key: cord-0015827-n5vxc71z
authors: Kerboua, Marwa; Ahmed, Monia Ali; Samba, Nsevolo; Aitfella-Lahlou, Radhia; Silva, Lucia; Boyero, Juan F.; Raposo, Cesar; Lopez Rodilla, Jesus Miguel
title: Phytochemical Investigation of New Algerian Lichen Species: Physcia Mediterranea Nimis
date: 2021-02-20
journal: Molecules
DOI: 10.3390/molecules26041121
sha: 2404e78deb7a46d946cdea7f460508fe7e525c5c
doc_id: 15827
cord_uid: n5vxc71z

The present study provides new data concerning the chemical characterisation of Physcia mediterranea Nimis, a rare Mediterranean species belonging to the family Physciaceae. The phytochemical screening was carried out using GC-MS, HPLC-ESI-MS-MS, and NMR techniques. Hot extraction of n-hexane was carried out, followed by separation of the part insoluble in methanol: wax (WA-hex), from the part soluble in methanol (ME-hex). GC-MS analysis of the ME-hex part revealed the presence of methylbenzoic acids such as sparassol and atraric acid and a diterpene with a kaurene skeleton which has never been detected before in lichen species. Out of all the compounds identified by HPLC-ESI-MS-MS, sixteen compounds are common between WA-hex and ME-hex. Most are aliphatic fatty acids, phenolic compounds and depsides. The wax part is characterised by the presence of atranorin, a depside of high biological value. Proton 1H and carbon 13C NMR have confirmed its identification. Atranol, chloroatranol (depsides compound), Ffukinanolide (sesquiterpene lactones), leprolomin (diphenyl ether), muronic acid (triterpenes), and ursolic acid (triterpenes) have also been identified in ME-hex. The results suggested that Physcia mediterranea Nimis is a valuable source of bioactive compounds that could be useful for several applications as functional foods, cosmetics, and pharmaceuticals.

From the Greek word, "leikhen" lichen was first used to designate a plant in the 4th century BC by Theophraste [1] . This small organism has been integrated into the fungal kingdom and results from the symbiotic association of a fungus called mycobiont and a green alga or a cyanobacterium called photobiont [2, 3] . An estimated more than 17,000 species of lichen exist today, extending from the tropics to the polar regions [4] . The symbiosis gives lichens a specific structure and reproduction to each constituent alone. Unlike higher plants, they have no root, stem, or leaf, but a rudimentary vegetative apparatus: the thallus [5] . Moreover, they grow on a wide variety of substrates including soil, bark, bare rock surfaces, leaves of vascular plants, barnacle shells, and other lichens [4] . organic lichen extracts from Physcia mediterranea Nimis using GC-MS, followed by HPLC-ESI-MS-MS and NMR analysis. 

The GC-MS analysis of ME-hex extract is shown in Figure 2 . The analysis allowed us to detect ten components. Nine products were identified by comparing their mass spectra with reference spectra from the NIST and Wiley databases ( Table 1 ). The major chemical compounds identified in ME-hex from Physcia mediterranea included derivatives of methylbenzoic acids (56%) and free fatty acids (36.1%). A diterpene with a Kauran skeleton ((−)-ent-Kauran-16a-ol) ( Figure 3 ) was also identified (3.8%) [18] . Only 2.2% of the entire extract was not identified. 

The GC-MS analysis of ME-hex extract is shown in Figure 2 . The analysis allowed us to detect ten components. Nine products were identified by comparing their mass spectra with reference spectra from the NIST and Wiley databases ( Table 1 ). The major chemical compounds identified in ME-hex from Physcia mediterranea included derivatives of methylbenzoic acids (56%) and free fatty acids (36.1%). A diterpene with a Kauran skeleton ((−)-ent-Kauran-16a-ol) ( Figure 3 ) was also identified (3.8%) [18] . Only 2.2% of the entire extract was not identified.

Molecules 2021, 26, x FOR PEER REVIEW 2 of 5 organic lichen extracts from Physcia mediterranea Nimis using GC-MS, followed by HPLC-ESI-MS-MS and NMR analysis. 

