Evaluation of the antioxidant activity and Citotoxic potential of lichen forming fungal species of the family Parmeliaceae (Ascomycota)

Evaluation of the antioxidant activity and Citotoxic potential of lichen forming fungal species of the family Parmeliaceae (Ascomycota) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the antioxidant activity and Citotoxic potential of lichen forming fungal species of the family Parmeliaceae (Ascomycota) Marta Sánchez, María Trento, Jose Antonio Valdés-González, María Victoria Naval, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6922462/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Lichens produce a variety of secondary metabolites with important biological properties. This study analyzed the antioxidant activity and cytotoxic potential of selected Parmeliaceae lichens using in vitro methods and HPLC analysis. Methanolic extracts were obtained from five species: Hypogymnia physodes (L.), Parmotrema pseudotinctorum (Abbayes), Usnea subfloridana (Stirt.), Lethariella canariensis (Ach.) and Lethariella intricate (Moris). Their antioxidant capacities were assessed through DPPH, FRAP, ORAC, and Folin tests, while cytotoxicity was examined in breast adenocarcinoma (MCF7) and hepatocellular carcinoma (HepG2) cell lines via MTT viability assay.Results showed significant antioxidant activity, with Parmotrema pseudotinctorum exhibiting the highest ORAC and Folin values. Regarding cytotoxicity, Usnea subfloridana and Hypogymnia physodes demonstrated notable growth inhibition in MCF7 and HepG2, particularly at intermediate concentrations. Parmotrema pseudotinctorum showed moderate inhibition, more pronounced at lower doses. HPLC analysis identified bioactive compounds such as physodic acid, lecanoric acid, and usnic acid, which correlate with the observed antioxidant and cytotoxic activities. These findings highlight lichens as a potential source of therapeutic secondary metabolites, warranting further research to explore their medicinal applications. cancer lichen antioxidant activity MCF7 HepG2 Figures Figure 1 Figure 2 Figure 3 1. Introduction Cancer is a group of diseases that have in common an uncontrolled multiplication of cells. This group of diseases has been affecting multicellular organisms for more than 200 million years (Hausman DM 2019). The incidence of cancer has increased throughout the 20th and 21st centuries as life expectancy has increased and living conditions have changed. Increased sedentary lifestyles, environmental factors and new eating habits are risk factors for developing cancer in the course of life (El-Sherif A 2021; Doll R 1981; Albuquerque RC 2014; Bertuccio P 2013; Friedenreich CM 2021; Liu X2014; Jochem C 2022; Magalhaes B 2021). Statistics say that one in four people will be at risk of developing cancer at some point in their lives (Roy PS 2016). Cancer does not only affect cells that have lost control over their replication. Recent studies show that cancer actually comprises a true organic biological ecosystem involving healthy cells around the cancer cells as well as the molecular interactions that take place in the tumor environment. The whole set of biological relationships around the tumor has been called "tumor microenvironment" (Visser KE 2023). Cancer therapy is based on three therapeutic strategies: surgery, chemotherapy (including innovative biological therapies) and radiotherapy (Roy PS 2016), but we are far from considering that we have an adequate solution. Research to discover new molecules with cancer protective or anticancer activity is therefore essential. Natural products have been a source of molecules with pharmacological bioactivity, especially against infectious diseases and cancer (Atanasov AG 2021). Despite this, the pharmaceutical industry has focused on synthetic drugs because of the intrinsic difficulties of natural products: difficulty in isolation, stability problems, and even complications in obtaining sufficient quantities of the natural product (Atanasov AG 2015). Lichens are the result of a symbiosis between a fungus (from Basidiomycota and Ascomycota filum) and a photobiont which contains chlorophyll such as a cyanobacterium or a green alga (Calcott MJ 2018). Traditionally, lichens have been used for their medicinal and culinary properties (Crawford SD 2015; Ivanova D 2009). Lichens produce a large number of secondary metabolites (Ureña-Vacas I 2022; Baczewska 2024; Phan 2025) More recently, more extensive studies on secondary metabolites have started to identify new substances such as depsides and depsidones, as well as other chemical groups (dibenzofurans, aliphatic or usnic acids, anthraquinones or xanthones) whose research has supported the potential of the different species of these families to produce unique natural compounds with different biological and physico-chemical properties (Cardile V 2017; Ren M 2023). In addition to their natural function, secondary compounds from lichens have demonstrated a number of useful biological activities in other organisms, such as antibiotic, antimycobacterial, antimutagenic, antioxidant, antiviral, antipyretic, analgesic and antitumor properties, and have therefore been used for the treatment of various diseases in traditional medicine. (Boustie J 2011). The biological functions of lichen compounds have motivated the study of their pharmacological activity, both in extracts and in isolated active compounds. However, this study, together with purification and identification of compounds, has not yet been able to fully realise their therapeutic potential. The most abundant group of compounds in lichens are the polyketides (depsides, depsidones, anthraquinones, dibenzofurans...) which are phenolic in nature. They arise from the polymerization and subsequent modification of acetyl-coenzyme A and malonyl-coenzyme A subunits. This results in polycyclic structures with several hydroxyl groups, which characterize molecules with antioxidant activity (Goga M 2020). Many studies have established the antioxidant activity of both extracts and compounds isolated from lichens corroborating that these organisms are an original source of these compounds (Shang 2018, Dwarakanath 2024). The vast majority have been assays on extracts that, depending on the genus, the antioxidant activity is found in the methanolic extract or in the acetonic extract. (Gómez-Serranillos MP 2014; Kosanić 2016) The lichen compounds that have been studied the most are the polyketides mentioned above, especially depsides, tridepsides and depsidones. Research over the last 10 years has focused on their potential antitumor, antimicrobial and antioxidant activity. Numerous studies have been conducted in in vitro models, although studies in in vivo models are scarce and no human clinical trials have been published (Ureña-Vacas I 2022; Rankovič B 2010; Kello M 2023; Ureña-Vacas I 2023; Solárová Z 2020). Several studies have investigated the anticancer potential of lichens (Varol 2020, Koopaie 2023). The anticancer activity of lichens has been previously established in multiple in vitro experiments. In these experiments, not only the activity of the lichen extracts but also the pharmacological activity of the isolated secondary metabolites has been demonstrated (Tripahi AH 2022; Ristic S 2016). These metabolites are responsible for a large part of the different biological activities of lichens (Molnár K 2010; Rankovic B 2015). Methanolic extracts of lichen (e.g. Roccella montagnei ), and in vitro assays showed significant cytotoxic activity against several human cancer cell lines such as colon (DLD-1, SW-620), breast (MCF-7) and head and neck (FaDu). With these activities, interesting compounds were isolated from the extract by chromatography (roccellic acid and everninic acid), so these findings highlight the promising anticancer activity of some lichens and their bioactive compounds (Mishra 2017) Oxidative stress is a damage factor resulting from the disruption of the balance between the generation of reactive oxygen or nitrogen species and the cell's antioxidant mechanisms (Prasad S 2017). Excess free radicals produce molecular damage that triggers loss of functionality of proteins by oxidation, lipid peroxidation and DNA damage leading to mutations and eventually cell death. Molecular damage due to oxidative stress accumulates over time until organs begin to experience organ failure leading to pathological conditions associated with, among others, cancer (Liguori I 2018). In this study, methanolic extracts of five lichen species belonging to the Parmeliaceae family, have been analysed: Hypogymnia physodes (L.), Parmotrema pseudotinctorum (Abbayes), Usnea subfloridana (Stirt.), Lethariella canariensis (Ach.) and Lethariella intricate (Moris), with the aim of determinate the cytotoxic bioactivity of the lichens extracts on cancer cells and their antioxidant potential. Subsequently, the secondary metabolite content of the three extracts with the highest biological activity was determined by High-Performance Liquid Chromatography (HPLC) and we found the presence of molecules such as physodalic acid, pshysodic acid, atranorin, lecanoric acid, salazinic acid and usnic acid. These secondary metabolites have shown bioactivity against cancer in many cell lines and cancer types. (Cardile V 2017; Kosanić M 2013; Stojanović IZ 2014; Stojanović IZ 2013; Correché ER 2004; Paluszczak J 2018; Manojlović N 2012; Roser LA 2022; Harikrishnan A 2021; Zhou R 2017; Majchrzak-Celińska A 2022; Sun TX 2021; Kumar K 2020; Pyrczak-Felczykowska A 2022) In this study, we aim to determinate the cytotoxicity of diverse lichens extracts on MCF-7 cells, as a model of breast cancer (Comşa Ş 2015), and in HepG2 cells, as a model of human hepatocellular carcinoma cells (Calcott MJ 2018). We also determinate the antioxidant bioactivity in vitro through different methods in order to select three lichen extracts with the best antioxidant potential. In the last instance, the three selected lichen extracts were subjected to HPLC with the aim of identifying the secondary metabolites most commonly present in each of them and thus attributing the bioactivity to well-defined chemical compounds. 2. Materials and Methods 2.1 Materials 2.1.1 Reagents 2,4,6-tri-2-pyridinyl-1,3,5-triiazine (TPTZ) were obtained from Alfasigma (Barcelona, Spain). Iron sulphate 7 hydrate (FeSO4 -7H2O) and sodium carbonate (Na2CO3) were purchased from PanReac (Barcelona, Spain). Iron chloride 6 hydrate (FeCl3–6H2O), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (TROLOX), 3,4,5-trihydroxybenzoic acid (gallic acid), Follin & Ciocalteu´s phenol reagent, fluorescein sodium salt and 2,2´-azobis (2-amidinopropano)-dihicloruro (AAPH) 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin and Triton X-100 were supplied from Sigma Aldrich-Merck KGaA (Darmstadt, Germany). Methanol and DMSO were obtained from Applichem (Darmstadt, Germany). RPMI 1640, Foetal bovine Serum (FBS), gentamicin and Phosphate -Buffered saline (PBS) were purchased from Gibco (Invitrogen, Paisley, UK) 2.1.2 Lichens For this study, we used five lichen species well identified by experts and already preserved in the MAF herbarium of the Faculty of Pharmacy of the Complutense University of Madrid. Subsequently, they were deposited in the herbarium of the Faculty of Pharmacy of the Complutense University of Madrid with the following details Hypogymnia physodes (L.) Nyl., Leon, Spain, November 2014 (MAF-Lich 19708) Lethariella canariensis (Ach.) Krog, Portugal, Madeira, November 1990, distributed by the Institut für Botanik, Karl-Franzens-Universität, Graz (GZU) (MAF-Lich 6865) Lethariella intricata (Moris) Krog, Navarra, Spain, September 2013 (MAF-Lich 18340) Parmotrema pseudotinctorum (Abbayes) Hale, La Palma, Spain, June 2009 (MAF-Lich 18178) Usnea subfloridana (Stirt.), Asturias, Spain, September 2012 (MAF-Lich 18040) Preparation of lichen extracts According to the method described by Amo De Paz et al. [ 118 ], 50 mg of the air-dried lichen thallus was weighed (exactly) and mixed with 2 ml of pure methanol, the samples were shaken for 1 minute every 30 minutes during the first 2 hours, then incubated 24 hours. After 24 hours, the extract was filtered (0.22µm) and left until completely evaporated at room temperature. The dried extracts were then weighed and re-dissolved in methanol or DMSO depending on the experiments to be performed. The extraction yield was calculated for each lichen species. The samples were kept cold (4ºC). 2.2. Methods 2.2.1. Determination of total phenolic compounds through Folin-Ciocalteu assay Following the method described by Saura-Calixto et al. [ 119 ] with some modifications we use the Folin-Ciocalteu assay to determinate the total amounts of phenolic compounds in the lichen extracts samples. 50 µl of Folin's reagent and 50 µl of each methanolic extract (1 mg/ml) were mixed and, after 5 min, 1 ml of Na 2 CO 3 (7%) was added dropwise to these. The solutions were incubated for 40 min at room temperature and shaking. The absorbance was measured at 595 nm using a microplate reader spectrophotometer (SPECTROstar Nano, BMG Labtech, Germany). Results were interpolated with those of a calibration line created using gallic acid (GA) at different concentrations (from 5 to 200 M) as a reference standard. It is performed in an alkaline medium and the reduction leads to the formation of a blue complex. 2.2.2. In vitro radical scaving activities 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay This method is based on the reduction of the DPPH reagent in radical form, in the presence of a hydrogen-donating antioxidant (Gulcin İ 2020). The method described by Amarowicz et al. (Amarowicz R 2004) was followed with slight modifications. In a 96-well plate 5 dilutions (from 100 to 900 µg/ml) of each methanolic lichen extract samples was added and a DPPH solution (550 µM) was placed to make up a final volume of 225 µl/well. the solutions were incubated at 37°C for 30 minutes. Then, the absorbance was read at 517 nm through a Spectrostar Nano microplate reader spectrophotometer (BMG Labtech, Germany). As a reference antioxidant, different dilutions of trolox were used to make a calibration curve. IC 50 was calculated to express DPPH scavenging activity. % inhibition = \(\:\frac{Absorbance\:sample}{Absorbance\:DPPH}\) · 100 Oxygen Radical Antioxidant Capacity (ORAC) assay This technique is based on a hydrogen atom transfer mechanism, in which peroxyl radicals, produced by thermal decomposition of the AAPH reagent, oxidize a fluorescent probe, i.e. fluorescein (Gulcin I 2020; Amarowicz R 2004; Nkhili E 2011). We followed the protocol previously described by Davalos et al. (Dávalos A 2004) with some modifications. In brief, 11 concentrations of lichen samples were prepared from 2 to 1000 µg/mL in methanol and subsequently incubated for 15 min at 37°C with a 70nM fluorescein solution in a 96-well plate. Finally, AAPH was added to all samples (12 mM) and the fluorescence was determined for 104 min at an excitation wavelength of 485nm and an emission wavelength 520 nm via FLUOstar OPTIMA, (BMG Labtech, Ortenberg, Germany). The area under the curve (AUC) was calculated for each sample and compared with Trolox. The results were expressed as µmol TE/mg sample. AUC = 1 + ∑ fi/f0 Ferric Reducing Antioxidant Power (FRAP) assay The assay is based on an electron transfer mechanism and on the principle of reduction of Fe 3 + to Fe 2 + by the action of an antioxidant present in the sample [ 120 ]. The occurrence of the reaction is detected using ,4,6-Tris(2-pyridyl-s-triazine (TPTZ), a compound capable of forming complexes with Fe 2 + and, as a consequence, imparting a purple color to the solution (Moon JK 2009). The method described by Sánchez-Muniz et al. (Sánchez-Muniz FJ 2012) was carried out, with some modifications. Lichen samples (1mg/ml in methanol) were mixed with the previously prepared FRAP reagent (TPTZ (10mM) in HCl (40mM), acetate buffer (ph 3.6) and FeCl 3 6 H 2 O (20mM) and incubated for 30 min at 37°C, and the absorbance was subsequently measured in a SPECTROstar Nano microplate reader at a wavelength of 595 nm. The results were expressed as µmol Fe 2 + eq/g sample. % inhibition = \(\:\frac{Abs\:Sample-Blank}{Abs\:Control-Blank}\) · 100 Antioxidant Index We calculate antioxidant index with the arithmetic mean of a normalized measure of the antioxidant power in each extract. To normalize these measurements, we take as 100% the species with the greatest antioxidant power and thus achieve comparable data. Subsequently, we find the arithmetic mean of the percentages of all the experiments which allows us to compare all the species with each other (Ureña-Vacas I 2022) \(\:\frac{Sample\:Score}{Best\:Score}\) · 100 2.2.3. Cytotoxic potential Cell Culture Breast adenocarcinoma cell line MCF7 and human hepatocellular carcinoma cell line HepG2 were obtained from the NCI-Frederick Cancer DCTD Tumor/Cell line Repository (Frederick National Laboratory for Cancer Research, National Cancer Institute) were used in this study. They were cultivated inside 25 cm 2 tissue culture flasks (Corning Incorporated, New York, USA) using RPMI-1640 medium supplemented with 10% FBS and 0.5% gentamicin at 37°C in a humidified environment with 5% CO 2 . Cell passaging was performed every 2–3 days, when 80–90% confluence was reached. Assessment of cell viability The cytotoxic potential was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, following the protocol described by Mosmann (Mosmann T 1983) with slight modifications. This colorimetric assay measures cell viability by exploiting the reduction of the MTT reagent to the purple formazan crystal performed by the mithocondrial activity of metabolically active cells (Van Meerloo J 2011; Ghasemi M 2021). 