Parietin, the Vibrant Natural Dye in Xanthoria parietina.

The use of natural dyes in several areas is regulated by current European and non-European legislation, due to various problems with synthetic dyes. The analysis revealed that the lichen studied: Xanthoria parietina has potential natural dye sources and provides bright colors for extraction solvents. Furthermore, dyed wool and toile fabric have good fastness properties in ammonia fermentation and boiling water, both with and without mordants. The sample dyes with Xanthoria parietina were characterized by several analytical techniques: high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization with tandem mass spectrometry (HPLC-ESI-Q-ToF). As compounds from Xanthoria parietina form a complex with mordants and tissues, it is impossible to identify the molecules responsible for coloring using chromatographic techniques. However, we have evaluated the dyeing power of their major molecule, parietin. To further confirm the coloring power of the isolated parietin molecule, we performed a dye test with pure parietin. Thus, CIALAB analyses have shown parietin is the molecule responsible for the coloring obtained by Xanthoria parietina. The utilization of parietin derived from lichens facilitates the development of sustainable dyes for textile coloring, presenting an environmentally friendly alternative to synthetic dyes while simultaneously enriching lichen biodiversity.

The Roles of the Anthraquinone Parietin in the Tolerance to Desiccation of the Lichen Xanthoria parietina: Physiology and Anatomy of the Pale and Bright-Orange Thalli.

Lichens are symbiotic organisms that effectively survive in harsh environments, including arid regions. Maintaining viability with an almost complete loss of water and the rapid restoration of metabolism during rehydration distinguishes lichens from most eukaryotic organisms. The lichen Xanthoria parietina is known to have high stress tolerance, possessing diverse defense mechanisms, including the presence of the bright-orange pigment parietin. While several studies have demonstrated the photoprotective and antioxidant properties of this anthraquinone, the role of parietin in the tolerance of lichens to desiccation is not clear yet. Thalli, which are exposed to solar radiation and become bright orange, may require enhanced desiccation tolerance. Here, we showed differences in the anatomy of naturally pale and bright-orange thalli of X. parietina and visualized parietin crystals on the surface of the upper cortex. Parietin was extracted from bright-orange thalli by acetone rinsing and quantified using HPLC. Although acetone rinsing did not affect PSII activity, thalli without parietin had higher levels of lipid peroxidation and a lower membrane stability index in response to desiccation. Furthermore, highly pigmented thalli possess thicker cell walls and, according to thermogravimetric analysis, higher water-holding capacities than pale thalli. Thus, parietin may play a role in desiccation tolerance by stabilizing mycobiont membranes, providing an antioxidative defense, and changing the morphology of the upper cortex of X. parietina.

Synthesis and Antioxidant Evaluation of O-Methylated Emodacidamides: Starting from Parietin, a Secondary Metabolite of Lichen Xanthoria parietina.

Polyhydroxy-anthraquinones bearing amino acids are found rather seldom in nature. Emodacidamides, isolated from a marine-derived fungus, Penicillium sp. SCSIO sof101 by Luo et al. (2017) are the first natural example of amino acid conjugated anthraquinone. In this study, O-methylated emodacidamides and emodinic acid-anilides were synthesized starting from parietin, extracted from the lichen Xanthoria parietina (L.) Th. Fr. The structural elucidations of prepared compounds were confirmed by 1D and 2D NMR analyses including HSQC and HMBC techniques. In addition, all newly synthesized compounds were evaluated for the antioxidant activities with free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging. The synthesized compounds showed low to moderate antioxidant and DPPH scavenging activities. The antioxidant activities were supported within quantum chemical calculations using the DFT-B3LYP/6-311++G(d,p) level of theory. It is observed that the antioxidant activity of emodacidamides mostly depends on the phenolic groups on anthraquinone ring. The phenolic groups on other substituents help to improve antioxidant activity and also the position of hydroxy group is a decisive factor for antioxidant ability.