The GC-MS analysis of ME-hex extract is shown in Figure 2 . The analysis allowed us to detect ten components. Nine products were identified by comparing their mass spectra with reference spectra from the NIST and Wiley databases ( Table 1 ). The major chemical compounds identified in ME-hex from Physcia mediterranea included derivatives of methylbenzoic acids (56%) and free fatty acids (36.1%). A diterpene with a Kauran skeleton ((−)-ent-Kauran-16a-ol) ( Figure 3 ) was also identified (3.8%) [18] . Only 2.2% of the entire extract was not identified. Derivatives of methylbenzoic acids are commonly present in different genera of lichen-like Stereocaulon halei [51] , Parmotrema mesotropum [52] , Cassipourea malosana [53] , Cetraria islandica [54] , Usnea longissima [55, 56] , Stereocaulon paschale [57] , and Parmelia sulcata Taylor [58] . Atraric acid (Figure 2 ), widely present in some species such as Hypogymnia physodes, Evernia prunastri, and Parmelia sulcata, growing on the same host tree (Prunus domestica) [59] , can be in the free or complexed form and serves as a basis for the composition of depsides and depsidones [60] . It is considered a specific antagonist of androgen receptors and therefore inhibits human prostate cancer growth [60, 61] . Atraric acid shows nematocidal, antioxidant, antimicrobial, and anti-inflammatory properties in vitro, and inhibits carrageenan-induced oedema and wound healing activity in vivo [62, 63] . Lichens contain many of the fatty acids commonly found in higher plants [64] . Indeed, many common lichen genera include species with multiple strains of fatty acids [65] [66] [67] . These lipid profiles, most often treated as chemotypes, have been used by many researchers to taxonomically classify certain lichens, such as Cladonia [68] , Lepraria [69] , Parmelia [70] , Tephromela s.lat [71] , and Mycoblastus sanguinarius [72] . Although they are widely present in some genera, fatty acids were of no taxonomic importance and were omitted during the first chemical studies on lichens [72] [73] [74] . By chemical and biochemical comparisons, a mechanistic relationship between polyketide and fatty acid biosynthesis has been recognised. The carbon backbones of the molecules are assembled by successive condensation of acyl units [75] . Another important fatty acid role was cell signal transduction [76] as well as chemical protection. Therefore, they allow the lichens to survive as environmental conditions change [8] [9] [10] .

In our study, palmitic acid is present in high concentration (24%) compared to oleic (4.2%), stearic acid (3.8%), and linoleic (3.2%) acid (Table 1 and Derivatives of methylbenzoic acids are commonly present in different genera of lichen-like Stereocaulon halei [51] , Parmotrema mesotropum [52] , Cassipourea malosana [53] , Cetraria islandica [54] , Usnea longissima [55, 56] , Stereocaulon paschale [57] , and Parmelia sulcata Taylor [58] . Atraric acid (Figure 2 ), widely present in some species such as Hypogymnia physodes, Evernia prunastri, and Parmelia sulcata, growing on the same host tree (Prunus domestica) [59] , can be in the free or complexed form and serves as a basis for the composition of depsides and depsidones [60] . It is considered a specific antagonist of androgen receptors and therefore inhibits human prostate cancer growth [60, 61] . Atraric acid shows nematocidal, antioxidant, antimicrobial, and anti-inflammatory properties in vitro, and inhibits carrageenan-induced oedema and wound healing activity in vivo [62, 63] .

Lichens contain many of the fatty acids commonly found in higher plants [64] . Indeed, many common lichen genera include species with multiple strains of fatty acids [65] [66] [67] . These lipid profiles, most often treated as chemotypes, have been used by many researchers to taxonomically classify certain lichens, such as Cladonia [68] , Lepraria [69] , Parmelia [70] , Tephromela s.lat [71] , and Mycoblastus sanguinarius [72] . Although they are widely present in some genera, fatty acids were of no taxonomic importance and were omitted during the first chemical studies on lichens [72] [73] [74] . By chemical and biochemical comparisons, a mechanistic relationship between polyketide and fatty acid biosynthesis has been recognised. The carbon backbones of the molecules are assembled by successive condensation of acyl units [75] . Another important fatty acid role was cell signal transduction [76] as well as chemical protection. Therefore, they allow the lichens to survive as environmental conditions change [8] [9] [10] .

