Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Biological activity of selected lichens and lichen-based Ag nanoparticles prepared by a green solid-state mechanochemical approach.

Lichens dispose a wide spectrum of bioactive compounds known as secondary metabolites. Their biological effects like antioxidant and antibacterial activities are widely studied. Green synthesis of silver nanoparticles (AgNPs) is a method where the compounds/substances present in plants are used for reduction of AgNO3instead of toxic chemicals. However, this methodology is usually a two-step process (extract preparation step and the synthesis step) performed under the elevated temperatures nad in the case of lichens, the redicing compounds are insoluble in water. These disadvantages can be overcome by a solid-state mechanochemical synthesis applied in the present study. As microorganisms are becoming more resistant to commercial antibiotics, AgNPs prepared in an environmentally friendly way represent an interesting alternative. In the present study, we compared the processing of lichen material of Pseudevernia furfuracea and Lobaria pulmonaria for extraction as well as for synthesis of AgNPs, and tested the antibacterial and antioxidant activity of the extracts. Both selected lichen species could be successfully used as reducing agents to produce AgNPs. Six different bacterial strains were tested for antibacterial activity of AgNPs-containing products and it was highly effective on all strains. However, the antioxidant activity of lichen extracts showed the lowest effect even if AgNPs are present which positively correlated with the content of total phenols and flavonoids. Both phenols and flavonoids are natural antioxidants and react with silver nitrate. Due to this fact, we observed a decrease of total phenols, total flavonoids as well as antioxidant activity when processing of lichen extracts with silver nitrate was used. We demonstrated that the formation of AgNPs increased the antibacterial activity but on the other hand reduced the antioxidant activity. Thus, antibacterial and antioxidant effects have to be treated differentially.

Application of liquid-phase microextraction to the analysis of plant and herbal samples.

INTRODUCTION: The analysis of plant and herbal samples is a challenging task for analytical chemists due to the complexity of the matrix combined with the low concentration of analytes. In recent years different liquid-phase microextraction (LPME) techniques coupled with a variety of analytical equipment have been developed for the determination of both organic and inorganic analytes. OBJECTIVE: Over the past few years, the number of research papers in this field has shown a markedly growing tendency. Therefore, the purpose of this review paper is to summarise and critically evaluate research articles focused on the application of LPME techniques for the analysis of plant and herbal samples. RESULTS: Due to the complex nature of the samples, the direct application of LPME techniques to the analysis of plants has not often been done. LPME techniques as well as their modalities have been commonly applied in combination with other pretreatment techniques, including a solid-liquid extraction technique supported by mechanical agitation or auxiliary energies for plant analysis. Applications and the most important parameters are summarised in the tables. CONCLUSION: This review summarises the application of the LPME procedure and shows the major benefits of LPME, such as the low volume of solvents used, high enrichment factor, simplicity of operation and wide selection of applicable detection techniques. We can expect further development of microextraction analytical methods that focus on direct sample analysis with the application of green extraction solvents while fully automating procedures for the analysis of plant materials.

Oxidative stress mediated by gyrophoric acid from the lichen Umbilicaria hirsuta affected apoptosis and stress/survival pathways in HeLa cells.

BACKGROUND: Lichens produce a huge diversity of bioactive compounds with several biological effects. Gyrophoric acid (GA) is found in high concentrations in the common lichen Umbilicaria hirsuta, however evidence for biological activity was limited to anti-proliferative activity described on several cancer cell lines. METHODS: We developed and validated a new protocol for GA isolation, resulting in a high yield of highly pure GA (validated by HPLC and NMR) in an easy and time saving manner. Anti-proliferative and pro-apoptotic activity, oxygen radicals formation and stress/survival proteins activity changes was study by flow cytometry. RESULTS: The highly purified GA showed anti-proliferative activity against HeLa (human cervix carcinoma) and other tumor cells. Moreover, GA threated cells showed a significant increase in caspase-3 activation followed by PARP cleavage, PS externalization and cell cycle changes mediated by oxidative stress. Production of oxygen radicals led to DNA damage and changes in stress/survival pathways activation. CONCLUSIONS: GA treatment on HeLa cells clearly indicates ROS production and apoptosis as form of occurred cell death. Moreover, DNA damage and changing activity of stress/survival proteins as p38MAPK, Erk1/2 and Akt mediated by GA treatment confirm pro-apoptotic potential. The pharmacological potential of U. hirsuta derived GA is discussed.

Lichen secondary metabolites affect growth of Physcomitrella patens by allelopathy.

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.