Green Extraction of Depsidones and Depsides from Hypogymnia physodes (L.) Nyl. Using Natural Deep Eutectic Solvents.

Various studies have shown that Hypogymnia physodes are a source of many biologically active compounds, including lichen acids. These lichen-specific compounds are characterized by antioxidant, antiproliferative, and antimicrobial properties, and they can be used in the cosmetic and pharmaceutical industries. The main aim of this study was to optimize the composition of natural deep eutectic solvents based on proline or betaine and lactic acid for the extraction of metabolites from H. physodes. The design of the experimental method and the response surface approach allowed the optimization of the extraction process of specific lichen metabolites. Based on preliminary research, a multivariate model of the experiment was developed. For optimization, the following parameters were employed in the experiment to confirm the model: a proline/lactic acid/water molar ratio of 1:2:2. Such a mixture allowed the efficient extraction of three depsidones (i.e., physodic acid, physodalic acid, 3-hydroyphysodic acid) and one depside (i.e., atranorin). The developed composition of the solvent mixtures ensured good efficiency when extracting the metabolites from the thallus of H. physodes with high antioxidant properties.

Lichen-derived caperatic acid and physodic acid inhibit Wnt signaling in colorectal cancer cells.

Lichens are a source of secondary metabolites which possess important biological activities, including antioxidant, antibacterial, anti-inflammatory, and cytotoxic effects. The anticancer activity of lichens was shown in many types of tumors, including colorectal cancers (CRC). Several studies revealed that the application of lichen extracts diminished the proliferation of CRC cells and induced apoptosis. Colon carcinogenesis is associated with aberrations in Wnt signaling. Elevated transcriptional activity of ß-catenin induces cell survival, proliferation, and migration. Thus, the inhibition of Wnt signaling is a promising therapeutic strategy in colorectal cancer. The aim of this study was the evaluation of the effects of lichen-derived depsides (atranorin, lecanoric acid, squamatic acid) and depsidones (physodic acid, salazinic acid) and a poly-carboxylic fatty acid-caperatic acid, on Wnt signaling in HCT116 and DLD-1 colorectal cancer cell lines. HCT116 cells were more sensitive to the modulatory effects of the compounds. PKF118-310, which was used as a reference ß-catenin inhibitor, dose-dependently reduced the expression of the classical ß-catenin target gene-Axin2 in both cell lines. Lecanoric acid slightly reduced Axin2 expression in HCT116 cells while caperatic acid tended to reduce Axin2 expression in both cell lines. Physodic acid much more potently decreased Axin2 expression in HCT116 cells than in DLD-1 cells. Physodic acid and caperatic acid also diminished the expression of survivin and MMP7 in a cell line and time-dependent manner. None of the compounds affected the nuclear translocation of ß-catenin. This is the first report showing the ability of caperatic acid and physodic acid to modulate ß-catenin-dependent transcription.

Physodic acid sensitizes LNCaP prostate cancer cells to TRAIL-induced apoptosis.

In spite of the extensive research for developing new therapies, prostate cancer is still one of the major human diseases with poor prognosis and high mortality. Therefore, with the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, in the present study, we evaluated the effect of lichen secondary metabolite physodic acid on cell growth in human prostate cancer cells. In addition, we tested the apoptotic activity of physodic acid on TRAIL-resistant LNCaP cells in combination with TRAIL. The cell viability was measured using MTT assay. LDH release, a marker of membrane breakdown, was also measured. For the detection of apoptosis, the evaluation of DNA fragmentation and caspase-3 activity assay were employed. The expression of proteins was detected by Western blot analysis. It was observed that physodic acid showed a dose-response relationship in the range of 12.5-50 µM concentrations in LNCaP and DU-145 cells, activating an apoptotic process. In addition, physodic acid sensitizes LNCaP cells to TRAIL-induced apoptosis. The combination of physodic acid with other anti-prostate cancer therapies could be considered a promising strategy that warrants further investigations.

Lichen Secondary Metabolites Inhibit the Wnt/ß-Catenin Pathway in Glioblastoma Cells and Improve the Anticancer Effects of Temozolomide.

Lichens are a source of secondary metabolites with significant pharmacological potential. Data regarding their possible application in glioblastoma (GBM) treatment are, however, scarce. The study aimed at analyzing the mechanism of action of six lichen secondary metabolites: atranorin, caperatic acid, physodic acid, squamatic acid, salazinic acid, and lecanoric acid using two- and three-dimensional GBM cell line models. The parallel artificial membrane permeation assay was used to predict the blood-brain barrier penetration ability of the tested compounds. Their cytotoxicity was analyzed using the MTT test on A-172, T98G, and U-138 MG cells. Flow cytometry was applied to the analysis of oxidative stress, cell cycle distribution, and apoptosis, whereas qPCR and microarrays detected the induced transcriptomic changes. Our data confirm the ability of lichen secondary metabolites to cross the blood-brain barrier and exert cytotoxicity against GBM cells. Moreover, the compounds generated oxidative stress, interfered with the cell cycle, and induced apoptosis in T98G cells. They also inhibited the Wnt/ß-catenin pathway, and this effect was even stronger in case of a co-treatment with temozolomide. Transcriptomic changes in cancer related genes induced by caperatic acid and temozolomide were the most pronounced. Lichen secondary metabolites, caperatic acid in particular, should be further analyzed as potential anti-GBM agents.

Pro-Apoptotic Potential of Pseudevernia furfuracea (L.) Zopf Extract and Isolated Physodic Acid in Acute Lymphoblastic Leukemia Model In Vitro.

Acute lymphoblastic leukemia (ALL) is the most frequently diagnosed type of leukemia among children. Although chemotherapy is a common treatment for cancer, it has a wide range of serious side effects, including myelo- and immunosuppression, hepatotoxicity and neurotoxicity. Combination therapies using natural substances are widely recommended to attenuate the adverse effects of chemotherapy. The aim of the present study was to investigate the anti-leukemic potential of extract from the lichen Pseudevernia furfuracea (L.) Zopf (PSE) and isolated physodic acid (Phy) in an in vitro ALL model. A screening assay, flow cytometry and Western blotting were used to analyze apoptosis occurrence, oxidative stress, DNA damage and stress/survival/apoptotic pathway modulation induced by the tested substances in Jurkat cells. We demonstrate for the first time that PSE and Phy treatment-induced intrinsic caspase-dependent cell death was associated with increased oxidative stress, DNA damage and cell cycle arrest with the activation of cell cycle checkpoint proteins p53, p21 and p27 and stress/survival kinases p38 MAPK, JNK and PI3K/Akt. Moreover, using peripheral T lymphocytes, we confirmed that PSE and Phy treatment caused minimal cytotoxicity in normal cells, and therefore, these naturally occurring lichen secondary metabolites could be promising substances for ALL therapy.