In our study, palmitic acid is present in high concentration (24%) compared to oleic (4.2%), stearic acid (3.8%), and linoleic (3.2%) acid (Table 1 and Figure 2 ). Various works using different growing conditions explain the variations in fatty acid in lichens [77, 78] . Molina et al. (2003) studied the lichen Physconia distorta and suggested a close relationship between the synthesis of secondary metabolites and fatty acid metabolism. Mycobiota grown in a glucose-enriched medium favoured the production of fatty acids [79] . Another important factor that could influence the production of fatty acids in lichens is temperature. According to several studies, the degree of unsaturation varies with the season and decreases with increasing temperature [80, 81] . In the thallus of Teloschistes flavicans, the saturated fatty acids, palmitic and stearic, were more abundant in February. In contrast, in August, when the average temperature was 23 • C, there was an increase in oleic and linoleic fatty acids [82] .

In addition to the stress due to the decrease in temperature, nitrogen deprivation and light intensity are also known to promote fatty acid accumulation [80, 82, 83] . However, these factors are not necessarily the unique parameters that determine the fatty acid content, but rather genetics, combined with environmental conditions (e.g., altitude, air pollution, seasonal effects), must also be taken into account [67, 84] . (Table 2) using LC/ESI/MS/MS in negative mode. Identifying major compounds was simplified by interpreting their MS/MS spectra, provided in our system resource and comparison with the literature. The 37 compounds identified were mainly paraconic and aliphatic acids, depsides (aromatic polyketides), phenolic compounds, and diterpenes. Only eight compounds could not be identified. The representative chemical structures are presented in Figure S2 . Twenty-nine paraconic and aliphatic acids were identified: peaks 1-2, 5-7, 9, 13-19, 21-24, 26-29, 31, 35, 37, 39, 40, 42 , and 44 using UHPLC-ESI-MS-MS analysis [13, 18] . Among these compounds is fumaric acid ( Figure S2 ), a valuable compound used in food, beverages, detergents, animal feed, pharmaceuticals, and various industrial products [85] [86] [87] [88] .

Similarly, for traumatic acid, a phytohormone belongs to the class of cytokinins, a study has demonstrated its positive influence on oxidative stress parameters in normal human fibroblasts [89] . It is also effective against breast cancer cells and has potential anticancer properties and tumour prevention activity. Traumatic acid leads to decreased cell proliferation and viability, GSH/GSSG ratio, and thiol group content. It increased caspase activity, membrane lipid peroxidation, and ROS content, simultaneously reducing breast cancer cell growth through the influence of oxidative stress on apoptosis [90] .

Three phenolic compounds were identified and assigned to peaks 4, 8, and 10 using UHPLC-ESI-MS-MS. Peak 4 and peak 10 were identified as p-Coumaric acid 6,7-Dihydroxycoumarin (Esculetin) ( Figure S2 ) whose molecular anions were at m/z 163.0395 and 177.0186, respectively. To our knowledge, this is the first time that compounds such as these have been found in lichen material. They possess diverse biological and pharmacological properties, including anti-asthma, anti-inflammatory, anti-nociceptive, antioxidative, antitumor, and antiviral activities [91] [92] [93] [94] [95] [96] [97] . Gingerol, a phenolic compound ( Figure S2 ), has been identified in the extract WA-hex. Figure S2 ). Atranorin, a derivative of β-orcinol, is one of the most common secondary metabolites of lichen [98] . It is particularly present in the lichen families Cladoniaceae, Lecanoraceae, Parmeliaceae, and Streocaulaceae [99] [100] [101] . In the Physcia genera, there are Physcia caesia [102] , Physcia aipolia [103, 104] , Physcia alnophila [105] , and Physcia sorediosa [106] . In recent years, atranorin has been the most extensively studied. Indeed, it has shown antioxidant [107] , antimicrobial [108] , anti-inflammatory [109] , antinociceptive [110] , wound healing [111] , and photoprotective properties [112] . Additionally, it exerted strong inhibitory effects on cancer cell proliferation, migration, and actin cytoskeleton organisation [113] . Figure S2 ). Likewise for asebotoxin I, a toxic diterpene has been identified at the peak 43 ([M − H] − ion at m/z 425.2581). It was initially and only discovered in the plant Pieris japonica [114] , but our results show the opposite, i.e., that the compound can be detected in lichen species.