Anti-Inflammatory Potential of Compounds Isolated from Tunisian Lichens Species.

The lichen's special symbiotic structure enables it to produce bioactive substances. They have historically been recognized for their aesthetic and medicinal benefits. Furthermore, in recent years, they have performed in various fields, including perfumery, dyeing, and pharmacology due to their rich secondary metabolites. From our study, four compounds were isolated from organic extracts of Parmotrema hypoleucinum, Roccella phycopsis, and Xanthoria parietina and identified by spectroscopic investigation as atranorin, (+)-iso-usnic acid, methyl orsellinate, and parietin, respectively. The anti-inflammatory effects of lichens extracts, and pure compounds were evaluated on RAW 264.7 macrophages cells at different concentrations. At 25 µg/mL all treated samples did not show any effect on cell viability. Atranorin and (+)-iso-usnic acid showed an inhibitory effect on nitric oxide (NO) levels in lipopolysaccharide (LPS)-stimulated macrophages. Nitric oxide (NO) production was measured using Griess reagent, atranorin and (+)-iso-usnic acid showed a high anti-inflammatory potential (75.99 % and 57.27 % at 25 µg/mL). On the other hand, methyl orsellinate and the organic extracts of three lichens showed good anti-inflammatory activity ranging from 29.16 % at 25 µg/mL to 86.91 % at 100 µg/mL.

Can Parietin Transfer Energy Radiatively to Photosynthetic Pigments?

The main role of lichen anthraquinones is in protection against biotic and abiotic stresses, such as UV radiation. These compounds are frequently deposited as crystals outside the fungal hyphae and most of them emit visible fluorescence when excited by UV. We wondered whether the conversion of UV into visible fluorescence might be photosynthetically used by the photobiont, thereby converting UV into useful energy. To address this question, thalli of Xanthoria parietina were used as a model system. In this species the anthraquinone parietin accumulates in the outer upper cortex, conferring the species its characteristic yellow-orange colouration. In ethanol, parietin absorbed strongly in the blue and UV-B and emitted fluorescence in the range 480⁻540 nm, which partially matches with the absorption spectra of photosynthetic pigments. In intact thalli, it was determined by confocal microscopy that fluorescence emission spectra shifted 90 nm towards longer wavelengths. Then, to study energy transfer from parietin, we compared the response to UV of untreated and parietin-free thalli (removed with acetone). A chlorophyll fluorescence kinetic assessment provided evidence of UV-induced electron transport, though independently of the presence of parietin. Thus, a role for anthraquinones in energy harvesting is not supported for X. parietina under presented experimental conditions.

Raman spectroscopic analysis of the effect of the lichenicolous fungus Xanthoriicola physciae on its lichen host.

Lichenicolous (lichen-dwelling) fungi have been extensively researched taxonomically over many years, and phylogenetically in recent years, but the biology of the relationship between the invading fungus and the lichen host has received limited attention, as has the effects on the chemistry of the host, being difficult to examine in situ. Raman spectroscopy is an established method for the characterization of chemicals in situ, and this technique is applied to a lichenicolous fungus here for the first time. Xanthoriicola physciae occurs in the apothecia of Xanthoria parietina, producing conidia at the hymenium surface. Raman spectroscopy of apothecial sections revealed that parietin and carotenoids were destroyed in infected apothecia. Those compounds protect healthy tissues of the lichen from extreme insolation and their removal may contribute to the deterioration of the apothecia. Scytonemin was also detected, but was most probably derived from associated cyanobacteria. This work shows that Raman spectroscopy has potential for investigating changes in the chemistry of a lichen by an invading lichenicolous fungus.

Secondary Metabolites in Ramalina terebrata Detected by UHPLC/ESI/MS/MS and Identification of Parietin as Tau Protein Inhibitor.