96-well plates were used, in which the cells were seeded at a density of 5x 10 3 and 7x 10 3 cells/well in MCF-7 and HepG2 cells line, respectively, and then incubated overnight. After reaching a confluence around 90%, they were treated with different concentrations of the 5 lichen species extracts (Nine different concentrations were used, ranging from 0.25 µg/ml to 200µg/ml) for 24h. After this time, a 2 mg/ml solution of MTT was added and the plates were incubated at 37°C for 1 hour in the dark. Finally, the medium was removed and 100 µl of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 550 nm through a Spectrostar Nano microplate reader spectrophotometer (BMG Labtech, Germany). Untreated cells (control) were only exposed to cell culture medium. Results are expressed as % of cell viability compared to the control, taken as 100%. Triton X-100 at 1% was used as a standard of comparison. Triton X-100 was used as a negative control. This compound generates excessive cytotoxicity, effectively reducing cell viability to around 20% viability. In this case the control is the group of cells that have not been treated with any extract. High-performance liquid chromatography HPLC The secondary lichen metabolites present in the extracts were identified using HPLC. The extracts were prepared at a concentration of 250 µg/ml in methanol and then filtered. Twenty microliters of the sample were injected into the Agilent 1200 Infinity chromatograph (Agilent Technology, California, USA). The mobile phase consisted of a mixture of 1% phosphoric acid in mili-Q water and HPLC-grade methanol, with the proportion gradually changing from 70% phosphoric acid at the initial time to 10% at 45 minutes. As the stationary phase, an analytical column in reverse phase Mediterranea Sea C18 (Tecknokroma, Barcelona, Spain) was employed. The analysis conditions included a temperature of 40°C and a flow rate of 0.6 ml/minute. The compounds eluted were detected using a photodiode array detector operating wavelength range from 190 to 800 nm. The selected wavelength was the one at which the lichen compounds exhibited their maximum absorbance (254 nm). Data processing was carried out using Agilent ChemStation software (Agilent Technology, California, USA), recording the retention times and maximum absorbance wavelengths for each of the compounds. The identification of the compounds was performed by comparing the data with references from the literature. In the case of usnic acid, a pure isolated compound was used as a standard (Sigma-Aldrich, Missouri, USA). Therefore, once the chromatographic spectra provided by the Agilent HPLC software connected to the instrument were obtained, we proceeded to identify the correspondence of each peak to a precise secondary compound/metabolite. This was mainly based on a comparison of the chromatographic spectra obtained experimentally with the data in the scientific bibliography. The instrument provided 8 chromatograms in the range of wavelengths from 273 to 210, in particular 210, 214, 230, 240, 250, 254, 260, 273 nm. For the identification of peaks, only those at 254 nm were considered for each of the three lichen species. To identify the extraction and production solvent in the spectrum of the extract, i.e. methanol, the HPLC measurement of this compound alone was carried out, in order to obtain a spectrum in which only one peak appeared, which inevitably correlated with methanol. The same was carried out injecting in the tool the pure usnic acid compound, in the research group's possession, which was known to be contained in one of the extracts, in particular in Usnea subfloridana , thanks to data from the scientific literature. Statistical analysis All the results are derived from 3 independent repetitions and are reported as mean ± standard deviation. Statistical analysis was conducted using GraphPad Prism 8 software, employing analysis of variance (ANOVA) with a significance level (p) lower than 0.05. Pearson's correlation coefficient ( R ) was calculated to determine the linear correlation between the different antioxidant activity techniques and the total phenolic compound content. 3. Results As mentioned in the introduction, methanolic extracts of five lichens have been an-alysed in this study: Hypogymnia physodes (L.), Parmotrema pseudotinctorum (Abbayes), Usnea subfloridana (Stirt.), Lethariella canariensis (Ach.) and Lethariella intricate (Moris). 3.1 Calculation of the Antioxidant index (%) From the results obtained in the four in vitro tests (Folin-Ciocalteu, FRAP, DPPH, ORAC) the antioxidant index was calculated. First, the results of each in vitro test were considered separately and the extract giving the best result was considered 100% (in the Folin-Ciocalteu, FRAP and ORAC tests the best result was the one giving the highest numerical value, in the DPPH test the one giving the lowest numerical value); then, at the value representing 100%, the results of all other extracts were compared with it. The arithmetic mean was then calculated considering the relative results of each extract in each test to obtain the antioxidant index expressed in %, which was included in Table 3.2. Antioxidant activity In this research we examined the total phenolic content (TPC) in the methanol extract at a concentration of 1/mg/ml of each lichen. A very high TPC has been found in P. psudotinctorum (348,67 ± 23,16 µg gallic acid (GA)/mg), the highest of the 5 plants studied. The TPC of U. subfloridana and H. physodes are also outstanding, and the contents of the Lethariellae studied are more discreet. The antioxidant capacity of our methanolic extracts was evaluated by a battery of three assays describing different antioxidant mechanisms. The antioxidant properties of some compounds are conditioned by pH, so in these assays, measurements were made at different pHs. The antioxidant activity is also influenced by the substrate that is reduced, so that not all assays feature the same lichens and, therefore, a combination of all of them is required to make a correct selection. Table 1 shows the results of the 3 antioxidant tests performed and phenolic content. The FRAP test in which the transfer of a single electron to the Fe3+-TPTZ complex to form Fe2+-TPTZ in acid medium (pH = 3.6) was assessed. The leading species in this test was U. subfloridana (69.36 ± 7.22 µmol eq Fe2+ /g E), well ahead of the immediately following H. physodes (30.36 ± 2.92) and L. intricata (30,09 ± 8,91). Table 1 Antioxidant assays of methanolic extracts Lichen species FRAP (µmol of Fe 2+ eq./g E) DPPH EC 50 (µg/ml) ORAC value (µmol of TE/mg E) Phenolic content (µg of GA eq./mg E) Antioxidant index (%) Hypogymnia physodes 30,36 ± 2,92 69,66 ± 6,50 5,77 ± 1,31 77,35 ± 11,96 56,20 Lethariella canariensis 22,59 ± 1,43 320,02 ± 42,98 5,39 ± 1,21 16,76 ± 2,63 43,91 Lethariella intricata 30,09 ± 8,91 466,86 ± 77,31 4,00 ± 1,59 29,73 ± 5,24 41,32 Parmotrema pseudotinctorum 14,96 ± 3,07 1527,61 ± 99,82 9,10 ± 2,00 348,67 ± 23,16 55,39 Usnea subfloridana 69,36 ± 7,22 84,71 ± 8,71 2,29 ± 0,06 113,92 ± 13,74 63,08 The DPPH assay results showed that the extracts requiring the lowest concentration to neutralise 50% of the DPPH were those of H. physodes (69.66 ± 6.50 µg/ml) and U. subfloridana (84.71 ± 8.71 µg/ml). The DPPH radical was mainly neutralised by lipophilic antioxidants whose mechanism consists of electron and/or proton transfer in an acidic medium (pH = 4.0). The ORAC assay was performed in a neutral medium (pH = 7.4) and the mechanism studied was proton transfer to neutralise the OH- radical. The results of the ORAC assay are shown in Table 1 ), where P. pseudotinctorium stands out from the rest of the species (9.10 H ± 2.00 µg eq Tx/mg E), quite a distance from the following, such as H. physodes (5,77 ± 1,31µg eq Tx/mg E) and L. canariensis (5,39 ± 1,21). The results obtained in the FRAP, DPPH, ORAC and Folin-Ciocalteu tests are grouped in Table 1 , in which the Antioxidant index was also calculated and, based on this value, a classification of the different species was established, with U. subfloridana (1st), P. pseudotinctorum (2nd), H. physodes (3rd), L. canariensis (4th) and L. intricata (5th). To determine whether there is a possible correlation between the content of phenolic compounds and the antioxidant activity of the five methanol extracts of the lichen species studied, a Pearson´s correlation study (r) was carried out to quantify the correlation between two quantitative variables. The results showed that there is a moderate correlation between DPPH and ORAC with the content of total phenolic compounds (0.71 < r < 0.85). 3.3. Cytotoxic potential 3.3.1. Assessment of cell viability in MCF7 cell cultures using the MTT colorimetric assay A marked effect was seen in the extracts of all the lichens studied, although the concentrations at which a significant decrease in cell viability was seen vary. In the case of P. pseudotinctorum , it stands out among all the extracts studied as it starts with significant activity at 1µg/ml (cell viability 84.84 ± 2.36% of control, p ≤ 0.05) maintaining its cytotoxicity at increasing concentrations, and in second place in H. physodes , marked activity was already observed at a concentration of 2.5 µg/ml (88.32 ± 2.03% cell viability, p ≤ 0.05), as well as L. intricata which at the same dose showed an interesting cytotoxicity (cell viability 93.48 ± 2.91% of control, p ≤ 0.05). In the case of U. subfloridana and L. canariensis , the onset of a significant decrease in cell viability occurs from a concentration of 5 µg/ml (cell viability 89.02 ± 4.76% and 91.76 ± 1.86%, p ≤ 0.05 respectively). All lichen extracts maintained a high cytotoxicity on MCF7 cells from the concentration of 5 µg/ml to 200 µg/ml, although it can be observed that U. subfloridana and H. physodes caused the greatest decrease in cell viability at the highest concentrations as it is shown in Fig. 1 . 3.3.2. Assessment of cell viability in HepG2 cell cultures using the MTT colorimetric assay Other natural products have shown biological activity on this cell line [ 48 ]. A marked effect was again observed in the extracts of all the lichens studied, especially at doses of 10 µg/ml or higher, where all of them showed a significant decrease in cell viability. However, some of them start to show significant cytotoxic activity at lower doses, as was the case of P. pseudotinctorum where significant differences in cytotoxicity start to appear from 0.5 µg/ml (cell viability 91.79 ± 1.48% of control, p ≤ 0.05), maintaining its cytotoxicity at increasing concentrations. The same occurred with U. subfloridana although significant differences was shown from 2.5 µg/ml (cell viability 79.63 ± 6.18, p ≤ 0.05), and L. intrincata and H. physodes from 5 µg/ml (cell viability 87.79 ± 3.57% and 88 ± 4.25%, p ≤ 0.05 respectively). L. canariensis starts with a significant decrease in cell viability from a concentration of 10 µg/ml (cell viability 82.10 ± 7.98%, p ≤ 0.05). All lichen extracts maintained high cytotoxicity on HepG2 cells from the concentration of 10 µg/ml to 150 µg/ml, although it can be observed that U. subfloridana and L. intrincata caused the greatest decrease in cell viability at the highest concentrations. These results are shown in Fig. 2 . As the methanol extracts showed cytotoxic activity in MCF7 and HepG2 cells, IC50s were calculated for each species in this cell line in order to make a better comparison between them as shown in Table 2 . A control was carried out with a drug with potent anticancer activity, doxorubicin, allowing us to compare the activity of the lichen extracts studied. Table 2 IC 50 data for each species in MCF7 and HepG2 cells (GraphPad Prism 8 software). MCF7 HepG2 IC50 (µg/ml) IC50 (µg/ml) Lethariella canariensis 612.6 62.42 Usnea subfloridana 41.25 90.88 Lethariella intricata 213.3 12.93 Parmotrema pseudotinctorum 151.8 120.8 Hypogymnia physodes 56.20 37.17 Doxorubicin 187.9 118.8 In terms of cytotoxicity, Usnea subfloridana and Hypogymnia physodes (L.) and Parmotrema pseudotinctorum showed a lower IC50 than doxorubicin in MCF7 cell line, but in the HepG2 cell line it was Lethariella canariensis , Usnea subfloridana, Lethariella intricata and Hypogymnia physodes that had a lower IC50 than doxorubicin, with that of Parmotrema pseudotinctorum being very similar to doxorubicin. Considering the results as a whole, extracts of Usnea subfloridana and Hypogymnia physodes (L.) showed a remarkable growth inhibition in MCF7 and HepG2 cell lines, indicating a good cytotoxic potential especially from the intermediate concentrations used, and Parmotrema pseudotinctorum (Abbayes) showed a moderate overall growth inhibition in both MCF7 and HepG2 cell lines, with a more interesting action at lower concentrations 3.4. Identification of lichen secondary metabolites by HPLC technique In the last instance, the best performing lichen extracts in bioactivity were subjected to High Performance Liquid Chromatography with the aim of identifying the secondary metabolites most commonly present in each of them and thus attributing the antioxidant activity to well-defined chemical compounds. The obtained chromatograms of one of the two measurements are shown in Fig. 3 . To trace the identity, in terms of lichen secondary metabolites, of each experimentally obtained chromatogram peak, we proceeded by comparing the wavelengths and retention times resulting from the experimental measurements of the 3 methanolic extracts considered, with wavelengths and retention times available in the bibliography. The compounds that were identified are displayed alongside their molecular structures, while the others remained unidentified due to insufficient bibliographic evidence. In the case of usnic acid present in U. subfloridana , it was identified using a commercial standard, verifying that the retention time and its UV absorption spectrum matched. For U. subfloridana , the identified compounds include salazinic acid and usnic acid. In P. pseudotinctorum , lecanoric acid and atranorin were identified, and in H. physodes, physodalic acid, physodic acid, and atranorin were identified. The retention times and maximum absorbances of the identified compounds are summarized in Table 3 . The average retention times for the identified compounds are as follows: Salazinic acid: 21.02 minutes, Lecanoric acid: 22.19 minutes, Physodalic acid: 26.69 minutes, Physodic acid: 34.39 minutes, Usnic acid: 39.69 minutes, and Atranorin: 41.56 minutes. Table 3 HPLC results: molecular formula, retention time, and ultraviolet spectrum data of the metabolites identified by HPLC. The retention time data corresponds to the average of four representative chromatograms ± the mean deviation. Molecule Molecular formula Retention time (minutes) λ max (nm) Salazinic acid C 18 H 12 O 10 21.02 ± 0.05 220/270/310 Usnic acid C 18 H 16 O 7 39.69 ± 0.12 232/282 Lecanoric acid C 16 H 14 O 7 22.19 ± 0.10 212/270/304 Atranorin C 19 H 18 O 8 41.56 ± 0.07 210/252/262/320 Physodalic acid C 20 H 16 O 10 26.69 ± 0.13 212/242/318 Physodic acid C 26 H 30 O 8 34.39 ± 0.05 218/258 4. Discussion Cancer is known as a group of pathologies in which cellular deregulation and un-controlled multiplication occur, and whose incidence is increasing, especially in developed countries. Natural products are a rich source of active compounds, yet a significant number remain unexplored and may contain metabolites with potential as valuable drugs. The pharmacology of lichens has not been adequately studied. Thus, the medicinal applications of these symbiotic organisms and their compounds represent a largely untapped field of research with immense potential. Most studies have focused on revealing the cytotoxic activity of secondary metabolites on carcinogenic processes. The molecular mechanisms by which metabolites may have cytotoxic activity are cell cycle arrest and induction of apoptotic cell death (extrinsic and intrinsic pathway). Also, against inflammation associated with the tumor process. Inflammation facilitates tumorigenesis and tumor progression by providing bioactive molecules including growth factors, cell death inhibitors, proangiogenic factors, extracellular matrix modifiers that facilitate invasion and metastasis. Lichen metabolites may also exert anticancer effects by regulating tu-mor-promoting inflammation and/or anti-tumor immunity, although most of the mechanisms underlying these effects remain to be defined. In this study, methanolic extracts of five lichens of the Parmeliaceae family have been analysed: Hypogymnia physodes (L.), Parmotrema pseudotinctorum (Abbayes), Usnea subfloridana (Stirt.), Lethariella canariensis (Ach.) and Lethariella intricate (Moris). Lichens are complex symbiotic organisms of fungi and algae. Traditional medicine has long used lichen-based products as plant material or their metabolites ( Thakur M 2021). Oxidative stress is considered to be a key factor in cancer´s origin and progression (Jelic MD 2021; Srinivas US 2019) and therefore an important part of its research focuses on antioxidant compounds of plant origin. The pharmacological activity of lichens is little studied. It is believed that the various secondary metabolites they possess, including a wide variety of polyphenolic compounds, are responsible for the various pharmacological activities, including antitumor activity. These same substances may contribute to their cytotoxic activity, as studies have shown their ability to induce apoptosis by altering the redox balance and activating pro-apoptotic signaling pathways. However, further studies on their activity are needed (Kosanić M 2013). For all the above-mentioned reasons, lichens, as a little explored source of active ingredients, could be an interesting option to counteract oxidative stress and its influence on the development of tumor diseases. (Thakur M 2023). They would also be an interesting proposal to alleviate the side effects of oncological therapies based on the generation of free radicals. (Jelic MD 2021) In order to carry out pharmacological prospecting studies on lichens and plants, the extraction of their active ingredients is necessary. In this case, extraction by maceration in methanol was used, since previous tests by our research group, supported by previous scientific evidence, showed that this solvent is the one that reported some of the best yields (Kalra R 2021). The fact that the maceration time is longer than in some of the reference studies has not significantly altered these yields. However, for future research it should also be considered that maceration with other solvents, despite the lower yields, would also allow the extraction of other bioactive compounds with low solubility in methanol. Regarding the composition of the five different lichen species, in this research we focused on the total phenolic content (TPC) in each methanolic extract (concentration of 1/mg/ml). A relevant TPC was found in all the species, but especially in P. pseudotictorum (348.67 ± 23.16 µg gallic acid (GA)/mg), the highest of the 5 species studied, followed by the TPCs of U. subfloridana (113.92 ± 13.74) and H. physodes (77.35 ± 11.96). This composition seemed to us to be a promising starting point for investigating the possible antioxidant activity of all our extracts. Phenolic compounds are natural products. Lichens contain a wide variety of phenolic compounds in their thalli, including polyketides, which, due to their structural characteristics, have an antioxidant action. In any case, this composition would not imply a priori antioxidant activity, since although