In the present study, the analysis of the phytochemical profile of ME-hex using UHPLC-ESI-MS/MS, in negative ion mode, resulted in the detection of 54 significant compounds indicated in Figure S3 . Only four compounds could not be identified. The identified compounds are of paraconic and aliphatic acids, aromatic polyketides (depsides, depsones, and phenyl ethers), phenolic acids, sesquiterpenes lactones, triterpenes, carboxybenzaldehyde, and carboxyphthalide types (Table 3) . [115] . In the lichens group, the sesquiterpene has only been identified in Cetraria islandica [116] .

Finally, two triterpenes, muronic acid at peak 32 and ursolic acid at peak 39, were also identified ( Figure 5 ) and produced major diagnostic MS ions at m/z 390.1314, 456.3605. Muronic acid was previously identified in Usnea rubicunda, Usnea subfloridana [117] , and Punctelia microsticta [118] . On the other hand, ursolic acid (3β-hydroxy-urs-12ene-28-oic acid) is widespread in the vegetable kingdom [119] . The lichenic species Ramalina hierrensis [16] , Ramalina hierrensis [120] , Stereocaulon evolutum [121] , and Pannaria tavaresii [122] also contain this compound. According to the results (Table 3) , four depsides compounds were identified corresponding to peak 4, 11, 23, and 35 in the ME-hex extract. Peak 4 was identified as atranol 

1H and C13 NMR analysed the two samples from WA-hex and ME-hex. The results we obtained reveal the predominance of the secondary metabolite atranorin only in the WA-hex sample. The structure has been characterised, and the NMR spectra are compared with those of a previously isolated sample of Physcia sorediosa [106, 123] , demonstrated in Figures S4 and S5 .

Atranorin ( Figure 6 ) is the most common secondary metabolite in lichens and is mainly found on lichens' surface (cortex) [124] . It acts as a photo-buffer because it reflects harmful UV rays to the thallus's surface and allows the lichens to live in areas receiving intense solar radiation [125] . Zhang et al. (2016) , fukinanolide, also called bakkenolide A, extracted from the plant Petasites tricholobus, showed antiinflammatory properties in the treatment of leukaemia [115] . In the lichens group, the sesquiterpene has only been identified in Cetraria islandica [116] .

Finally, two triterpenes, muronic acid at peak 32 and ursolic acid at peak 39, were also identified ( Figure 5 ) and produced major diagnostic MS ions at m/z 390.1314, 456.3605. Muronic acid was previously identified in Usnea rubicunda, Usnea subfloridana [117] , and Punctelia microsticta [118] . On the other hand, ursolic acid (3β-hydroxy-urs-12-ene-28-oic acid) is widespread in the vegetable kingdom [119] . The lichenic species Ramalina hierrensis [16] , Ramalina hierrensis [120] , Stereocaulon evolutum [121] , and Pannaria tavaresii [122] also contain this compound.

1H and C13 NMR analysed the two samples from WA-hex and ME-hex. The results we obtained reveal the predominance of the secondary metabolite atranorin only in the WA-hex sample. The structure has been characterised, and the NMR spectra are compared with those of a previously isolated sample of Physcia sorediosa [106, 123] , demonstrated in Figures S4 and S5 .