Liquid chromatography coupled with mass spectrometry is an outstanding methodology for fast analysis of phenolic compounds in biological samples. Twenty two compounds were quickly and accurately identified in the methanolic extract of the Antarctic lichen Ramalina terebrata for the first time using ultra high pressure liquid chromatography coupled with photodiode array detector and high resolution mass spectrometry (UHPLC-PDA-Q/Orbitrap/MS/MS). In addition, the extract and the four compounds isolated from this species were tested for the inhibitory activity of tau protein aggregation, which is a protein involved in Alzheimer's disease (AD). All compounds showed null activity with the exception of parietin, which it was able to inhibit aggregation process of tau in a concentration range between 3 µg/mL (10 µM) to 28 µg/mL (100 µM). In addition, we show how parietin interact with tau (306)VQIVYK(311) hexapeptide inside of the microtubule binding domain (4R) with the help of molecular docking experiments. Finally, the constituents present in the methanolic extract could possibly contribute to the established anti-aggregation activity for this extract and this in-depth analysis of the chemical composition of R. terebrata could guide further research into its medicinal properties and potential uses.

UV photo-degradation of the secondary lichen substance parietin: A multi-spectroscopic analysis in astrobiology perspective.

The cortical anthraquinone yellow-orange pigment parietin is a secondary lichen substance providing UV-shielding properties that is produced by several lichen species. In our work, the secondary metabolite has been extracted from air-dried thalli of Xanthoria parietina. The aims of this study were to characterize parietin absorbance through UV-VIS spectrophotometry and with IR spectroscopy and to evaluate its photodegradability under UV radiation through in situ reflectance IR spectroscopy to understand to what extent the substance may have a photoprotective role. This allows us to relate parietin photo-degradability to the lichen UV tolerance in its natural terrestrial habitat and in extreme environments relevant for astrobiology such as Mars. Extracted crystals were UV irradiated for 5.59 h under N2 flux. After the UV irradiation, we assessed relevant degradations in the 1614, 1227, 1202, 1160 and 755 cm-1 bands. However, in light of Xanthoria parietina survivability in extreme conditions such as space- and Mars-simulated ones, we highlight parietin UV photo-resistance and its relevance for astrobiology as photo-protective substance and possible bio-hint.

DNA protection, molecular docking, antioxidant, antibacterial, enzyme inhibition, and enzyme kinetic studies for parietin, isolated from Xanthoria parietina (L.) Th. Fr.

Parietin was isolated from Xanthoria parietina (L.) Th. Fr.' (methanol:chloroform) extract, using a silica column. 13 C NMR and 1H NMR were used to confirm the structure of the isolated parietin. For the first time, parietin was investigated for its antioxidant, antibacterial and DNA protective activities. Molecular docking was carried out to determine the binding affinity and interactions between the enzymes and our molecule. Inhibition and kinetic mechanism studies for the action of the enzymes were performed too. Parietin exhibited high metal chelating activity. The MIC values of parietin were sufficient to inhibit different bacterial strains; E. coli, P. aeruginosa, K. pneumoniae and S. aureus. Molecular docking applications exhibited that acetylcholinesterase (AChE), butyrylcholinesterase (BChE), lipase, and tyrosinase have high potential for binding with the parietin. Especially, the parietin's highest binding affinity was recorded with AChE and tyrosinase. These results were confirmed by the inhibition and kinetics results, where, parietin observed a potent inhibition with an IC50 values between 0.013-0.003 µM. Moreover, parietin acts' as a non-competitive inhibitor against AChE, BChE, and lipase, and as a competitive inhibitor against tyrosinase with a high rate of inhibition stability. The promising biological properties of parietin revealed its effectiveness in terms of suitability in the food and pharmaceutical industries.Communicated by Ramaswamy H. Sarma.

Photodynamic and antiangiogenic activities of parietin liposomes in triple negative breast cancer.