some studies point to a correlation between phenolic content and antioxidant capacity, there is not always a parallelism between the two, because such activity may be due to the presence of other compounds of a different nature. On the other hand, it has been seen that some of the compounds in lichens may have a dual mechanism, such as atranorin, since on the one hand it has free radical scavenging power and is a cytoprotectant, but also a pro-oxidant. It increases the survival of cells exposed to oxidative damage by hydrogen peroxide but can generate an increase in lipid peroxidation (Fernández-Moriano C 2016) As described in Results, the antioxidant capacity of our methanolic extracts was assessed by a battery of three assays based on different antioxidant mechanisms and, therefore, a combination of all of them is required to make a correct selection. Table 1 shows the graphs of the results of the 3 antioxidant tests performed. The FRAP test results shown the species that stands out is U. subfloridana (69.36 ± 7.22 µmol eq Fe2+ /g E). The DPPH assay results show that the extracts requiring the lowest concentration to neutralise 50% of the DPPH are those of H. physodes (69.66 ± 6.50 µg/ml) and U. subfloridana (84.71 ± 8.71 µg/ml). The results of the ORAC assay shown, that P. pseudotinctorum stands out from the rest of the species (9.10 ± 2.00 µg eq Tx/mg E), despite having the highest concentration in the DPPH assay and, therefore, the lowest reduction capacity. Although all species showed antioxidant activity, as revealed by the different measurement methods, the predominant species in each assay were different. Methods to determine antioxidant activity are based on verifying how an oxidizing agent induces oxidative damage to an oxidizable substrate, damage that is inhibited or reduced in the presence of an antioxidant. This inhibition is proportional to the antioxidant activity of the compound or sample. On the other hand, there are tests that are based on the quantification of the products formed after the oxidative process. The different methods differ in the oxidizing agent, the substrate used, the end point measurement, the instrumental technique used and the possible interactions of the sample with the reaction medium. Furthermore, the objectives of the different measurement methods are diverse. Within the chosen tests are two Electron transfer tests (DPPH and FRAP) and a Hydrogen atom transfer test (ORAC). The DPPH radical is mainly neutralised by lipophilic antioxidants whose mechanism consists of electron and/or proton transfer in an acidic medium (pH = 4.0) and this test is usually used to check the antioxidant activity of phenols, foods or drinks. The FRAP test in which the transfer of a single electron to the Fe3+-TPTZ complex to form Fe2+-TPTZ in acid medium (pH = 3.6) is assessed, it can be used to check the antioxidant activity of foods or drinks and of biological samples. The ORAC assay is performed in a neutral medium (pH = 7.4) and this method is considered the most similar to the physiological mechanisms in living beings, since it can be used to check the antioxidant activity of phenols, foods or biological samples, since it can be used to check the antioxidant activity of phenols, foods or biological samples. This justifies the need to use several methods to select the most antioxidant extracts. The results obtained in the FRAP, DPPH, ORAC and Folin-Ciocalteu tests are grouped in Table 1 , in which the Antioxidant index was also calculated and, based on this value, a classification of the different species was established, with U. subfloridana (1st), P. pseudotinctorum (2nd), H. physodes (3rd), L. canariensis (4th) and L. intricata (5th). In view of these results, it seems that Usnea subfloridana would be the most active as an antioxidant: is second in phenolic compound content or in DPPH and excels in FRAP (69,36 ± 7,22 µmol of Fe2 + eq./g E) and antioxidant index (63,08%) although is at the bottom in ORAC. The fact that there is different activity regarding the amount of antioxidant activity according to each test for each plant study may mean that there are different molecules responsible and that they act by different mechanisms revealed in each test. As can be seen, the strongest correlation found between phenolic content and antioxidant activity is observed in the ORAC method, which is based on the transfer of hydrogen atoms, where free radicals are stabilised by the donation of a hydrogen atom by an antioxidant molecule. This would help to explain the high activity of lichen extracts in this test, since phenolic compounds can relatively easily donate the hydrogen atom of their aromatic hydroxyls, as the delocalization of the charge on the benzene ring is favored. In vitro methods are useful for comparing the antioxidant activity of different samples, such as plant extracts. The results are limited from a biological point of view since they do not reproduce the physiological situation. On the other hand, the antioxidant activity of an extract or an isolated compound in vitro differs from its antioxidant effect in vivo, since the metabolic transformations that antioxidant compounds undergo in the body modify their activity. Certain polymeric phenolic compounds that have low in vitro activity can, however, contribute to the antioxidant capacity of the target organism after their metabolic transformation into simpler compounds. Therefore, the next step in our research will be to carry out the relevant checks in more complex biological systems. The importance of several mechanisms in the development of certain pathological changes in the body is well known. Oxidative stress, by itself or in synergy with other related processes such as inflammation, apoptosis and mitochondrial dysfunction, is considered to be the cause of very frequent diseases in the body, especially in the central nervous system. Metabolic transformations of the body's own molecules such as neurotransmitters can lead to the development of Reactive Oxygen Species (ROS), which in turn can attack glial cells and sensitive neurons, causing damage to CNS structures (Thanan R 2014). Degradation of some CNS components, such as hyaluronic acid, produces isoforms that have been associated with pro-inflammatory properties as well as an increased ability of cancer cells to proliferate and invade. In addition, most brain tumors, including glial tumors of different grades, express high levels of cyclooxygenase-2 (COX-2), and these correlate with many aggressive aspects of the disease and poor prognosis (Studzińska-Sroka E 2021). Having established the antioxidant capacity of the extracts under study, our research has gone further to test their antitumor activity by testing their effect on the viability of a breast cancer cell line such as MCF7 and against hepatocellular carcinoma cells like HepG2. A large body of recent literature uses the cytotoxicity assay methodology in MCF7 and/or HepG2 cell lines to test for anti-tumour activity (Fernandez-Moriano C 2016; Tas I 2019; Alexandrino CA 2019; Nugraha A 2019; Bézivin C 2003). These and other articles complement the results with assays on their composition or other related activities, such as antioxidant activity as it influences the tumour microenvironment (Nguyen TTH 2019; Mitrović T 2011) Until a few years ago, the cytotoxic properties of Parmeliaceae lichens had hardly been evaluated. Some investigations (Gómez-Serranillos MP 2014; Fernandez-Moriano C 2016; Fernandez-Moriano C 2015) reported antiproliferative activity of methanol extracts of some Parmeliaceae species, including Hypogymnia physodes , but against the colon cancer cell lines. In addition, other groups studied the anticancer activity of other extracts obtained from Parmeliaceae spp. in human and murine cancer lines. However, so far the species included in our study had not been previously evaluated against MCF-7 and HepG2 cell lines. Our study group chose these cell lines as it is a well-established model for this purpose and there are precedents in the scientific literature for research with MCF7 or Hepg2 on anti-tumor activity of lichens and their isolated compounds the research indicates that lichen extracts exhibit cytotoxic effects on HepG2 cells, showing strong cytotoxicity (Studzińska-Sroka E 2016; Kumar J 2014). Therefore, we evaluated the cytotoxic effects of methanol extracts of the five Parmeliaceae species studied, as an approach to their anticancer potential. Their effects on cell viability were analysed and quantified by MTT assay after 24 hours of treatment with a wide range of concentrations of our extracts. According to our results, as can be seen in Figs. 1 and 2 , a marked effect is seen in the extracts of all the lichens studied, although the concentrations at which a significant decrease in cell viability is seen vary. In the light of the data obtained, it appears that P. pseudotinctorum would stand out among all the extracts studied as it starts with a significant decrease in % cell viability of MCF7 at the concentration of 1 µg/ml. The next species showing marked activity at a relatively low dose were H. physodes and L. intricata , as a marked decrease in cell viability in tumor line cell viability was observed at a concentration of 2.5 µg/ml. In the case of the other extracts, in L. canariensis and U. subfloridana there is the onset of a significant decrease in cell viability from a concentration of 5 µg/ml, although it seems more noticeable in the latter. Finally, all lichen extracts maintained a high cytotoxicity on MCF7 cells from the concentration of 5 µg/ml to 200 µg/ml, although it can be observed that U. subfloridana and H. physodes caused the greatest decrease in cell viability at the highest concentrations. In the case of HepG2 cell line, a marked effect is again observed in the extracts of all the lichens studied, especially at doses of 10 µg/ml or higher, where all of them show a significant decrease in cell viability. However, some of them start to show significant cytotoxic activity at lower doses, as is the case of P. pseudotinctorum where significant differences in cytotoxicity start to appear from 0.5 µg/ml, maintaining its cytotoxicity at increasing concentrations. The same occurs with U. subfloridana although significant differences are shown from 2.5 µg/ml, and L. intrincata and H. physodes from 5 µg/ml. L. canariensis starts with a significant decrease in cell viability from a concentration of 10 µg/ml. All lichen extracts maintained high cytotoxicity on HepG2 cells from the concentration of 10 µg/ml to 150 µg/ml, although it can be observed that U. subfloridana and L. intrincata caused the greatest decrease in cell viability at the highest concentrations. As the methanol extracts showed a remarkable cytotoxic activity, IC50s were calculated for each species in order to make a better comparison between them, for each cell line as shown in Table 2 . From these results it can be concluded that Usnea subfloridana and Hypogymnia physodes have the lowest IC50 for MCF7 (41.25 and 56.20 µg/ml, respectively) and that Lethariella intricata and Hypogymnia physodes have the lowest IC50 for HepG2 (12.93 and 37.17 µg/ml, respectively). If we look at the two cell lines together we can see that H. physodes would have the highest overall cytotoxicity profile in both as an individual extract. Parmotrema pseudotinctorum (Abbayes) showed a moderate overall growth inhibition in both MCF7 and HepG2 cell lines, with a more interesting action at lower concentrations. Therefore, this may be of great importance to avoid the occurrence of side effects. In view of this evident cytotoxicity in tumor cell lines, it is clear that the extracts studied have interesting potential for development as anticancer treatments. The differences between the percentages of cell viability of each species on each of the cancer types may be due to their different composition, as can be elucidated from the HPLC analyses shown, or their influence on the microenvironment due to their antioxidant action. There is a relationship between antioxidant activity and anti-cancerigenous protection either by influencing the microenvironment, in prevention or to avoid proliferation and invasiveness. The most studied secondary metabolites with antioxidant activity are dibenzofurans such as usnic acid; depsidones such as atranorin, diffractaic acid and lecanoric acid; depsidones such as stictic acid, lobaric acid, protocetraric acid, fumarprotocetraric acid, salazinic acid, physodic acid; simple phenolic compounds such as methyl orselinate and orselinic acid, methyl haematomate, orcinol and methyl β-orselinate. This relationship has already been seen in other plant species and metabolites might be utilised to treat cancer-related oxidative stress (Prasad S 2017). On the other hand, antioxidants also serve to minimize the side effects of oncological therapies (Fuchs-Tarlovsky V 2013) Although it may seem like a contradiction that an extract or metabolite with anti-oxidant capacity is both cytotoxic and anti-tumor, since the most common oncological therapies are based on the pro-oxidant capacity of plant active ingredients to be used as anti-tumor (Goga M 2019), there is more and more evidence in which both activities coexist and can be used together therapeutically or directed at different pathways that complement each other.(Cakmak K 2019). In addition, there is growing scientific evidence supporting the implication of ROS and OE in the aetiology of neoplasms such as breast, colon or rectal cancer, which are ROS and OE in the aetiology of neoplasms such as breast, colon or rectal cancer, which are related to lipid peroxidation and toxic aldehyde formation. The study on the potential of lichens as antioxidants and anticancer is beginning to have great potential as seen in the study of Kosanić et al (2023), that analyzed the antioxidant, antimicrobial, cytotoxic and anti-inflammatory properties of the acetone extract of the lichen Platismatia glauca (PGAE). Seven bioactive compounds were identified, including salazinic acid, physodic acid and atranorin. Their antioxidant capacity was evaluated with the DPPH method. Regarding its cytotoxic activity, the extract showed the highest efficacy against human epithelial carcinoma cells. These findings highlight the potential of lichens as a source of bioactive compounds with possible therapeutic applications. In this respect, phenolic compounds may attenuate carcinogenic potential by uptake of ROS and arrest of lipid peroxidation. In fact, a positive correlation between phenolic content and cytotoxic activities against carcinogenic cell lines has been described for some cell lines. Moreover, some of them (such as usnic acid) might show variable antioxidant or pro-oxidant properties, depending on different system conditions and/or cellular environment. Therefore, the cytotoxic actions of our extracts, related to their composition with active principles especially as usnic acid or physodic acid could be explained through their antioxidant activities as previously evaluated (Paluszczak J 2018; Studzińska-Sroka E 2021; Studzińska-Sroka E 2016; Cakmak K 2019; Maulidiyah M 2020; Mitrovic T 2011; Araújo AAS 2015; Rabelo TK 2012). Our findings seem to expand the information that is beginning to exist on the cytotoxic, antioxidant and tumor effect that is being discovered as part of the activity of various lichen species. It is important to be able to standardize the composition of lichen ex-tracts since in the case of phenolic compounds derived from lichens such as depsides and depsidones but also anthraquinones, dibenzofurans and xanthones, it seems that they could interfere with several cancer cell survival pathways and exert cytotoxic effects against said cells or modulate cellular interactions in the tumor microenvironment (Kello M 2023; Bačkorová M 2012; Petrova K 2021; Petrová K 2022). This composition could explain the bioactivity manifested by the extracts we have studied, since the presence of certain components of different extracts, similar or identical to those studied in our work, have been related to their antioxidant activity (Toledo Marante FJ 2016) as well as to different anticancer capacities( (Toledo Marante FJ 2003). As mentioned at the beginning, the Parmeliaceae family is attracting increasing attention for its possible anti-cancer activity, even in breast cancer cell lines (Nugraha AS 2020; Gandhi AD 2021; Ari F 2015). However, there is still not much information about the anti-cancer effects of pure compounds obtained from lichens. Among the most investigated is usnic acid, on which in vitro and research animal studies have allowed it to begin to be taken into account for its use as an anticancer agent (Yildirim M 2022; Bessadottir M 2012; Bačkorová M 2011; Galanty A 2017; Dinçsoy AB 2017). There is also a large literature that looks at the composition of whole extracts to see how the different components could be held responsible for the activity. HPLC and TLC are commonly used methods in the study of natural product extracts, especially for those with a chemical composition that is not extraordinarily complex. There is also a large literature that analyses the composition of whole extracts to see how the different components of the activity could be held accountable (Fernandez-Moriano C 2015; Hawrył A 2020; Manojlović NT 2010; Bhattarai HD 2008). The secondary metabolite content of our most biologically active extracts has been studied by different analytical methods. Our research group determined the chemical composition of the most promising extracts in terms of antioxidant activity by high-performance liquid chromatography (HPLC). The presence of interesting molecules consistent with previous literature was observed, even using alternative methods, such as physodic acid, physodalic acid, atranorin, lecanoric acid, salazinic acid and usnic acid (Gómez-Serranillos MP 2014; Fernandez-Moriano C 2016; Fernandez-Moriano 2015). Hypogymnia physodes revealed that the most prominent components in its composition are physodalic acid, physodic acid and atranorin, which may help explain the origin of its protective activity ( Studzińska-Sroka E 2021; Studzińska-Sroka E 2016; Toledo Marante FJ 2016; Bessadottir M 2012; Bačkorová M 2011; Galanty A 2017; Dinçsoy AB 2017; Studzińska-Sroka E 2019; Meysurova AF 2020; Stojanović IŽ 2014; Kosanić M 2019). As can be seen in the bibliography, physodalic and physodic acids have demonstrated anticancer activity in various experiments with different cell lines (Cardile V 2017; Kosanic M 2013; Stojanović IZ 2014; Stojanović IZ 2013), as well as the antioxidant and antitumor activity of these components, mainly physodic acid (Cardile V 2017; Studzińska-Sroka E 2016; Petrova K 2021; Stojanović IŽ 2014; Paluszczak J 2018; Emsen B 2016; Poulsen-Silva E 2023; Yildirim M 2022; Bessadottir M 2012; Bačkorová M 2011). On the other hand, atranorin showed significant antineoplastic effects in the 4T1 breast cancer allograft model in BALB/c mice. Since it significantly increased the survival time of tumor-bearing animals, reduced tumor volume and had a more direct proapoptotic than antiproliferative effect on tumor cells. This study is especially relevant to our hypotheses since this metabolite protected the livers of mice with tumors against oxidative stress (Solár P 2016)]. In the case of Parmotrema pseudotinctorum , our analyzes corroborated the bibliographic evidence on its composition, among which it stands out for containing lecanoric acid and atranorin has been seen in other species of the same genus, where its antitumor action even on breast cancer has been proven (Harikrishnan A 2021; Ghate NB 2013), as well as its antioxidant activity (Shameera Ahamed TK 2019). Lecanoric acid has shown anticancer activities in cell lines such as colon cancer cells (Goga M 2019) and atranorin against breast cancer (Verma S 2023), lung cancer (Bačkorová M 2012) or glioblastoma cells (Petrova K 2021)]. Antitumoral activity of same genus appears in recent publications (Mallavadhani UV 2019) as well as new components [Huynh BLC 2021; Linh B 2016; Saha S 2021; Duong TH 2015). Our species under study, P. pseudotinctorum , showed antioxidant activity thanks to its content of atranorin and lecanoric acid.