Atranorin ( Figure 6 ) is the most common secondary metabolite in lichens and is mainly found on lichens' surface (cortex) [124] . It acts as a photo-buffer because it reflects harmful UV rays to the thallus's surface and allows the lichens to live in areas receiving intense solar radiation [125] . Several factors can influence atranorin concentrations in lichens. They fluctuate with the seasons [126, 127] and vary according to the habitat [128] . The method of preparation and extraction of lichens can also influence the concentration of this metabolite. Conventional organic solvents (such as hexane and acetone) are commonly used for its extraction as hydrophobic [129] . Our study used n-hexane with Soxhlet extraction method, which is of choice in studying organic analytes extracted from lichens [130] . It is still used to date to extract organic air pollutants, organochlorinated pesticides and insecticides from the lichen matrix [131, 132] .

According to Komaty et al. (2015) , the lichen grinding method can affect the extraction efficiency and even be used to selectively increase the extraction efficiency of certain secondary metabolites such as atranorin [133] . To achieve a higher yield, use a blender instead of a ball mill, as it selectively grinds the cortex into a fine powder, which can be recovered from the larger medulla pieces. A ball mill or a mortar and pestle technique will reduce the whole lichen to powder, which will reduce the extraction efficiency of the atranorin [129] . Pseudevernia furfuracea is a lichen widely used as a raw material in the perfume and cosmetics industries due to its richness in aromatic compounds [33, [134] [135] [136] [137] . Microwave-assisted extraction of this lichen has increased atranorin extraction efficiency by a factor of five [133] .

The saxicolous lichen specimen Physcia mediterranea Nimis was collected at Ain Tebib (Sector Oum tboul) on the rock, at an altitude of 120 m above sea level, coordinate 36°49′ 09″N; 08°31′ 33″E in June 2017 (Figure 7) . Ain Tebib station is located in the national park of El Kala (80,000 ha). The collected lichen samples were packed in polyethene bags and stored at 4 °C until further processed. Professor Monia Ali Ahmed has identified Physcia mediterranea Nimis (Figure 1 ), lichenologist and research director of the Pathology of Ecosystems team at the University of Badji-Mokhtar, Annaba, Algeria. Botanical description of Physcia mediterranea Nimis is in Figure S6 . The identification was confirmed by Pr Jean Michel Sussey, lichenologist at the French Association of Lichenology (AFL). This sample has been deposited in Badji-Mokhtar University, Annaba, code AAM-1. Several factors can influence atranorin concentrations in lichens. They fluctuate with the seasons [126, 127] and vary according to the habitat [128] . The method of preparation and extraction of lichens can also influence the concentration of this metabolite. Conventional organic solvents (such as hexane and acetone) are commonly used for its extraction as hydrophobic [129] . Our study used n-hexane with Soxhlet extraction method, which is of choice in studying organic analytes extracted from lichens [130] . It is still used to date to extract organic air pollutants, organochlorinated pesticides and insecticides from the lichen matrix [131, 132] .

According to Komaty et al. (2015) , the lichen grinding method can affect the extraction efficiency and even be used to selectively increase the extraction efficiency of certain secondary metabolites such as atranorin [133] . To achieve a higher yield, use a blender instead of a ball mill, as it selectively grinds the cortex into a fine powder, which can be recovered from the larger medulla pieces. A ball mill or a mortar and pestle technique will reduce the whole lichen to powder, which will reduce the extraction efficiency of the atranorin [129] . Pseudevernia furfuracea is a lichen widely used as a raw material in the perfume and cosmetics industries due to its richness in aromatic compounds [33, [134] [135] [136] [137] . Microwave-assisted extraction of this lichen has increased atranorin extraction efficiency by a factor of five [133] .

The saxicolous lichen specimen Physcia mediterranea Nimis was collected at Ain Tebib (Sector Oum tboul) on the rock, at an altitude of 120 m above sea level, coordinate 36 • 

The lichens were washed with tap water to remove the dust and other foreign materials. The washed samples were dried under shade for a week. The lichen was ground using a grinder. The preparations were then pulverised into powdered form by using heavy-duty blender.