Parietin (PTN) is an anthraquinone with promising efficacy in the inhibition of cancer cell proliferation and tumor growth. Due to its hydrophobicity, PTN is sparingly soluble under physiological conditions and has a low bioavailability. Hence, we presented PTN in liposomes to overcome these drawbacks. The prepared liposomes were characterized and their stability was also assessed in serum. Singlet oxygen quantum yield of PTN loaded liposomes was indirectly quantified using uric acid. The intracellular uptake of liposomes was studied by CLSM which indicated the perinuclear localization of PTN liposomes. Cellular viability assay and live/dead staining demonstrated both light and dose-dependent phototoxicity of PTN on the human breast cancer cell line. The mechanism of cellular uptake was investigated using different pathway inhibitors and the results showed that clathrin-mediated endocytosis is predominant. The colocalization experiment indicated that PTN is localized in both mitochondria and lysosomes. These findings together with flow cytometry analysis elucidated that apoptosis is the main mechanism underlying cell death post-PDT. Finally, the antiangiogenic effect of PTN liposomes was further evaluated in the chorioallantoic membrane (CAM) model and the results indicated that PDT induced vascular response was confined to the irradiated area leaving the non-irradiated unscathed.

The Raman Active Vibrational Modes of Anthraquinones.

Anthraquinones are a family of natural products with useful bioactivity and optical properties. An anthraquinone called parietin is produced by extremophiles to protect against solar ultraviolet B radiation, so it is a potential biosignature in astrobiology. Raman spectroscopy, which is now used in space environments, can detect molecules such as parietin based on molecular vibrations. In this study, we show that time-dependent density functional theory (TDDFT) can accurately calculate the Raman spectra of three dihydroxyanthraquinones: parietin, emodin, and chrysophanol. By comparing calculated spectra to measured Raman spectra from purified powders, 10 vibrational modes are identified. The detailed molecular motions of these fused ring vibrations are described, and vibrations modes that are common to all three molecules are highlighted. In addition to powder spectra, Raman measurements from the thallus of Xanthoria parietina, a lichen that produces parietin, are reported, with excellent agreement to both the parietin powder and calculated Raman spectra. These results show that TDDFT calculations could make significant contributions to spectral analysis in the search for biotic organic materials beyond Earth.

The Effect of Parietin Isolated From Rheum ribes L on In Vitro Wound Model Using Human Dermal Fibroblast Cells.

Parietin is one of the well-known anthraquinone compounds that can be extracted from Rheum ribes L. In this study, we aimed to investigate the effects of parietin isolated from Rheum ribes L on an in vitro wound model using human dermal fibroblast cells and compare its effectiveness against zinc. The antioxidant effect of parietin was determined by using the 1,1-diphenyl-2-picrylhydrazine (DPPH) method. Human dermal fibroblast cells were cultured in proculture medium and were kept until 100% confluence was achieved. The wound model was created by using a pipette tip. After that, different concentrations of parietin and zinc (final concentrations in the well to be 5-250 µM and 25-200 µM, respectively) were added into the medium. The proliferation-inducing effect on cell viability was determined by using MTT assay. Images of cells were taken at 0, 12, and 24 hours. According to the DPPH method, parietin exhibited have antioxidant activity. According to the MTT results, parietin exhibited significant proliferation-inducing effect on cell viability in a dose range of 5 to 10 M, and zinc showed significant proliferation-inducing effect on cell viability at dose 50 µM ( P < .05). In addition, the image of cell proliferation was also shown at the same doses at 24 hours. In this study, we claim that parietin induces cell proliferation at low doses in cases of dermal fibroblast loss. In conclusion, parietin as an alternative to zinc in wound healing could be used by clinicians in the future with more extensive studies.

Physiological and ultrastructural effects of acute ozone fumigation in the lichen Xanthoria parietina: the role of parietin and hydration state.