[Kekuda TR 2009] In the composition of Usnea subfloridana , the most important compounds are usnic acid and salazinic acid. The genus Usnea has been one of the most studied genera among the lichens and its various biological and pharmacological properties, including antioxidant and cytotoxicity, have been investigated (Petrová K 2022; Bui VM 2022; Tram NTT 2020; Tuong TL 2020; Salgado F 2017; Londoñe-Bailon P 2019). Among the most studied species is Usnea barbata , with numerous publications to verify its composition, and more recently its antioxidant and cytotoxic activities (Popovici V 2022; Tang JY 2020; Popovici V 2021; Popovici 2021b). As mentioned above, usnic acid has been the subject of considerable attention especially in anticancer treatments (Petrová K 2022; Bessadottir M 2012; Bačkorová M 2011; Galanty A 2017; Dinçsoy AB 2017; Kumari 2023), it has shown cytotoxic bioactivity against cancer in many types of cancer (cervical cancer cells (Petrová K 2022), gastric cancer cells (Toledo Marante FJ 2016) and breast cancer cells in experiments with usnic acid derivatives ( Toledo Marante FJ 2003). Salazinic acid includes antibacterial, antioxidant and cytotoxic properties, measured in different lichens and extracts (Gómez-Serranillos MP 2014), and has demonstrated its ability to exert cytotoxic bioactivity against many types of cancer, such as colorectal cell lines and melanoma cell lines (Studzińska-Sroka E 2016; Kumar J 2014; Fuchs-Tarloysky V 2013). However, there are few studies that include our species under study ( Usnea subfloridana ), except in inflammation [Nguyen TT 2021], but its phylogenetic proximity makes us suspect its possible involvement in one or more properties already found in its genus. Our research has shown that this hypothesis was true for the antioxidant activity and cytotoxic action against cancer cells. On Lethariella , there is still little research on them, especially on anti-cancer activity [Ren MR 2009]. The studies have focused more on L. canariensis where its composition has been further investigated (canarione, atranol, chloroatranol, hematommic acid, chlorohematommic acid, methylhematommate, methylchlorohematommate, ethylhematommate, ethylchlorohematommate, methyl-orsellinate, atranorin, chloroatranorin and usnic acid) (Toledo Marante FJ 2016; Toledo Marante FJ 2003) and, because of these findings, its possible biological activities have been postulated. Our findings in L. intricata and L. canariensis represent a breakthrough in the study of the pharmacological properties of this genus. However, it should not be forgotten that natural products are complex mixtures that include compounds that can act synergistically and whose activity is not only due to the sum of the activities of the isolated compounds. It is extremely interesting to find biological activities in complete extracts, especially if, when their composition is subsequently analyzed, it is found that they include molecules with already proven activity on what is being studied, since it reinforces the reasoning that would explain their bioactivity. On other occasions, we manage to isolate and identify new components whose study represents an important advance in the search for compounds of natural origin with pharmacological activity that can be a source of new drugs. Taking all the above results together, the antioxidant and anticarcinogenic activity of the species studied is well represented and provides an important starting point for further studies to determine the mechanisms and pathways by which these are produced. 5. Conclusions In the present study, methanolic extracts of five lichens species have been studied by evaluating their antioxidant and free radical scavenging capacity and their cytotoxic activity on human carcinoma cell lines. This study revealed that these lichen species have a high antioxidant capacity. The lichen extracts were found to contain a considerable amount of phenolic compounds responsible for their high antioxidant and anti-free radical power, and as a natural source of antioxidants to ameliorate oxidative stress-related disorders. Their cytotoxic action was investigated in MCF7 and HepG2 cell lines, with all extracts showing clear cytotoxicity at a wide range of concentrations tested. This opens an interesting field for further studies to clarify whether the % decrease in cell viability is due to an increase in cell lethality due to the effect of the extracts, a decrease in cell proliferation or a combination of both. Ultimately, the three lichen extracts with the best results in bioactivity were subjected to high performance liquid chromatography in order to identify the secondary metabolites most commonly present in each of them, thus allowing attributing the antioxidant activity of well-defined chemical compounds. Declarations Funding Sources This study was supported by the Spanish Ministry of Science, Innovation and Universities (PID2019-105312GB-100) Conflicts of Interest: The authors declare no conflicts of interest. Author Contributions Conceptualization, M.P.G.-S., M.S; methodology, M.S.; validation M.S, M.V.N.; and M.P.G.-S.; data curation, M.S and M.T.; writing—original draft preparation, M.S, M.V.N; J.A.V.-G. and M.T; writing—review and editing, M.P.G.-S., M.V.N., P.K.D. and M.S.; visualization, M.P.G.-S.; supervision, M.P.G.-S. and M.S.; project administration, P.K.D and M.P.G.-S.;. All authors have read and agreed to the published version of the manuscript. 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Rev Bras Farmacogn 29:728–734 Verma S, Suman P, Mandal S, Kumar R, Sahana N, Siddiqui N, Chakdar H (2023) Assessment and identification of bioactive metabolites from terrestrial Lyngbya spp. responsible for antioxidant, antifungal, and anticancer activities. Braz J Microbiol Visser KE, Joyce JA (2023) The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell 41:374–403 Yildirim M, Degirmenci U, Akkapulu M, Gungor M, Oztornacı RO, Berkoz M, Comelekoglu U, Yalın AE, Yalın S (2022) Anti-inflammatory effects of usnic acid in breast cancer. Russ J Bioorg Chem 48:S110–S114 Zhou R, Yang Y, Park SY, Nguyen TT, Seo YW, Lee KH, Lee JH, Kim KK, Hur JS, Kim H (2017) The lichen secondary metabolite atranorin suppresses lung cancer cell motility and tumorigenesis. Sci Rep 7:8136 Supplementary Files Graphicalabstractlichens.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 26 Jun, 2025 Reviewers invited by journal 26 Jun, 2025 Editor assigned by journal 20 Jun, 2025 First submitted to journal 18 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6922462","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":476946863,"identity":"a16455e8-65d1-4ed9-8dc0-4ca569834205","order_by":0,"name":"Marta Sánchez","email":"","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Sánchez","suffix":""},{"id":476946864,"identity":"216bd42a-7790-4efa-9b8b-60d49cac1a58","order_by":1,"name":"María Trento","email":"","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Trento","suffix":""},{"id":476946865,"identity":"53abaea2-d5ae-4821-8f71-bf8f442842d7","order_by":2,"name":"Jose Antonio Valdés-González","email":"","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"Antonio","lastName":"Valdés-González","suffix":""},{"id":476946866,"identity":"92aa4eea-f52c-46b7-8016-5e847e157f11","order_by":3,"name":"María Victoria Naval","email":"","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Victoria","lastName":"Naval","suffix":""},{"id":476946867,"identity":"424b813f-b455-4101-a453-21b52324ccff","order_by":4,"name":"Pradeep K. Divakar","email":"","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Pradeep","middleName":"K.","lastName":"Divakar","suffix":""},{"id":476946868,"identity":"dabe10e0-f3b8-4a26-8f46-5e9bed535180","order_by":5,"name":"Pilar Gómez-Serranillos","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAn0lEQVRIiWNgGAWjYPACG9K1pJGu5TAJas35Dx/7XFFzPrF/2gHGhz+I0WI5Iy155pljtxNn3E5gNuYhRovBDR5jxga224kbpBPYpIlymMH5858ZG/6dA2lh/0mUwwwO5DAzNrYdANvCQJTDgH4xZmzsSzaecTuxWZooLcAQe8zY8M1Otn928sGPxDkMwWRsIEYDipZRMApGwSgYBTgAAKhSMTL1y5l3AAAAAElFTkSuQmCC","orcid":"","institution":"Complutense University of Madrid: Universidad Complutense de Madrid","correspondingAuthor":true,"prefix":"","firstName":"Pilar","middleName":"","lastName":"Gómez-Serranillos","suffix":""}],"badges":[],"createdAt":"2025-06-18 11:15:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6922462/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6922462/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85757454,"identity":"ac22dcd8-59f7-4b89-a902-5feb04aadcef","added_by":"auto","created_at":"2025-07-01 10:55:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44501,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic potential in MCF7 cells. The cytotoxic potential was evaluated by MTT colorimetric assay. Results are expressed as % of cell viability compared to the control, taken as 100%. GraphPad Prism 8 software, employing analysis of variance (ANOVA) with a significance level (p) lower than 0.05: *= p ≤ 0.05\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6922462/v1/1e997370ad6ac3ce6b237c45.png"},{"id":85757463,"identity":"71b5a480-8905-497c-a5c3-3d30f9dfb76b","added_by":"auto","created_at":"2025-07-01 10:55:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37650,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic potential in HepG2 cells. The cytotoxic potential was evaluated by MTT colorimetric assay. Results are expressed as % of cell viability compared to the control, taken as 100%. GraphPad Prism 6 software, employing analysis of variance (ANOVA) with a significance level (p) lower than 0.05: *= p ≤ 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6922462/v1/4a3c25d9c670627ff3641514.png"},{"id":85757457,"identity":"7facadc4-2672-413c-9a41-aca73ef00978","added_by":"auto","created_at":"2025-07-01 10:55:41","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":347387,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC chromatograms of the methanolic extracts from \u003cem\u003eHypogymnia physodes\u003c/em\u003e, \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e and \u003cem\u003eUsnea subfloridana\u003c/em\u003e. (λ = 254 nm).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6922462/v1/8f825f4d809bc9f8d79c6c59.jpeg"},{"id":85759273,"identity":"07f8dfca-56c8-4ca7-88f0-3e95ee0c27fc","added_by":"auto","created_at":"2025-07-01 11:11:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1523359,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6922462/v1/813916b6-f4a7-440e-8d3d-2fd468efa0b9.pdf"},{"id":85757470,"identity":"350b4774-7568-4bbc-855b-63b1af19cb5e","added_by":"auto","created_at":"2025-07-01 10:55:42","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":107007,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstractlichens.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6922462/v1/1c9aecf5d18a17f687b75e56.pdf"}],"financialInterests":"","formattedTitle":"Evaluation of the antioxidant activity and Citotoxic potential of lichen forming fungal species of the family Parmeliaceae (Ascomycota)","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer is a group of diseases that have in common an uncontrolled multiplication of cells. This group of diseases has been affecting multicellular organisms for more than 200\u0026nbsp;million years (Hausman DM 2019). The incidence of cancer has increased throughout the 20th and 21st centuries as life expectancy has increased and living conditions have changed. Increased sedentary lifestyles, environmental factors and new eating habits are risk factors for developing cancer in the course of life (El-Sherif A 2021; Doll R 1981; Albuquerque RC 2014; Bertuccio P 2013; Friedenreich CM 2021; Liu X2014; Jochem C 2022; Magalhaes B 2021). Statistics say that one in four people will be at risk of developing cancer at some point in their lives (Roy PS 2016).\u003c/p\u003e \u003cp\u003eCancer does not only affect cells that have lost control over their replication. Recent studies show that cancer actually comprises a true organic biological ecosystem involving healthy cells around the cancer cells as well as the molecular interactions that take place in the tumor environment. The whole set of biological relationships around the tumor has been called \"tumor microenvironment\" (Visser KE 2023).\u003c/p\u003e \u003cp\u003eCancer therapy is based on three therapeutic strategies: surgery, chemotherapy (including innovative biological therapies) and radiotherapy (Roy PS 2016), but we are far from considering that we have an adequate solution. Research to discover new molecules with cancer protective or anticancer activity is therefore essential. Natural products have been a source of molecules with pharmacological bioactivity, especially against infectious diseases and cancer (Atanasov AG 2021). Despite this, the pharmaceutical industry has focused on synthetic drugs because of the intrinsic difficulties of natural products: difficulty in isolation, stability problems, and even complications in obtaining sufficient quantities of the natural product (Atanasov AG 2015).\u003c/p\u003e \u003cp\u003eLichens are the result of a symbiosis between a fungus (from Basidiomycota and Ascomycota filum) and a photobiont which contains chlorophyll such as a cyanobacterium or a green alga (Calcott MJ 2018). Traditionally, lichens have been used for their medicinal and culinary properties (Crawford SD 2015; Ivanova D 2009). Lichens produce a large number of secondary metabolites (Ure\u0026ntilde;a-Vacas I 2022; Baczewska 2024; Phan 2025)\u003c/p\u003e \u003cp\u003eMore recently, more extensive studies on secondary metabolites have started to identify new substances such as depsides and depsidones, as well as other chemical groups (dibenzofurans, aliphatic or usnic acids, anthraquinones or xanthones) whose research has supported the potential of the different species of these families to produce unique natural compounds with different biological and physico-chemical properties (Cardile V 2017; Ren M 2023). In addition to their natural function, secondary compounds from lichens have demonstrated a number of useful biological activities in other organisms, such as antibiotic, antimycobacterial, antimutagenic, antioxidant, antiviral, antipyretic, analgesic and antitumor properties, and have therefore been used for the treatment of various diseases in traditional medicine. (Boustie J 2011). The biological functions of lichen compounds have motivated the study of their pharmacological activity, both in extracts and in isolated active compounds. However, this study, together with purification and identification of compounds, has not yet been able to fully realise their therapeutic potential.\u003c/p\u003e \u003cp\u003eThe most abundant group of compounds in lichens are the polyketides (depsides, depsidones, anthraquinones, dibenzofurans...) which are phenolic in nature. They arise from the polymerization and subsequent modification of acetyl-coenzyme A and malonyl-coenzyme A subunits. This results in polycyclic structures with several hydroxyl groups, which characterize molecules with antioxidant activity (Goga M 2020).\u003c/p\u003e \u003cp\u003eMany studies have established the antioxidant activity of both extracts and compounds isolated from lichens corroborating that these organisms are an original source of these compounds (Shang 2018, Dwarakanath 2024). The vast majority have been assays on extracts that, depending on the genus, the antioxidant activity is found in the methanolic extract or in the acetonic extract. (G\u0026oacute;mez-Serranillos MP 2014; Kosanić 2016)\u003c/p\u003e \u003cp\u003eThe lichen compounds that have been studied the most are the polyketides mentioned above, especially depsides, tridepsides and depsidones. Research over the last 10 years has focused on their potential antitumor, antimicrobial and antioxidant activity. Numerous studies have been conducted in \u003cem\u003ein vitro\u003c/em\u003e models, although studies in \u003cem\u003ein vivo\u003c/em\u003e models are scarce and no human clinical trials have been published (Ure\u0026ntilde;a-Vacas I 2022; Rankovič B 2010; Kello M 2023; Ure\u0026ntilde;a-Vacas I 2023; Sol\u0026aacute;rov\u0026aacute; Z 2020).\u003c/p\u003e \u003cp\u003eSeveral studies have investigated the anticancer potential of lichens (Varol 2020, Koopaie 2023). The anticancer activity of lichens has been previously established in multiple in vitro experiments. In these experiments, not only the activity of the lichen extracts but also the pharmacological activity of the isolated secondary metabolites has been demonstrated (Tripahi AH 2022; Ristic S 2016). These metabolites are responsible for a large part of the different biological activities of lichens (Moln\u0026aacute;r K 2010; Rankovic B 2015).