The powder samples (24 g) of Physcia mediterranea Nimis were extracted with the solvent n-hexane (500 mL) using a Soxhlet extractor 24 h. After complete extraction, the solvent was evaporated using a rotary evaporator under reduced pressure to obtain n-hexane extract (1.026 g). It was then extracted with hot methanol (60 °C) to obtain two parts: insoluble precipitate representing lichen wax (WA-hex) and the methanol soluble part (ME-hex). Both extracts (WA-hex/ME-hex) were completely evaporated using a rotary evaporator under reduced pressure to obtain dry extracts (0.552 g/0.300 g), respectively.

In this present study, the chemical composition of the two fractions WA-hex and MEhex of Algerian Physcia mediterranea Nimis was analysed using the HPLC-ESI-MS-MS method. Moreover, we combined GC-MS's ability with the targeted metabolomics of HPLC-ESI-MS-MS methods to characterise the composition of n-hexane extract (ME-hex) for the first time. In addition, most compounds potentially present in the wax (WA-hex) are detected and characterised using the NMR method.

For the GC-MS analysis, an Agilent MS220 (Varian, Inc. Walnut Creek, CA, USA) mass spectrometer coupled to a 7890A GC. The oven temperature was initially set to 50 °C, held for 5 min, then a ramp of 30 °C/min was applied up to 270 °C that was held for an additional5 min. MS spectra were acquired in EI mode with a mass range from 50 to 600 a.m.u. Before being injected into the GC-MS system, the ME-hex fraction was preesterified with diazomethane, in order to identify eventual less polar compounds in this fraction. It was then solubilised in dichloromethane and injected into the apparatus.

Proton 1H and carbon 13C NMR spectroscopy were recorded on a Brüker Advance III 400 MHz spectrometer (Brüker Scientific Inc, Billerica, MA, USA) at 400 MHz for proton and 100 MHz for carbon. The recovered WA was dissolved in deuterated solvent's (CDCl3) (5 mg/mL), at room temperature. The solution was transferred to 5 mm outside diameter tubes, and the spectra were acquired at room temperature. The deuterated solvent's residual peak signal was for 1H spectra at 7.26 ppm and 13C spectra at 77.2 ppm. 

The lichens were washed with tap water to remove the dust and other foreign materials. The washed samples were dried under shade for a week. The lichen was ground using a grinder. The preparations were then pulverised into powdered form by using heavyduty blender.

The powder samples (24 g) of Physcia mediterranea Nimis were extracted with the solvent n-hexane (500 mL) using a Soxhlet extractor 24 h. After complete extraction, the solvent was evaporated using a rotary evaporator under reduced pressure to obtain nhexane extract (1.026 g). It was then extracted with hot methanol (60 • C) to obtain two parts: insoluble precipitate representing lichen wax (WA-hex) and the methanol soluble part (ME-hex). Both extracts (WA-hex/ME-hex) were completely evaporated using a rotary evaporator under reduced pressure to obtain dry extracts (0.552 g/0.300 g), respectively.

In this present study, the chemical composition of the two fractions WA-hex and ME-hex of Algerian Physcia mediterranea Nimis was analysed using the HPLC-ESI-MS-MS method. Moreover, we combined GC-MS's ability with the targeted metabolomics of HPLC-ESI-MS-MS methods to characterise the composition of n-hexane extract (ME-hex) for the first time. In addition, most compounds potentially present in the wax (WA-hex) are detected and characterised using the NMR method.

For the GC-MS analysis, an Agilent MS220 (Varian, Inc. Walnut Creek, CA, USA) mass spectrometer coupled to a 7890A GC. The oven temperature was initially set to 50 • C, held for 5 min, then a ramp of 30 • C/min was applied up to 270 • C that was held for an additional5 min. MS spectra were acquired in EI mode with a mass range from 50 to 600 a.m.u. Before being injected into the GC-MS system, the ME-hex fraction was pre-esterified with diazomethane, in order to identify eventual less polar compounds in this fraction. It was then solubilised in dichloromethane and injected into the apparatus.

