analysis using HPLC-PDA. Shikonin extraction was performed using VNADESs based on thymol, camphor, menthol and benzyl alcohol. The feasibility of removing the VNADES from the extracts via freeze-drying was assessed. The cytotoxic, antioxidant, anti-inflammatory and antimicrobial activities of the hexane extract and the selected VNADES-based extract (TBa 2:8) were compared. Results: Eight shikonin derivatives were identified in the extracts. VNADES extracts contained comparable amounts of shikonin to hexane extracts; however, freeze-drying resulted in significant shikonin content loss. TBa 2:8 extract exhibited noticeably lower cytotoxicity than the hexane extract while its antioxidant potential depended on the assay applied. In contrast to the hexane extract, TBa 2:8 demonstrated the ability to reduce intracellular ROS and NO levels. However, the hexane extract exhibited stronger antimicrobial activity. Conclusions: VNADES systems enable efficient extraction of shikonin derivatives with performance comparable to hexane. Although the resulting extracts exhibit multidirectional biological activity, it remains challenging to remove the VNADESs effectively without losing the shikonins.