\u003c/p\u003e \u003cp\u003eMethanolic extracts of lichen (e.g. \u003cem\u003eRoccella montagnei\u003c/em\u003e), and in vitro assays showed significant cytotoxic activity against several human cancer cell lines such as colon (DLD-1, SW-620), breast (MCF-7) and head and neck (FaDu). With these activities, interesting compounds were isolated from the extract by chromatography (roccellic acid and everninic acid), so these findings highlight the promising anticancer activity of some lichens and their bioactive compounds (Mishra 2017)\u003c/p\u003e \u003cp\u003eOxidative stress is a damage factor resulting from the disruption of the balance between the generation of reactive oxygen or nitrogen species and the cell's antioxidant mechanisms (Prasad S 2017). Excess free radicals produce molecular damage that triggers loss of functionality of proteins by oxidation, lipid peroxidation and DNA damage leading to mutations and eventually cell death. Molecular damage due to oxidative stress accumulates over time until organs begin to experience organ failure leading to pathological conditions associated with, among others, cancer (Liguori I 2018). In this study, methanolic extracts of five lichen species belonging to the \u003cem\u003eParmeliaceae\u003c/em\u003e family, have been analysed: \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.), \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes), \u003cem\u003eUsnea subfloridana\u003c/em\u003e (Stirt.), \u003cem\u003eLethariella canariensis\u003c/em\u003e(Ach.) and \u003cem\u003eLethariella intricate\u003c/em\u003e (Moris), with the aim of determinate the cytotoxic bioactivity of the lichens extracts on cancer cells and their antioxidant potential. Subsequently, the secondary metabolite content of the three extracts with the highest biological activity was determined by High-Performance Liquid Chromatography (HPLC) and we found the presence of molecules such as physodalic acid, pshysodic acid, atranorin, lecanoric acid, salazinic acid and usnic acid. These secondary metabolites have shown bioactivity against cancer in many cell lines and cancer types. (Cardile V 2017; Kosanić M 2013; Stojanović IZ 2014; Stojanović IZ 2013; Correch\u0026eacute; ER 2004; Paluszczak J 2018; Manojlović N 2012; Roser LA 2022; Harikrishnan A 2021; Zhou R 2017; Majchrzak-Celińska A 2022; Sun TX 2021; Kumar K 2020; Pyrczak-Felczykowska A 2022) In this study, we aim to determinate the cytotoxicity of diverse lichens extracts on MCF-7 cells, as a model of breast cancer (Comşa Ş 2015), and in HepG2 cells, as a model of human hepatocellular carcinoma cells (Calcott MJ 2018). We also determinate the antioxidant bioactivity in vitro through different methods in order to select three lichen extracts with the best antioxidant potential. In the last instance, the three selected lichen extracts were subjected to HPLC with the aim of identifying the secondary metabolites most commonly present in each of them and thus attributing the bioactivity to well-defined chemical compounds.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1 Reagents\u003c/h2\u003e \u003cp\u003e2,4,6-tri-2-pyridinyl-1,3,5-triiazine (TPTZ) were obtained from Alfasigma (Barcelona, Spain). Iron sulphate 7 hydrate (FeSO4 -7H2O) and sodium carbonate (Na2CO3) were purchased from PanReac (Barcelona, Spain). Iron chloride 6 hydrate (FeCl3\u0026ndash;6H2O), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (TROLOX), 3,4,5-trihydroxybenzoic acid (gallic acid), Follin \u0026amp; Ciocalteu\u0026acute;s phenol reagent, fluorescein sodium salt and 2,2\u0026acute;-azobis (2-amidinopropano)-dihicloruro (AAPH) 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), doxorubicin and Triton X-100 were supplied from Sigma Aldrich-Merck KGaA (Darmstadt, Germany). Methanol and DMSO were obtained from Applichem (Darmstadt, Germany). RPMI 1640, Foetal bovine Serum (FBS), gentamicin and Phosphate -Buffered saline (PBS) were purchased from Gibco (Invitrogen, Paisley, UK)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2 Lichens\u003c/h2\u003e \u003cp\u003eFor this study, we used five lichen species well identified by experts and already preserved in the MAF herbarium of the Faculty of Pharmacy of the Complutense University of Madrid. Subsequently, they were deposited in the herbarium of the Faculty of Pharmacy of the Complutense University of Madrid with the following details\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.) Nyl., Leon, Spain, November 2014 (MAF-Lich 19708)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eLethariella canariensis\u003c/em\u003e (Ach.) Krog, Portugal, Madeira, November 1990, distributed by the Institut f\u0026uuml;r Botanik, Karl-Franzens-Universit\u0026auml;t, Graz (GZU) (MAF-Lich 6865)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eLethariella intricata\u003c/em\u003e (Moris) Krog, Navarra, Spain, September 2013 (MAF-Lich 18340)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes) Hale, La Palma, Spain, June 2009 (MAF-Lich 18178)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003eUsnea subfloridana\u003c/em\u003e (Stirt.), Asturias, Spain, September 2012 (MAF-Lich 18040)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003ePreparation of lichen extracts\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAccording to the method described by Amo De Paz et al. [\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e], 50 mg of the air-dried lichen thallus was weighed (exactly) and mixed with 2 ml of pure methanol, the samples were shaken for 1 minute every 30 minutes during the first 2 hours, then incubated 24 hours. After 24 hours, the extract was filtered (0.22\u0026micro;m) and left until completely evaporated at room temperature. The dried extracts were then weighed and re-dissolved in methanol or DMSO depending on the experiments to be performed. The extraction yield was calculated for each lichen species. The samples were kept cold (4\u0026ordm;C).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Methods\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Determination of total phenolic compounds through Folin-Ciocalteu assay\u003c/h2\u003e \u003cp\u003eFollowing the method described by Saura-Calixto et al. [\u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e] with some modifications we use the Folin-Ciocalteu assay to determinate the total amounts of phenolic compounds in the lichen extracts samples. 50 \u0026micro;l of Folin's reagent and 50 \u0026micro;l of each methanolic extract (1 mg/ml) were mixed and, after 5 min, 1 ml of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (7%) was added dropwise to these. The solutions were incubated for 40 min at room temperature and shaking. The absorbance was measured at 595 nm using a microplate reader spectrophotometer (SPECTROstar Nano, BMG Labtech, Germany). Results were interpolated with those of a calibration line created using gallic acid (GA) at different concentrations (from 5 to 200 M) as a reference standard. It is performed in an alkaline medium and the reduction leads to the formation of a blue complex.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. \u003cem\u003eIn vitro\u003c/em\u003e radical scaving activities\u003c/h2\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003e2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay\u003c/h3\u003e\n\u003cp\u003eThis method is based on the reduction of the DPPH reagent in radical form, in the presence of a hydrogen-donating antioxidant (Gulcin İ 2020). The method described by Amarowicz et al. (Amarowicz R 2004) was followed with slight modifications. In a 96-well plate 5 dilutions (from 100 to 900 \u0026micro;g/ml) of each methanolic lichen extract samples was added and a DPPH solution (550 \u0026micro;M) was placed to make up a final volume of 225 \u0026micro;l/well. the solutions were incubated at 37\u0026deg;C for 30 minutes. Then, the absorbance was read at 517 nm through a Spectrostar Nano microplate reader spectrophotometer (BMG Labtech, Germany). As a reference antioxidant, different dilutions of trolox were used to make a calibration curve. IC\u003csub\u003e50\u003c/sub\u003e was calculated to express DPPH scavenging activity.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e% inhibition = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Absorbance\\:sample}{Absorbance\\:DPPH}\\)\u003c/span\u003e\u003c/span\u003e \u0026middot; 100\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eOxygen Radical Antioxidant Capacity (ORAC) assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis technique is based on a hydrogen atom transfer mechanism, in which peroxyl radicals, produced by thermal decomposition of the AAPH reagent, oxidize a fluorescent probe, i.e. fluorescein (Gulcin I 2020; Amarowicz R 2004; Nkhili E 2011). We followed the protocol previously described by Davalos et al. (D\u0026aacute;valos A 2004) with some modifications. In brief, 11 concentrations of lichen samples were prepared from 2 to 1000 \u0026micro;g/mL in methanol and subsequently incubated for 15 min at 37\u0026deg;C with a 70nM fluorescein solution in a 96-well plate. Finally, AAPH was added to all samples (12 mM) and the fluorescence was determined for 104 min at an excitation wavelength of 485nm and an emission wavelength 520 nm via FLUOstar OPTIMA, (BMG Labtech, Ortenberg, Germany). The area under the curve (AUC) was calculated for each sample and compared with Trolox. The results were expressed as \u0026micro;mol TE/mg sample.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAUC\u0026thinsp;=\u0026thinsp;1 + \u0026sum; fi/f0\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eFerric Reducing Antioxidant Power (FRAP) assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe assay is based on an electron transfer mechanism and on the principle of reduction of Fe\u003csup\u003e3\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;to Fe\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;by the action of an antioxidant present in the sample [\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e]. The occurrence of the reaction is detected using ,4,6-Tris(2-pyridyl-s-triazine (TPTZ), a compound capable of forming complexes with Fe\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;and, as a consequence, imparting a purple color to the solution (Moon JK 2009). The method described by S\u0026aacute;nchez-Muniz et al. (S\u0026aacute;nchez-Muniz FJ 2012) was carried out, with some modifications.\u003c/p\u003e \u003cp\u003eLichen samples (1mg/ml in methanol) were mixed with the previously prepared FRAP reagent (TPTZ (10mM) in HCl (40mM), acetate buffer (ph 3.6) and FeCl\u003csub\u003e3\u003c/sub\u003e 6 H\u003csub\u003e2\u003c/sub\u003eO (20mM) and incubated for 30 min at 37\u0026deg;C, and the absorbance was subsequently measured in a SPECTROstar Nano microplate reader at a wavelength of 595 nm. The results were expressed as \u0026micro;mol Fe\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;eq/g sample.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e% inhibition = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Abs\\:Sample-Blank}{Abs\\:Control-Blank}\\)\u003c/span\u003e\u003c/span\u003e \u0026middot; 100\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eAntioxidant Index\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe calculate antioxidant index with the arithmetic mean of a normalized measure of the antioxidant power in each extract. To normalize these measurements, we take as 100% the species with the greatest antioxidant power and thus achieve comparable data. Subsequently, we find the arithmetic mean of the percentages of all the experiments which allows us to compare all the species with each other (Ure\u0026ntilde;a-Vacas I 2022)\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Sample\\:Score}{Best\\:Score}\\)\u003c/span\u003e \u003c/span\u003e \u0026middot; 100\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.2.3. Cytotoxic potential\u003c/h2\u003e \u003cp\u003e \u003cb\u003eCell Culture\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBreast adenocarcinoma cell line MCF7 and human hepatocellular carcinoma cell line HepG2 were obtained from the NCI-Frederick Cancer DCTD Tumor/Cell line Repository (Frederick National Laboratory for Cancer Research, National Cancer Institute) were used in this study. They were cultivated inside 25 cm\u003csup\u003e2\u003c/sup\u003e tissue culture flasks (Corning Incorporated, New York, USA) using RPMI-1640 medium supplemented with 10% FBS and 0.5% gentamicin at 37\u0026deg;C in a humidified environment with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cell passaging was performed every 2\u0026ndash;3 days, when 80\u0026ndash;90% confluence was reached.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssessment of cell viability\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe cytotoxic potential was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, following the protocol described by Mosmann (Mosmann T 1983) with slight modifications. This colorimetric assay measures cell viability by exploiting the reduction of the MTT reagent to the purple formazan crystal performed by the mithocondrial activity of metabolically active cells (Van Meerloo J 2011; Ghasemi M 2021). 96-well plates were used, in which the cells were seeded at a density of 5x 10\u003csup\u003e3\u003c/sup\u003e and 7x 10\u003csup\u003e3\u003c/sup\u003e cells/well in MCF-7 and HepG2 cells line, respectively, and then incubated overnight. After reaching a confluence around 90%, they were treated with different concentrations of the 5 lichen species extracts (Nine different concentrations were used, ranging from 0.25 \u0026micro;g/ml to 200\u0026micro;g/ml) for 24h. After this time, a 2 mg/ml solution of MTT was added and the plates were incubated at 37\u0026deg;C for 1 hour in the dark. Finally, the medium was removed and 100 \u0026micro;l of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 550 nm through a Spectrostar Nano microplate reader spectrophotometer (BMG Labtech, Germany). Untreated cells (control) were only exposed to cell culture medium. Results are expressed as % of cell viability compared to the control, taken as 100%. Triton X-100 at 1% was used as a standard of comparison. Triton X-100 was used as a negative control. This compound generates excessive cytotoxicity, effectively reducing cell viability to around 20% viability. In this case the control is the group of cells that have not been treated with any extract.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh-performance liquid chromatography HPLC\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe secondary lichen metabolites present in the extracts were identified using HPLC. The extracts were prepared at a concentration of 250 \u0026micro;g/ml in methanol and then filtered. Twenty microliters of the sample were injected into the Agilent 1200 Infinity chromatograph (Agilent Technology, California, USA). The mobile phase consisted of a mixture of 1% phosphoric acid in mili-Q water and HPLC-grade methanol, with the proportion gradually changing from 70% phosphoric acid at the initial time to 10% at 45 minutes. As the stationary phase, an analytical column in reverse phase Mediterranea Sea C18 (Tecknokroma, Barcelona, Spain) was employed. The analysis conditions included a temperature of 40\u0026deg;C and a flow rate of 0.6 ml/minute.\u003c/p\u003e \u003cp\u003eThe compounds eluted were detected using a photodiode array detector operating wavelength range from 190 to 800 nm. The selected wavelength was the one at which the lichen compounds exhibited their maximum absorbance (254 nm).\u003c/p\u003e \u003cp\u003eData processing was carried out using Agilent ChemStation software (Agilent Technology, California, USA), recording the retention times and maximum absorbance wavelengths for each of the compounds.\u003c/p\u003e \u003cp\u003eThe identification of the compounds was performed by comparing the data with references from the literature. In the case of usnic acid, a pure isolated compound was used as a standard (Sigma-Aldrich, Missouri, USA).\u003c/p\u003e \u003cp\u003eTherefore, once the chromatographic spectra provided by the Agilent HPLC software connected to the instrument were obtained, we proceeded to identify the correspondence of each peak to a precise secondary compound/metabolite.\u003c/p\u003e \u003cp\u003eThis was mainly based on a comparison of the chromatographic spectra obtained experimentally with the data in the scientific bibliography.\u003c/p\u003e \u003cp\u003eThe instrument provided 8 chromatograms in the range of wavelengths from 273 to 210, in particular 210, 214, 230, 240, 250, 254, 260, 273 nm. For the identification of peaks, only those at 254 nm were considered for each of the three lichen species.\u003c/p\u003e \u003cp\u003eTo identify the extraction and production solvent in the spectrum of the extract, i.e. methanol, the HPLC measurement of this compound alone was carried out, in order to obtain a spectrum in which only one peak appeared, which inevitably correlated with methanol. The same was carried out injecting in the tool the pure usnic acid compound, in the research group's possession, which was known to be contained in one of the extracts, in particular in \u003cem\u003eUsnea subfloridana\u003c/em\u003e, thanks to data from the scientific literature.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAll the results are derived from 3 independent repetitions and are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Statistical analysis was conducted using GraphPad Prism 8 software, employing analysis of variance (ANOVA) with a significance level (p) lower than 0.05. Pearson's correlation coefficient ( R ) was calculated to determine the linear correlation between the different antioxidant activity techniques and the total phenolic compound content.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eAs mentioned in the introduction, methanolic extracts of five lichens have been an-alysed in this study: \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.), \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes), \u003cem\u003eUsnea subfloridana\u003c/em\u003e (Stirt.), \u003cem\u003eLethariella canariensis\u003c/em\u003e (Ach.) \u003cem\u003eand Lethariella intricate\u003c/em\u003e (Moris).