Proton 1H and carbon 13C NMR spectroscopy were recorded on a Brüker Advance III 400 MHz spectrometer (Brüker Scientific Inc, Billerica, MA, USA) at 400 MHz for proton and 100 MHz for carbon. The recovered WA was dissolved in deuterated solvent's (CDCl 3 ) (5 mg/mL), at room temperature. The solution was transferred to 5 mm outside diameter tubes, and the spectra were acquired at room temperature. The deuterated solvent's residual peak signal was for 1H spectra at 7.26 ppm and 13C spectra at 77.2 ppm. The chemical deviations (δ) are expressed in parts per million (ppm) and the coupling constants (J) in Hertz. The data was processed using TOPSPIN 3.5 software (Brüker Scientific Inc.).

The method was carried out on an orbitrap Thermo q-Exactive mass spectrometer coupled to a Vanquish HPLC. A Kinetex XB-C18 (Phenomenex) with a particle size of 2.6 microns, 100 mm of length, and a diameter of 2.1 mm was used as a column. The mobile phases were 0.1% formic aqueous solution (A) and, acetonitrile (B). The gradient program (time (min), % B) was: (0.00, 50); (20.00, 100); (25.00, 100); (26.00, 50). The flow rate was 0.200 mL min −1 and the injection volume was 10 µL.

The ionisation electrospray in negative mode was used. The following analysis parameters were: electrospray voltage −3.8 kV, sheath gas flow rate, 30; auxiliary gas unit flow rate, 10; drying gas temperature, 310 • C; capillary temperature, 320 • C; S-lens and RF level, 55. The acquisition was performed in a mass range from 100 to 1000 a.m.u. An auto MS2 program was used with a fragmentation voltage of 30.

Knowledge of the chemical constituents of lichens is invaluable as this information will be useful for the synthesis of potential new chemical substances. Many researchers report such phytochemical screening of various lichens [103, 106, 116, 117, 123, 124] . A growing body of evidence indicates that lichens' secondary metabolites play an essential role in human health and may be nutritionally important [24, 26, 27, [29] [30] [31] 58, 112, [118] [119] [120] . In the present study, we have identified and chemically characterised Algerian Physcia mediterranea Nimis for the first time. The extraction of these metabolites was carried out with hot hexane. A methanol-crystallisation process allowed us to characterise and identify the atranorine depside, as a major component of lichen wax. In this work, several aromatic acids, a kaurane, and fatty acids have been identified by GC-MS of ME-hex. The UHPLC-ESI-MS-MS technique has been used to analyse the crystallised fraction, WA-hex, whose major product is atranorin and chloroatranorin with the minority products identified. In MEhex fraction, this technique identifies paraconic and aliphatic acids, depsides, depsones, phenolic compounds, sesquiterpenes, triterpenes, and phenyl ethers in addition to other minority derivatives. This study reveals for the first time the different compounds of Physcia medeterranea considered as a rare international species; furthermore, it highlights the importance of the lichens of Algeria as a promising source of bioactive molecules.

Supplementary Materials: The following are available online, Figure S1 (UHPLC/ESI/MS/MS chromatogram of Physcia mediterranea (WA-hex). Figure S2 : Chemical structure of Compounds identified in WA-hex of Physcia mediterranea by UHPLC/ESI/MS/MS. Figure S3 : UHPLC/ESI/MS/MS chromatogram of Physcia mediterranea (ME-hex). Figure S4 : 1H NMR spectrum of WA-hex, 400 MHz. Figure S5 : C13 NMR spectrum of WA-hex, 100 MHz. Figure 

The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata

Fungi and lichens

Fossil Plants: Fungi and Lichens

Cyanobacterial-plant symbioses

Identity and genetic structure of the photobiont of the epiphytic lichen Ramalina menziesii on three oak species in southern California

Guide des Lichens de France: Lichens Des Arbres; Belin

Extremotolerance and Resistance of Lichens: Comparative Studies on Five Species Used in Astrobiological Research I. Morphological and Anatomical Characteristics

Antioxidants and photoprotection in a lichen as compared with its isolated symbiotic partners

Relationship between Isozymic Variability and Envir-onmental Conditions in the Lichen Xanthoria parietina