The physiological and ultrastructural effects induced by acute exposure to ozone (O3) were investigated in the lichen Xanthoria parietina. Our working hypothesis was that parietin content and hydration of the thalli may play a role in the modulation of the effects of O3 exposure. Four batches of X. parietina samples, dry and wet, with (P+) and without (P-) parietin, were fumigated for 1 h with 3 ppm O3. The effects of O3 were assessed immediately after the fumigation and after one week of recovery under controlled conditions. O3 fumigation caused physiological and ultrastructural impairment both to the photobiont and the mycobiont, irrespective if samples were fumigated wet or dry, and P+ or P-. However, one week after fumigation, a recovery was observed in P+ samples for the photobiont and in dry samples for the mycobiont. We suggest that the hydration state may play a major role in determining the severity of the damage, while the presence of parietin may promote the recovery. Our results provide physiological and ultrastructural basis to explain the ecological insensitivity of lichens to high environmental levels of ozone occurring during dry Mediterranean summers.

Detection and aggregation of the antitumoral drug parietin in ethanol/water mixture and on plasmonic metal nanoparticles studied by surface-enhanced optical spectroscopy: Effect of pH and ethanol concentration.

In the present paper, we have investigated the effect of ethanol in aqueous media, the pH and the presence of Ag nanoparticles (NPs) on the aggregation processes of the antitumoral anthraquinone parietin in aqueous media and on the metal surface. UV-visible absorption, fluorescence and Raman spectra of parietin were used for such purpose. The present study provides information about the deprotonation and molecular aggregation processes occurring in parietin under different environments: ethanol/water mixture and when adsorbed onto Ag nanoparticles. The effect of ethanol on the optical properties of parietin in alcohol-water mixtures was also investigated at different ethanol concentrations with the time. For the case of the adsorption and organization of parietin molecules on the surface of Ag NPs, special attention was paid to the use of surface-enhanced optical techniques, SEF (surface-enhanced fluorescence) and SERS (surface-enhanced Raman scattering), for the characterization of the parietin aggregates and the ionization of the molecule on the surface. In particular, we have studied the variation of the SEF signal with the pH, which depends on the molecular organization of the molecule on the surface. Furthermore, a detailed analysis of the SERS spectra at different pH was accomplished and the main Raman bands of the protonated, mono-deprotonated and di-deprotonated parietin were identified. Finally, the second ionization pK of parietin on metal NPs was deduced from the SERS spectra.

Parietin as an efficient and promising anti-angiogenic and apoptotic small-molecule from Xanthoria parietina

ABSTRACT Lichens that are exclusive symbiotic organisms composed of fungus and alga, are considered as a wealthy source of biologically and pharmacologically active small-molecules thanks to the tight metabolic relationship between symbiotic partners. We herein report cytotoxic, anti-angiogenic and apoptotic profile of a lichen derived small-molecule named parietin. Parietin was isolated from the acetone extract of Xanthoria parietina (L.) Th.Fr (1860), Teloschistaceae, which was gathered from Afyon, Turkey. AlamarBlue™ cell viability, lactate dehydrogenase membrane leakage and PicoGreen™ dsDNA quantitation assays were used to determine the cytotoxic concentrations of parietin on cisplatin-resistant BRCA2-mutated human breast TNM stage IV adenocarcinoma (HCC1428), human breast ductal carcinoma (T-47D), and human umbilical vein endothelial (HUVEC) cells. Additionally, cell adhesion, endothelial tube formation, reactive oxygen species accumulation and active caspase 3 determination assays were employed to identify the anti-angiogenic and apoptotic efficiency of parietin. Low concentrations of parietin such as 50 and 100 µM showed a significant anti-angiogenic and apoptotic activity though the half-maximal inhibitory concentration (IC50) values were higher than 600 µM on the cells. On the other hand, it was observed that parietin shows less cytotoxic and membrane degradative activities on healthy HUVEC cells than the HCC1428 and T-47D breast cancer cells. Parietin seems to be a promising anti-angiogenic and apoptotic lichen metabolite for the further investigations.