\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Calculation of the Antioxidant index (%)\u003c/h2\u003e \u003cp\u003eFrom the results obtained in the four in vitro tests (Folin-Ciocalteu, FRAP, DPPH, ORAC) the antioxidant index was calculated. First, the results of each in vitro test were considered separately and the extract giving the best result was considered 100% (in the Folin-Ciocalteu, FRAP and ORAC tests the best result was the one giving the highest numerical value, in the DPPH test the one giving the lowest numerical value); then, at the value representing 100%, the results of all other extracts were compared with it. The arithmetic mean was then calculated considering the relative results of each extract in each test to obtain the antioxidant index expressed in %, which was included in Table\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Antioxidant activity\u003c/h2\u003e \u003cp\u003eIn this research we examined the total phenolic content (TPC) in the methanol extract at a concentration of 1/mg/ml of each lichen. A very high TPC has been found in P. \u003cem\u003epsudotinctorum\u003c/em\u003e (348,67\u0026thinsp;\u0026plusmn;\u0026thinsp;23,16 \u0026micro;g gallic acid (GA)/mg), the highest of the 5 plants studied. The TPC of \u003cem\u003eU. subfloridana\u003c/em\u003e and \u003cem\u003eH. physodes\u003c/em\u003e are also outstanding, and the contents of the Lethariellae studied are more discreet.\u003c/p\u003e \u003cp\u003eThe antioxidant capacity of our methanolic extracts was evaluated by a battery of three assays describing different antioxidant mechanisms. The antioxidant properties of some compounds are conditioned by pH, so in these assays, measurements were made at different pHs. The antioxidant activity is also influenced by the substrate that is reduced, so that not all assays feature the same lichens and, therefore, a combination of all of them is required to make a correct selection.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the results of the 3 antioxidant tests performed and phenolic content. The FRAP test in which the transfer of a single electron to the Fe3+-TPTZ complex to form Fe2+-TPTZ in acid medium (pH\u0026thinsp;=\u0026thinsp;3.6) was assessed. The leading species in this test was \u003cem\u003eU. subfloridana\u003c/em\u003e (69.36\u0026thinsp;\u0026plusmn;\u0026thinsp;7.22 \u0026micro;mol eq Fe2+ /g E), well ahead of the immediately following \u003cem\u003eH. physodes\u003c/em\u003e (30.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92) and \u003cem\u003eL. intricata\u003c/em\u003e (30,09\u0026thinsp;\u0026plusmn;\u0026thinsp;8,91).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntioxidant assays of methanolic extracts\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLichen species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFRAP (\u0026micro;mol of Fe\u003csup\u003e2+\u003c/sup\u003eeq./g E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDPPH EC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eORAC value (\u0026micro;mol of TE/mg E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePhenolic content (\u0026micro;g of GA eq./mg E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAntioxidant index (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHypogymnia physodes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30,36\u0026thinsp;\u0026plusmn;\u0026thinsp;2,92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e69,66\u0026thinsp;\u0026plusmn;\u0026thinsp;6,50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5,77\u0026thinsp;\u0026plusmn;\u0026thinsp;1,31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e77,35\u0026thinsp;\u0026plusmn;\u0026thinsp;11,96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56,20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLethariella canariensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22,59\u0026thinsp;\u0026plusmn;\u0026thinsp;1,43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e320,02\u0026thinsp;\u0026plusmn;\u0026thinsp;42,98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5,39\u0026thinsp;\u0026plusmn;\u0026thinsp;1,21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e16,76\u0026thinsp;\u0026plusmn;\u0026thinsp;2,63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43,91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLethariella intricata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e30,09\u0026thinsp;\u0026plusmn;\u0026thinsp;8,91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e466,86\u0026thinsp;\u0026plusmn;\u0026thinsp;77,31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4,00\u0026thinsp;\u0026plusmn;\u0026thinsp;1,59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e29,73\u0026thinsp;\u0026plusmn;\u0026thinsp;5,24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41,32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14,96\u0026thinsp;\u0026plusmn;\u0026thinsp;3,07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1527,61\u0026thinsp;\u0026plusmn;\u0026thinsp;99,82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9,10\u0026thinsp;\u0026plusmn;\u0026thinsp;2,00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e348,67\u0026thinsp;\u0026plusmn;\u0026thinsp;23,16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55,39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eUsnea subfloridana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e69,36\u0026thinsp;\u0026plusmn;\u0026thinsp;7,22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e84,71\u0026thinsp;\u0026plusmn;\u0026thinsp;8,71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2,29\u0026thinsp;\u0026plusmn;\u0026thinsp;0,06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e113,92\u0026thinsp;\u0026plusmn;\u0026thinsp;13,74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e63,08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe DPPH assay results showed that the extracts requiring the lowest concentration to neutralise 50% of the DPPH were those of \u003cem\u003eH. physodes\u003c/em\u003e (69.66\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50 \u0026micro;g/ml) and \u003cem\u003eU. subfloridana\u003c/em\u003e (84.71\u0026thinsp;\u0026plusmn;\u0026thinsp;8.71 \u0026micro;g/ml). The DPPH radical was mainly neutralised by lipophilic antioxidants whose mechanism consists of electron and/or proton transfer in an acidic medium (pH\u0026thinsp;=\u0026thinsp;4.0).\u003c/p\u003e \u003cp\u003eThe ORAC assay was performed in a neutral medium (pH\u0026thinsp;=\u0026thinsp;7.4) and the mechanism studied was proton transfer to neutralise the OH- radical. The results of the ORAC assay are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), where \u003cem\u003eP. pseudotinctorium\u003c/em\u003e stands out from the rest of the species (9.10 H\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 \u0026micro;g eq Tx/mg E), quite a distance from the following, such as \u003cem\u003eH. physodes\u003c/em\u003e (5,77\u0026thinsp;\u0026plusmn;\u0026thinsp;1,31\u0026micro;g eq Tx/mg E) and \u003cem\u003eL. canariensis\u003c/em\u003e (5,39\u0026thinsp;\u0026plusmn;\u0026thinsp;1,21).\u003c/p\u003e \u003cp\u003eThe results obtained in the FRAP, DPPH, ORAC and Folin-Ciocalteu tests are grouped in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, in which the Antioxidant index was also calculated and, based on this value, a classification of the different species was established, with \u003cem\u003eU. subfloridana\u003c/em\u003e (1st), \u003cem\u003eP. pseudotinctorum\u003c/em\u003e (2nd), \u003cem\u003eH. physodes\u003c/em\u003e (3rd), \u003cem\u003eL. canariensis\u003c/em\u003e (4th) and \u003cem\u003eL. intricata\u003c/em\u003e (5th).\u003c/p\u003e \u003cp\u003eTo determine whether there is a possible correlation between the content of phenolic compounds and the antioxidant activity of the five methanol extracts of the lichen species studied, a Pearson\u0026acute;s correlation study (r) was carried out to quantify the correlation between two quantitative variables. The results showed that there is a moderate correlation between DPPH and ORAC with the content of total phenolic compounds (0.71\u0026thinsp;\u0026lt;\u0026thinsp;r\u0026thinsp;\u0026lt;\u0026thinsp;0.85).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Cytotoxic potential\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. Assessment of cell viability in MCF7 cell cultures using the MTT colorimetric assay\u003c/h2\u003e \u003cp\u003eA marked effect was seen in the extracts of all the lichens studied, although the concentrations at which a significant decrease in cell viability was seen vary. In the case of \u003cem\u003eP. pseudotinctorum\u003c/em\u003e, it stands out among all the extracts studied as it starts with significant activity at 1\u0026micro;g/ml (cell viability 84.84\u0026thinsp;\u0026plusmn;\u0026thinsp;2.36% of control, p\u0026thinsp;\u0026le;\u0026thinsp;0.05) maintaining its cytotoxicity at increasing concentrations, and in second place in \u003cem\u003eH. physodes\u003c/em\u003e, marked activity was already observed at a concentration of 2.5 \u0026micro;g/ml (88.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03% cell viability, p\u0026thinsp;\u0026le;\u0026thinsp;0.05), as well as \u003cem\u003eL. intricata\u003c/em\u003e which at the same dose showed an interesting cytotoxicity (cell viability 93.48\u0026thinsp;\u0026plusmn;\u0026thinsp;2.91% of control, p\u0026thinsp;\u0026le;\u0026thinsp;0.05). In the case of \u003cem\u003eU. subfloridana\u003c/em\u003e and \u003cem\u003eL. canariensis\u003c/em\u003e, the onset of a significant decrease in cell viability occurs from a concentration of 5 \u0026micro;g/ml (cell viability 89.02\u0026thinsp;\u0026plusmn;\u0026thinsp;4.76% and 91.76\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86%, p\u0026thinsp;\u0026le;\u0026thinsp;0.05 respectively). All lichen extracts maintained a high cytotoxicity on MCF7 cells from the concentration of 5 \u0026micro;g/ml to 200 \u0026micro;g/ml, although it can be observed that \u003cem\u003eU. subfloridana\u003c/em\u003e and \u003cem\u003eH. physodes\u003c/em\u003e caused the greatest decrease in cell viability at the highest concentrations as it is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. Assessment of cell viability in HepG2 cell cultures using the MTT colorimetric assay\u003c/h2\u003e \u003cp\u003eOther natural products have shown biological activity on this cell line [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. A marked effect was again observed in the extracts of all the lichens studied, especially at doses of 10 \u0026micro;g/ml or higher, where all of them showed a significant decrease in cell viability. However, some of them start to show significant cytotoxic activity at lower doses, as was the case of \u003cem\u003eP. pseudotinctorum\u003c/em\u003e where significant differences in cytotoxicity start to appear from 0.5 \u0026micro;g/ml (cell viability 91.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48% of control, p\u0026thinsp;\u0026le;\u0026thinsp;0.05), maintaining its cytotoxicity at increasing concentrations. The same occurred with \u003cem\u003eU. subfloridana\u003c/em\u003e although significant differences was shown from 2.5 \u0026micro;g/ml (cell viability 79.63\u0026thinsp;\u0026plusmn;\u0026thinsp;6.18, p\u0026thinsp;\u0026le;\u0026thinsp;0.05), and L. \u003cem\u003eintrincata\u003c/em\u003e and \u003cem\u003eH. physodes\u003c/em\u003e from 5 \u0026micro;g/ml (cell viability 87.79\u0026thinsp;\u0026plusmn;\u0026thinsp;3.57% and 88\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25%, p\u0026thinsp;\u0026le;\u0026thinsp;0.05 respectively). \u003cem\u003eL. canariensis\u003c/em\u003e starts with a significant decrease in cell viability from a concentration of 10 \u0026micro;g/ml (cell viability 82.10\u0026thinsp;\u0026plusmn;\u0026thinsp;7.98%, p\u0026thinsp;\u0026le;\u0026thinsp;0.05). All lichen extracts maintained high cytotoxicity on HepG2 cells from the concentration of 10 \u0026micro;g/ml to 150 \u0026micro;g/ml, although it can be observed that U. \u003cem\u003esubfloridana\u003c/em\u003e and L. \u003cem\u003eintrincata\u003c/em\u003e caused the greatest decrease in cell viability at the highest concentrations. These results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs the methanol extracts showed cytotoxic activity in MCF7 and HepG2 cells, IC50s were calculated for each species in this cell line in order to make a better comparison between them as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A control was carried out with a drug with potent anticancer activity, doxorubicin, allowing us to compare the activity of the lichen extracts studied.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIC 50 data for each species in MCF7 and HepG2 cells (GraphPad Prism 8 software).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMCF7\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHepG2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC50 (\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIC50 (\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLethariella canariensis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e612.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eUsnea subfloridana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLethariella intricata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e213.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e151.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e120.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHypogymnia physodes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoxorubicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e187.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e118.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn terms of cytotoxicity, \u003cem\u003eUsnea subfloridana\u003c/em\u003e and \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.) and \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e showed a lower IC50 than doxorubicin in MCF7 cell line, but in the HepG2 cell line it was \u003cem\u003eLethariella canariensis\u003c/em\u003e, \u003cem\u003eUsnea subfloridana, Lethariella intricata\u003c/em\u003e and \u003cem\u003eHypogymnia physodes\u003c/em\u003e that had a lower IC50 than doxorubicin, with that of \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e being very similar to doxorubicin.\u003c/p\u003e \u003cp\u003eConsidering the results as a whole, extracts of \u003cem\u003eUsnea subfloridana\u003c/em\u003e and \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.) showed a remarkable growth inhibition in MCF7 and HepG2 cell lines, indicating a good cytotoxic potential especially from the intermediate concentrations used, and \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes) showed a moderate overall growth inhibition in both MCF7 and HepG2 cell lines, with a more interesting action at lower concentrations\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Identification of lichen secondary metabolites by HPLC technique\u003c/h2\u003e \u003cp\u003eIn the last instance, the best performing lichen extracts in bioactivity were subjected to High Performance Liquid Chromatography with the aim of identifying the secondary metabolites most commonly present in each of them and thus attributing the antioxidant activity to well-defined chemical compounds. The obtained chromatograms of one of the two measurements are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo trace the identity, in terms of lichen secondary metabolites, of each experimentally obtained chromatogram peak, we proceeded by comparing the wavelengths and retention times resulting from the experimental measurements of the 3 methanolic extracts considered, with wavelengths and retention times available in the bibliography.\u003c/p\u003e \u003cp\u003eThe compounds that were identified are displayed alongside their molecular structures, while the others remained unidentified due to insufficient bibliographic evidence. In the case of usnic acid present in \u003cem\u003eU. subfloridana\u003c/em\u003e, it was identified using a commercial standard, verifying that the retention time and its UV absorption spectrum matched.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eU. subfloridana\u003c/em\u003e, the identified compounds include salazinic acid and usnic acid. In \u003cem\u003eP. pseudotinctorum\u003c/em\u003e, lecanoric acid and atranorin were identified, and in H. physodes, physodalic acid, physodic acid, and atranorin were identified.\u003c/p\u003e \u003cp\u003eThe retention times and maximum absorbances of the identified compounds are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The average retention times for the identified compounds are as follows: Salazinic acid: 21.02 minutes, Lecanoric acid: 22.19 minutes, Physodalic acid: 26.69 minutes, Physodic acid: 34.39 minutes, Usnic acid: 39.69 minutes, and Atranorin: 41.56 minutes.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHPLC results: molecular formula, retention time, and ultraviolet spectrum data of the metabolites identified by HPLC. The retention time data corresponds to the average of four representative chromatograms\u0026thinsp;\u0026plusmn;\u0026thinsp;the mean deviation.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMolecule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRetention time (minutes)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eλ\u003csub\u003emax\u003c/sub\u003e (nm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSalazinic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.02 \u0026plusmn; 0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e220/270/310\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUsnic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e39.69 \u0026plusmn; 0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e232/282\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLecanoric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.19 \u0026plusmn; 0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e212/270/304\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAtranorin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e41.56 \u0026plusmn; 0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e210/252/262/320\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhysodalic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e26.69 \u0026plusmn; 0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e212/242/318\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhysodic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eC\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e34.39 \u0026plusmn; 0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e218/258\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eCancer is known as a group of pathologies in which cellular deregulation and un-controlled multiplication occur, and whose incidence is increasing, especially in developed countries.\u003c/p\u003e\n\u003cp\u003eNatural products are a rich source of active compounds, yet a significant number remain unexplored and may contain metabolites with potential as valuable drugs. The pharmacology of lichens has not been adequately studied. Thus, the medicinal applications of these symbiotic organisms and their compounds represent a largely untapped field of research with immense potential. Most studies have focused on revealing the cytotoxic activity of secondary metabolites on carcinogenic processes. The molecular mechanisms by which metabolites may have cytotoxic activity are cell cycle arrest and induction of apoptotic cell death (extrinsic and intrinsic pathway). Also, against inflammation associated with the tumor process. Inflammation facilitates tumorigenesis and tumor progression by providing bioactive molecules including growth factors, cell death inhibitors, proangiogenic factors, extracellular matrix modifiers that facilitate invasion and metastasis. Lichen metabolites may also exert anticancer effects by regulating tu-mor-promoting inflammation and/or anti-tumor immunity, although most of the mechanisms underlying these effects remain to be defined.\u003c/p\u003e\n\u003cp\u003eIn this study, methanolic extracts of five lichens of the Parmeliaceae family have been analysed: \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.), \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes), \u003cem\u003eUsnea subfloridana\u003c/em\u003e (Stirt.), \u003cem\u003eLethariella canariensis\u003c/em\u003e(Ach.) and \u003cem\u003eLethariella intricate\u003c/em\u003e (Moris).\u003c/p\u003e\n\u003cp\u003eLichens are complex symbiotic organisms of fungi and algae. Traditional medicine has long used lichen-based products as plant material or their metabolites ( Thakur M 2021).\u003c/p\u003e\n\u003cp\u003eOxidative stress is considered to be a key factor in cancer\u0026acute;s origin and progression (Jelic MD 2021; Srinivas US 2019) and therefore an important part of its research focuses on antioxidant compounds of plant origin.\u003c/p\u003e\n\u003cp\u003eThe pharmacological activity of lichens is little studied. It is believed that the various secondary metabolites they possess, including a wide variety of polyphenolic compounds, are responsible for the various pharmacological activities, including antitumor activity. These same substances may contribute to their cytotoxic activity, as studies have shown their ability to induce apoptosis by altering the redox balance and activating pro-apoptotic signaling pathways. However, further studies on their activity are needed (Kosanić M 2013).\u003c/p\u003e\n\u003cp\u003eFor all the above-mentioned reasons, lichens, as a little explored source of active ingredients, could be an interesting option to counteract oxidative stress and its influence on the development of tumor diseases. (Thakur M 2023). They would also be an interesting proposal to alleviate the side effects of oncological therapies based on the generation of free radicals. (Jelic MD 2021)\u003c/p\u003e\n\u003cp\u003eIn order to carry out pharmacological prospecting studies on lichens and plants, the extraction of their active ingredients is necessary. In this case, extraction by maceration in methanol was used, since previous tests by our research group, supported by previous scientific evidence, showed that this solvent is the one that reported some of the best yields (Kalra R 2021). The fact that the maceration time is longer than in some of the reference studies has not significantly altered these yields.\u003c/p\u003e\n\u003cp\u003eHowever, for future research it should also be considered that maceration with other solvents, despite the lower yields, would also allow the extraction of other bioactive compounds with low solubility in methanol.\u003c/p\u003e\n\u003cp\u003eRegarding the composition of the five different lichen species, in this research we focused on the total phenolic content (TPC) in each methanolic extract (concentration of 1/mg/ml). A relevant TPC was found in all the species, but especially in \u003cem\u003eP. pseudotictorum\u003c/em\u003e (348.67\u0026thinsp;\u0026plusmn;\u0026thinsp;23.16 \u0026micro;g gallic acid (GA)/mg), the highest of the 5 species studied, followed by the TPCs of \u003cem\u003eU. subfloridana\u003c/em\u003e (113.92\u0026thinsp;\u0026plusmn;\u0026thinsp;13.74) and \u003cem\u003eH. physodes\u003c/em\u003e (77.35\u0026thinsp;\u0026plusmn;\u0026thinsp;11.96). This composition seemed to us to be a promising starting point for investigating the possible antioxidant activity of all our extracts.\u003c/p\u003e\n\u003cp\u003ePhenolic compounds are natural products. Lichens contain a wide variety of phenolic compounds in their thalli, including polyketides, which, due to their structural characteristics, have an antioxidant action. In any case, this composition would not imply a priori antioxidant activity, since although some studies point to a correlation between phenolic content and antioxidant capacity, there is not always a parallelism between the two, because such activity may be due to the presence of other compounds of a different nature.\u003c/p\u003e\n\u003cp\u003eOn the other hand, it has been seen that some of the compounds in lichens may have a dual mechanism, such as atranorin, since on the one hand it has free radical scavenging power and is a cytoprotectant, but also a pro-oxidant. It increases the survival of cells exposed to oxidative damage by hydrogen peroxide but can generate an increase in lipid peroxidation (Fern\u0026aacute;ndez-Moriano C 2016)\u003c/p\u003e\n\u003cp\u003eAs described in Results, the antioxidant capacity of our methanolic extracts was assessed by a battery of three assays based on different antioxidant mechanisms and, therefore, a combination of all of them is required to make a correct selection. Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e shows the graphs of the results of the 3 antioxidant tests performed.\u003c/p\u003e\n\u003cp\u003eThe FRAP test results shown the species that stands out is \u003cem\u003eU. subfloridana\u003c/em\u003e (69.36\u0026thinsp;\u0026plusmn;\u0026thinsp;7.22 \u0026micro;mol eq Fe2+ /g E).\u003c/p\u003e\n\u003cp\u003eThe DPPH assay results show that the extracts requiring the lowest concentration to neutralise 50% of the DPPH are those of \u003cem\u003eH. physodes\u003c/em\u003e (69.66\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50 \u0026micro;g/ml) and \u003cem\u003eU. subfloridana\u003c/em\u003e (84.71\u0026thinsp;\u0026plusmn;\u0026thinsp;8.71 \u0026micro;g/ml).\u003c/p\u003e\n\u003cp\u003eThe results of the ORAC assay shown, that \u003cem\u003eP. pseudotinctorum\u003c/em\u003e stands out from the rest of the species (9.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 \u0026micro;g eq Tx/mg E), despite having the highest concentration in the DPPH assay and, therefore, the lowest reduction capacity.\u003c/p\u003e\n\u003cp\u003eAlthough all species showed antioxidant activity, as revealed by the different measurement methods, the predominant species in each assay were different.\u003c/p\u003e\n\u003cp\u003eMethods to determine antioxidant activity are based on verifying how an oxidizing agent induces oxidative damage to an oxidizable substrate, damage that is inhibited or reduced in the presence of an antioxidant. This inhibition is proportional to the antioxidant activity of the compound or sample. On the other hand, there are tests that are based on the quantification of the products formed after the oxidative process. The different methods differ in the oxidizing agent, the substrate used, the end point measurement, the instrumental technique used and the possible interactions of the sample with the reaction medium. Furthermore, the objectives of the different measurement methods are diverse.\u003c/p\u003e\n\u003cp\u003eWithin the chosen tests are two Electron transfer tests (DPPH and FRAP) and a Hydrogen atom transfer test (ORAC). The DPPH radical is mainly neutralised by lipophilic antioxidants whose mechanism consists of electron and/or proton transfer in an acidic medium (pH\u0026thinsp;=\u0026thinsp;4.0) and this test is usually used to check the antioxidant activity of phenols, foods or drinks. The FRAP test in which the transfer of a single electron to the Fe3+-TPTZ complex to form Fe2+-TPTZ in acid medium (pH\u0026thinsp;=\u0026thinsp;3.6) is assessed, it can be used to check the antioxidant activity of foods or drinks and of biological samples. The ORAC assay is performed in a neutral medium (pH\u0026thinsp;=\u0026thinsp;7.4) and this method is considered the most similar to the physiological mechanisms in living beings, since it can be used to check the antioxidant activity of phenols, foods or biological samples, since it can be used to check the antioxidant activity of phenols, foods or biological samples. This justifies the need to use several methods to select the most antioxidant extracts.\u003c/p\u003e\n\u003cp\u003eThe results obtained in the FRAP, DPPH, ORAC and Folin-Ciocalteu tests are grouped in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, in which the Antioxidant index was also calculated and, based on this value, a classification of the different species was established, with \u003cem\u003eU. subfloridana\u003c/em\u003e (1st), \u003cem\u003eP. pseudotinctorum\u003c/em\u003e (2nd), \u003cem\u003eH. physodes\u003c/em\u003e (3rd), \u003cem\u003eL. canariensis\u003c/em\u003e (4th) and \u003cem\u003eL. intricata\u003c/em\u003e (5th).\u003c/p\u003e\n\u003cp\u003eIn view of these results, it seems that \u003cem\u003eUsnea subfloridana\u003c/em\u003e would be the most active as an antioxidant: is second in phenolic compound content or in DPPH and excels in FRAP (69,36\u0026thinsp;\u0026plusmn;\u0026thinsp;7,22 \u0026micro;mol of Fe2\u0026thinsp;+\u0026thinsp;eq./g E) and antioxidant index (63,08%) although is at the bottom in ORAC.\u003c/p\u003e\n\u003cp\u003eThe fact that there is different activity regarding the amount of antioxidant activity according to each test for each plant study may mean that there are different molecules responsible and that they act by different mechanisms revealed in each test.\u003c/p\u003e\n\u003cp\u003eAs can be seen, the strongest correlation found between phenolic content and antioxidant activity is observed in the ORAC method, which is based on the transfer of hydrogen atoms, where free radicals are stabilised by the donation of a hydrogen atom by an antioxidant molecule. This would help to explain the high activity of lichen extracts in this test, since phenolic compounds can relatively easily donate the hydrogen atom of their aromatic hydroxyls, as the delocalization of the charge on the benzene ring is favored.\u003c/p\u003e\n\u003cp\u003eIn vitro methods are useful for comparing the antioxidant activity of different samples, such as plant extracts. The results are limited from a biological point of view since they do not reproduce the physiological situation. On the other hand, the antioxidant activity of an extract or an isolated compound in vitro differs from its antioxidant effect in vivo, since the metabolic transformations that antioxidant compounds undergo in the body modify their activity. Certain polymeric phenolic compounds that have low in vitro activity can, however, contribute to the antioxidant capacity of the target organism after their metabolic transformation into simpler compounds.\u003c/p\u003e\n\u003cp\u003eTherefore, the next step in our research will be to carry out the relevant checks in more complex biological systems.\u003c/p\u003e\n\u003cp\u003eThe importance of several mechanisms in the development of certain pathological changes in the body is well known. Oxidative stress, by itself or in synergy with other related processes such as inflammation, apoptosis and mitochondrial dysfunction, is considered to be the cause of very frequent diseases in the body, especially in the central nervous system.\u003c/p\u003e\n\u003cp\u003eMetabolic transformations of the body\u0026apos;s own molecules such as neurotransmitters can lead to the development of Reactive Oxygen Species (ROS), which in turn can attack glial cells and sensitive neurons, causing damage to CNS structures (Thanan R 2014). Degradation of some CNS components, such as hyaluronic acid, produces isoforms that have been associated with pro-inflammatory properties as well as an increased ability of cancer cells to proliferate and invade. In addition, most brain tumors, including glial tumors of different grades, express high levels of cyclooxygenase-2 (COX-2), and these correlate with many aggressive aspects of the disease and poor prognosis (Studzińska-Sroka E 2021).\u003c/p\u003e\n\u003cp\u003eHaving established the antioxidant capacity of the extracts under study, our research has gone further to test their antitumor activity by testing their effect on the viability of a breast cancer cell line such as MCF7 and against hepatocellular carcinoma cells like HepG2. A large body of recent literature uses the cytotoxicity assay methodology in MCF7 and/or HepG2 cell lines to test for anti-tumour activity (Fernandez-Moriano C 2016; Tas I 2019; Alexandrino CA 2019; Nugraha A 2019; B\u0026eacute;zivin C 2003). These and other articles complement the results with assays on their composition or other related activities, such as antioxidant activity as it influences the tumour microenvironment (Nguyen TTH 2019; Mitrović T 2011)\u003c/p\u003e\n\u003cp\u003eUntil a few years ago, the cytotoxic properties of Parmeliaceae lichens had hardly been evaluated. Some investigations (G\u0026oacute;mez-Serranillos MP 2014; Fernandez-Moriano C 2016; Fernandez-Moriano C 2015) reported antiproliferative activity of methanol extracts of some Parmeliaceae species, including \u003cem\u003eHypogymnia physodes\u003c/em\u003e, but against the colon cancer cell lines. In addition, other groups studied the anticancer activity of other extracts obtained from Parmeliaceae spp. in human and murine cancer lines. However, so far the species included in our study had not been previously evaluated against MCF-7 and HepG2 cell lines.\u003c/p\u003e\n\u003cp\u003eOur study group chose these cell lines as it is a well-established model for this purpose and there are precedents in the scientific literature for research with MCF7 or Hepg2 on anti-tumor activity of lichens and their isolated compounds the research indicates that lichen extracts exhibit cytotoxic effects on HepG2 cells, showing strong cytotoxicity (Studzińska-Sroka E 2016; Kumar J 2014).\u003c/p\u003e\n\u003cp\u003eTherefore, we evaluated the cytotoxic effects of methanol extracts of the five Parmeliaceae species studied, as an approach to their anticancer potential. Their effects on cell viability were analysed and quantified by MTT assay after 24 hours of treatment with a wide range of concentrations of our extracts.\u003c/p\u003e\n\u003cp\u003eAccording to our results, as can be seen in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, a marked effect is seen in the extracts of all the lichens studied, although the concentrations at which a significant decrease in cell viability is seen vary. In the light of the data obtained, it appears that \u003cem\u003eP. pseudotinctorum\u003c/em\u003e would stand out among all the extracts studied as it starts with a significant decrease in % cell viability of MCF7 at the concentration of 1 \u0026micro;g/ml. The next species showing marked activity at a relatively low dose were \u003cem\u003eH. physodes\u003c/em\u003e and \u003cem\u003eL. intricata\u003c/em\u003e, as a marked decrease in cell viability in tumor line cell viability was observed at a concentration of 2.5 \u0026micro;g/ml. In the case of the other extracts, in \u003cem\u003eL. canariensis\u003c/em\u003e and \u003cem\u003eU. subfloridana\u003c/em\u003e there is the onset of a significant decrease in cell viability from a concentration of 5 \u0026micro;g/ml, although it seems more noticeable in the latter. Finally, all lichen extracts maintained a high cytotoxicity on MCF7 cells from the concentration of 5 \u0026micro;g/ml to 200 \u0026micro;g/ml, although it can be observed that \u003cem\u003eU. subfloridana\u003c/em\u003e and \u003cem\u003eH. physodes\u003c/em\u003e caused the greatest decrease in cell viability at the highest concentrations. In the case of HepG2 cell line, a marked effect is again observed in the extracts of all the lichens studied, especially at doses of 10 \u0026micro;g/ml or higher, where all of them show a significant decrease in cell viability. However, some of them start to show significant cytotoxic activity at lower doses, as is the case of \u003cem\u003eP. pseudotinctorum\u003c/em\u003e where significant differences in cytotoxicity start to appear from 0.5 \u0026micro;g/ml, maintaining its cytotoxicity at increasing concentrations. The same occurs with \u003cem\u003eU. subfloridana\u003c/em\u003e although significant differences are shown from 2.5 \u0026micro;g/ml, and \u003cem\u003eL. intrincata\u003c/em\u003e and \u003cem\u003eH. physodes\u003c/em\u003e from 5 \u0026micro;g/ml. \u003cem\u003eL. canariensis\u003c/em\u003e starts with a significant decrease in cell viability from a concentration of 10 \u0026micro;g/ml. All lichen extracts maintained high cytotoxicity on HepG2 cells from the concentration of 10 \u0026micro;g/ml to 150 \u0026micro;g/ml, although it can be observed that U. subfloridana and \u003cem\u003eL. intrincata\u003c/em\u003e caused the greatest decrease in cell viability at the highest concentrations.\u003c/p\u003e\n\u003cp\u003eAs the methanol extracts showed a remarkable cytotoxic activity, IC50s were calculated for each species in order to make a better comparison between them, for each cell line as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. From these results it can be concluded that \u003cem\u003eUsnea subfloridana\u003c/em\u003e and \u003cem\u003eHypogymnia physodes\u003c/em\u003e have the lowest IC50 for MCF7 (41.25 and 56.20 \u0026micro;g/ml, respectively) and that Lethariella intricata and \u003cem\u003eHypogymnia physodes\u003c/em\u003e have the lowest IC50 for HepG2 (12.93 and 37.17 \u0026micro;g/ml, respectively). If we look at the two cell lines together we can see that \u003cem\u003eH. physodes\u003c/em\u003e would have the highest overall cytotoxicity profile in both as an individual extract. \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes) showed a moderate overall growth inhibition in both MCF7 and HepG2 cell lines, with a more interesting action at lower concentrations. Therefore, this may be of great importance to avoid the occurrence of side effects.\u003c/p\u003e\n\u003cp\u003eIn view of this evident cytotoxicity in tumor cell lines, it is clear that the extracts studied have interesting potential for development as anticancer treatments. The differences between the percentages of cell viability of each species on each of the cancer types may be due to their different composition, as can be elucidated from the HPLC analyses shown, or their influence on the microenvironment due to their antioxidant action.\u003c/p\u003e\n\u003cp\u003eThere is a relationship between antioxidant activity and anti-cancerigenous protection either by influencing the microenvironment, in prevention or to avoid proliferation and invasiveness. The most studied secondary metabolites with antioxidant activity are dibenzofurans such as usnic acid; depsidones such as atranorin, diffractaic acid and lecanoric acid; depsidones such as stictic acid, lobaric acid, protocetraric acid, fumarprotocetraric acid, salazinic acid, physodic acid; simple phenolic compounds such as methyl orselinate and orselinic acid, methyl haematomate, orcinol and methyl \u0026beta;-orselinate. This relationship has already been seen in other plant species and metabolites might be utilised to treat cancer-related oxidative stress (Prasad S 2017). On the other hand, antioxidants also serve to minimize the side effects of oncological therapies (Fuchs-Tarlovsky V 2013)\u003c/p\u003e\n\u003cp\u003eAlthough it may seem like a contradiction that an extract or metabolite with anti-oxidant capacity is both cytotoxic and anti-tumor, since the most common oncological therapies are based on the pro-oxidant capacity of plant active ingredients to be used as anti-tumor (Goga M 2019), there is more and more evidence in which both activities coexist and can be used together therapeutically or directed at different pathways that complement each other.(Cakmak K 2019). In addition, there is growing scientific evidence supporting the implication of ROS and OE in the aetiology of neoplasms such as breast, colon or rectal cancer, which are ROS and OE in the aetiology of neoplasms such as breast, colon or rectal cancer, which are related to lipid peroxidation and toxic aldehyde formation.\u003c/p\u003e\n\u003cp\u003eThe study on the potential of lichens as antioxidants and anticancer is beginning to have great potential as seen in the study of Kosanić et al (2023), that analyzed the antioxidant, antimicrobial, cytotoxic and anti-inflammatory properties of the acetone extract of the lichen \u003cem\u003ePlatismatia glauca\u003c/em\u003e (PGAE). Seven bioactive compounds were identified, including salazinic acid, physodic acid and atranorin. Their antioxidant capacity was evaluated with the DPPH method. Regarding its cytotoxic activity, the extract showed the highest efficacy against human epithelial carcinoma cells. These findings highlight the potential of lichens as a source of bioactive compounds with possible therapeutic applications.\u003c/p\u003e\n\u003cp\u003eIn this respect, phenolic compounds may attenuate carcinogenic potential by uptake of ROS and arrest of lipid peroxidation. In fact, a positive correlation between phenolic content and cytotoxic activities against carcinogenic cell lines has been described for some cell lines. Moreover, some of them (such as usnic acid) might show variable antioxidant or pro-oxidant properties, depending on different system conditions and/or cellular environment. Therefore, the cytotoxic actions of our extracts, related to their composition with active principles especially as usnic acid or physodic acid could be explained through their antioxidant activities as previously evaluated (Paluszczak J 2018; Studzińska-Sroka E 2021; Studzińska-Sroka E 2016; Cakmak K 2019; Maulidiyah M 2020; Mitrovic T 2011; Ara\u0026uacute;jo AAS 2015; Rabelo TK 2012).\u003c/p\u003e\n\u003cp\u003eOur findings seem to expand the information that is beginning to exist on the cytotoxic, antioxidant and tumor effect that is being discovered as part of the activity of various lichen species. It is important to be able to standardize the composition of lichen ex-tracts since in the case of phenolic compounds derived from lichens such as depsides and depsidones but also anthraquinones, dibenzofurans and xanthones, it seems that they could interfere with several cancer cell survival pathways and exert cytotoxic effects against said cells or modulate cellular interactions in the tumor microenvironment (Kello M 2023; Bačkorov\u0026aacute; M 2012; Petrova K 2021; Petrov\u0026aacute; K 2022).\u003c/p\u003e\n\u003cp\u003eThis composition could explain the bioactivity manifested by the extracts we have studied, since the presence of certain components of different extracts, similar or identical to those studied in our work, have been related to their antioxidant activity (Toledo Marante FJ 2016) as well as to different anticancer capacities( (Toledo Marante FJ 2003).\u003c/p\u003e\n\u003cp\u003eAs mentioned at the beginning, the Parmeliaceae family is attracting increasing attention for its possible anti-cancer activity, even in breast cancer cell lines (Nugraha AS 2020; Gandhi AD 2021; Ari F 2015). However, there is still not much information about the anti-cancer effects of pure compounds obtained from lichens. Among the most investigated is usnic acid, on which in vitro and research animal studies have allowed it to begin to be taken into account for its use as an anticancer agent (Yildirim M 2022; Bessadottir M 2012; Bačkorov\u0026aacute; M 2011; Galanty A 2017; Din\u0026ccedil;soy AB 2017). There is also a large literature that looks at the composition of whole extracts to see how the different components could be held responsible for the activity. HPLC and TLC are commonly used methods in the study of natural product extracts, especially for those with a chemical composition that is not extraordinarily complex. There is also a large literature that analyses the composition of whole extracts to see how the different components of the activity could be held accountable (Fernandez-Moriano C 2015; Hawrył A 2020; Manojlović NT 2010; Bhattarai HD 2008). The secondary metabolite content of our most biologically active extracts has been studied by different analytical methods. Our research group determined the chemical composition of the most promising extracts in terms of antioxidant activity by high-performance liquid chromatography (HPLC). The presence of interesting molecules consistent with previous literature was observed, even using alternative methods, such as physodic acid, physodalic acid, atranorin, lecanoric acid, salazinic acid and usnic acid (G\u0026oacute;mez-Serranillos MP 2014; Fernandez-Moriano C 2016; Fernandez-Moriano 2015).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cem\u003eHypogymnia physodes\u003c/em\u003e revealed that the most prominent components in its composition are physodalic acid, physodic acid and atranorin, which may help explain the origin of its protective activity ( Studzińska-Sroka E 2021; Studzińska-Sroka E 2016; Toledo Marante FJ 2016; Bessadottir M 2012; Bačkorov\u0026aacute; M 2011; Galanty A 2017; Din\u0026ccedil;soy AB 2017; Studzińska-Sroka E 2019; Meysurova AF 2020; Stojanović IŽ 2014; Kosanić M 2019). As can be seen in the bibliography, physodalic and physodic acids have demonstrated anticancer activity in various experiments with different cell lines (Cardile V 2017; Kosanic M 2013; Stojanović IZ 2014; Stojanović IZ 2013), as well as the antioxidant and antitumor activity of these components, mainly physodic acid (Cardile V 2017; Studzińska-Sroka E 2016; Petrova K 2021; Stojanović IŽ 2014; Paluszczak J 2018; Emsen B 2016; Poulsen-Silva E 2023; Yildirim M 2022; Bessadottir M 2012; Bačkorov\u0026aacute; M 2011). On the other hand, atranorin showed significant antineoplastic effects in the 4T1 breast cancer allograft model in BALB/c mice. Since it significantly increased the survival time of tumor-bearing animals, reduced tumor volume and had a more direct proapoptotic than antiproliferative effect on tumor cells. This study is especially relevant to our hypotheses since this metabolite protected the livers of mice with tumors against oxidative stress (Sol\u0026aacute;r P 2016)].\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eIn the case of \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e, our analyzes corroborated the bibliographic evidence on its composition, among which it stands out for containing lecanoric acid and atranorin has been seen in other species of the same genus, where its antitumor action even on breast cancer has been proven (Harikrishnan A 2021; Ghate NB 2013), as well as its antioxidant activity (Shameera Ahamed TK 2019). Lecanoric acid has shown anticancer activities in cell lines such as colon cancer cells (Goga M 2019) and atranorin against breast cancer (Verma S 2023), lung cancer (Bačkorov\u0026aacute; M 2012) or glioblastoma cells (Petrova K 2021)]. Antitumoral activity of same genus appears in recent publications (Mallavadhani UV 2019) as well as new components [Huynh BLC 2021; Linh B 2016; Saha S 2021; Duong TH 2015). Our species under study, \u003cem\u003eP. pseudotinctorum\u003c/em\u003e, showed antioxidant activity thanks to its content of atranorin and lecanoric acid.[Kekuda TR 2009]\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eIn the composition of \u003cem\u003eUsnea subfloridana\u003c/em\u003e, the most important compounds are usnic acid and salazinic acid. The genus Usnea has been one of the most studied genera among the lichens and its various biological and pharmacological properties, including antioxidant and cytotoxicity, have been investigated (Petrov\u0026aacute; K 2022; Bui VM 2022; Tram NTT 2020; Tuong TL 2020; Salgado F 2017; Londo\u0026ntilde;e-Bailon P 2019). Among the most studied species is \u003cem\u003eUsnea barbata\u003c/em\u003e, with numerous publications to verify its composition, and more recently its antioxidant and cytotoxic activities (Popovici V 2022; Tang JY 2020; Popovici V 2021; Popovici 2021b). As mentioned above, usnic acid has been the subject of considerable attention especially in anticancer treatments (Petrov\u0026aacute; K 2022; Bessadottir M 2012; Bačkorov\u0026aacute; M 2011; Galanty A 2017; Din\u0026ccedil;soy AB 2017; Kumari 2023), it has shown cytotoxic bioactivity against cancer in many types of cancer (cervical cancer cells (Petrov\u0026aacute; K 2022), gastric cancer cells (Toledo Marante FJ 2016) and breast cancer cells in experiments with usnic acid derivatives ( Toledo Marante FJ 2003). Salazinic acid includes antibacterial, antioxidant and cytotoxic properties, measured in different lichens and extracts (G\u0026oacute;mez-Serranillos MP 2014), and has demonstrated its ability to exert cytotoxic bioactivity against many types of cancer, such as colorectal cell lines and melanoma cell lines (Studzińska-Sroka E 2016; Kumar J 2014; Fuchs-Tarloysky V 2013). However, there are few studies that include our species under study (\u003cem\u003eUsnea subfloridana\u003c/em\u003e), except in inflammation [Nguyen TT 2021], but its phylogenetic proximity makes us suspect its possible involvement in one or more properties already found in its genus. Our research has shown that this hypothesis was true for the antioxidant activity and cytotoxic action against cancer cells.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eOn \u003cem\u003eLethariella\u003c/em\u003e, there is still little research on them, especially on anti-cancer activity [Ren MR 2009]. The studies have focused more on \u003cem\u003eL. canariensis\u003c/em\u003e where its composition has been further investigated (canarione, atranol, chloroatranol, hematommic acid, chlorohematommic acid, methylhematommate, methylchlorohematommate, ethylhematommate, ethylchlorohematommate, methyl-orsellinate, atranorin, chloroatranorin and usnic acid) (Toledo Marante FJ 2016; Toledo Marante FJ 2003) and, because of these findings, its possible biological activities have been postulated. Our findings in \u003cem\u003eL. intricata\u003c/em\u003e and \u003cem\u003eL. canariensis\u003c/em\u003e represent a breakthrough in the study of the pharmacological properties of this genus.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eHowever, it should not be forgotten that natural products are complex mixtures that include compounds that can act synergistically and whose activity is not only due to the sum of the activities of the isolated compounds. It is extremely interesting to find biological activities in complete extracts, especially if, when their composition is subsequently analyzed, it is found that they include molecules with already proven activity on what is being studied, since it reinforces the reasoning that would explain their bioactivity. On other occasions, we manage to isolate and identify new components whose study represents an important advance in the search for compounds of natural origin with pharmacological activity that can be a source of new drugs.\u003c/p\u003e\n\u003cp\u003eTaking all the above results together, the antioxidant and anticarcinogenic activity of the species studied is well represented and provides an important starting point for further studies to determine the mechanisms and pathways by which these are produced.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn the present study, methanolic extracts of five lichens species have been studied by evaluating their antioxidant and free radical scavenging capacity and their cytotoxic activity on human carcinoma cell lines. This study revealed that these lichen species have a high antioxidant capacity. The lichen extracts were found to contain a considerable amount of phenolic compounds responsible for their high antioxidant and anti-free radical power, and as a natural source of antioxidants to ameliorate oxidative stress-related disorders. Their cytotoxic action was investigated in MCF7 and HepG2 cell lines, with all extracts showing clear cytotoxicity at a wide range of concentrations tested. This opens an interesting field for further studies to clarify whether the % decrease in cell viability is due to an increase in cell lethality due to the effect of the extracts, a decrease in cell proliferation or a combination of both.\u003c/p\u003e \u003cp\u003eUltimately, the three lichen extracts with the best results in bioactivity were subjected to high performance liquid chromatography in order to identify the secondary metabolites most commonly present in each of them, thus allowing attributing the antioxidant activity of well-defined chemical compounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Sources\u003c/h2\u003e \u003cp\u003eThis study was supported by the Spanish Ministry of Science, Innovation and Universities (PID2019-105312GB-100)\u003c/p\u003e \u003cp\u003eConflicts of Interest: The authors declare no conflicts of interest.\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eConceptualization, M.P.G.-S., M.S; methodology, M.S.; validation M.S, M.V.N.; and M.P.G.-S.; data curation, M.S and M.T.; writing\u0026mdash;original draft preparation, M.S, M.V.N; J.A.V.-G. and M.T; writing\u0026mdash;review and editing, M.P.G.-S., M.V.N., P.K.D. and M.S.; visualization, M.P.G.-S.; supervision, M.P.G.-S. and M.S.; project administration, P.K.D and M.P.G.-S.;. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlbuquerque RC, Baltar VT, Marchioni DM (2014) Breast cancer and dietary patterns: a systematic review. Nutr Rev 72:1\u0026ndash;17\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlexandrino CA (2019) Antitumor effect of depsidones from lichens on tumor cell lines and experimental murine melanoma. Rev Bras Farmacogn 29:449\u0026ndash;456\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmarowicz R, Pegg RB, Rahimi-Moghaddam P, Barl B, Weil JA (2004) Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. 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Sci Rep 7:8136\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cancer, lichen, antioxidant activity, MCF7, HepG2","lastPublishedDoi":"10.21203/rs.3.rs-6922462/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6922462/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLichens produce a variety of secondary metabolites with important biological properties. This study analyzed the antioxidant activity and cytotoxic potential of selected \u003cem\u003eParmeliaceae\u003c/em\u003e lichens using in vitro methods and HPLC analysis. Methanolic extracts were obtained from five species: \u003cem\u003eHypogymnia physodes\u003c/em\u003e (L.), \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e (Abbayes), \u003cem\u003eUsnea subfloridana\u003c/em\u003e (Stirt.), \u003cem\u003eLethariella canariensis\u003c/em\u003e(Ach.) and \u003cem\u003eLethariella intricate\u003c/em\u003e (Moris). Their antioxidant capacities were assessed through DPPH, FRAP, ORAC, and Folin tests, while cytotoxicity was examined in breast adenocarcinoma (MCF7) and hepatocellular carcinoma (HepG2) cell lines via MTT viability assay.Results showed significant antioxidant activity, with \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e exhibiting the highest ORAC and Folin values. Regarding cytotoxicity, \u003cem\u003eUsnea subfloridana\u003c/em\u003e and \u003cem\u003eHypogymnia physodes\u003c/em\u003e demonstrated notable growth inhibition in MCF7 and HepG2, particularly at intermediate concentrations. \u003cem\u003eParmotrema pseudotinctorum\u003c/em\u003e showed moderate inhibition, more pronounced at lower doses. HPLC analysis identified bioactive compounds such as physodic acid, lecanoric acid, and usnic acid, which correlate with the observed antioxidant and cytotoxic activities. These findings highlight lichens as a potential source of therapeutic secondary metabolites, warranting further research to explore their medicinal applications.\u003c/p\u003e","manuscriptTitle":"Evaluation of the antioxidant activity and Citotoxic potential of lichen forming fungal species of the family Parmeliaceae (Ascomycota)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-01 10:55:36","doi":"10.21203/rs.3.rs-6922462/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-06-26T18:00:31+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-26T13:27:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-20T05:20:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-06-19T03:47:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"87b5bc78-3f2e-4d48-9f9c-f403582e33e6","owner":[],"postedDate":"July 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-01T10:55:36+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-01 10:55:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6922462","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6922462","identity":"rs-6922462","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}