Lichen Metabolites: An Overview of Some Secondary Metabolites and Their Biological Potential. In Co-Evolution of Secondary Metabolites

Lichen-Derived Products

Advances in Lichenology: Modern Methods and Approaches in Lichen Systematics and Culture Techniques

A zearalenone derivative from the liquid culture of the lichen, Baeomyces placophyllus

Chemical and Botanical Guide to Lichen Products

Chemistry and biological activity of Ramalina lichenised fungi

Ecological and biotechnological aspects of lichens

Identification of Lichen Substances

Secondary metabolism in the lichen symbiosis

Algal carbohydrates affect polyketide synthesis of the lichen-forming fungus Cladonia rangiferina

Occurrence of terpenes, polyketides, and tannins in some Japanese lichens and green mosses

Anticancer potential of lichens' secondary metabolites

Investigation of antioxidant, antimicrobial and toxicity activities of lichens from high altitude regions of Nepal

Potential of Lichen Compounds as Antidiabetic Agents with Antioxidative Properties: A Review

Lichens: Unexpected anti-prion agents? Prion

Antifungal and anti-biofilm activities of acetone lichen extracts against Candida albicans

Biopharmaceutical potential of lichens

Antioxidant activity and mechanisms of action of natural compounds isolated from lichens: A systematic review

Lichens as a potential natural source of bioactive compounds: A review

Antioxidative and cardiovascular-protective activities of metabolite usnic acid and psoromic acid produced by lichen species Usnea complanata under submerged fermentation

Lichens used in traditional medicine

Lichens Used in Traditional Medicine

Sample preparation for an optimised extraction of localised metabolites in lichens: Application to Pseudevernia furfuracea

Antibacterial Activity of the Chloroform, Acetone, Methanol and queous Extracts of Algerian Lichens

Lichen diversity in the edough peninsula

Chemical and biological investigation of Algerian lichens

Chemical Composition, Antibacterial and Antioxidant Activities of Thyme Essential Oil (Thymus vulgaris)

Contribution to the lichens;inventory from the Oubeira Lake (NE Algeria)

Lichenological exploration of Algeria: Historical overview and annotated bibliography

Inventaire des lichens du Parc national de Gouraya

Mise à jour de l'inventaire des lichens d

Cartographie de la pollution plombique dans la région d'Alger en utilisant un lichen (Xanthoria parientina) comme bioaccumulateur

Macrolichens New to Algeria and Other Interesting Species from Theniet-el-Had National Park

UHPLC-HRMS/MS Based Profiling of Algerian Lichens and Their Antimicrobial Activities

Contribution a l'etude de la flore lichenique dans la zeenaie de Bougous

Air quality in the metropolitan area of Sidi Bel Abbes (Algeria) through the lichens species as bio-indicators

Biosurveillance par SIG de la qualité de l'air d'une cité méditerraneene (Alger), diversité lichénique, indice de pureté atmosphérique (IPA) et indice d'impact. Revue

Jumelage des bio-indicateurs et d'un réseau de surveillance de la qualité de l'air pour la détection de la pollution par le SO 2 dans la région de Annaba (Algérie)

Responses of antioxidants in Flavoparmelia caperata (L.) Hale to the atmospheric pollution air at two urban and semi-urban areas in the region of Annaba (East of Algeria)

Biological diversity of the National Park of El-Kala (Algeria), valorisation and protection

Ismed, F. Structure, activity, and drug-likeness of pure compounds of Sumatran lichen (Stereocaulon halei) for the targeted ACE2 protein in COVID-19 disease

Isolation, Chemical Modification, and Anticancer Activity of Major Metabolites of the Lichen Parmotrema mesotropum

Structure of constituents isolated from the bark of Cassipourea malosana and their cytotoxicity against a human ovarian cell line

Extracted compounds from Cetraria islandica

Ach) Articus/Parmelia perlata Esch. (Parmeliaceae)

Phenolic constituents from lichen Usnea longissima

Dibenzofurans and pseudodepsidones from the lichen Stereocaulon paschale collected in northern Quebec

Promising anticancer activity of a lichen, Parmelia sulcata Taylor, against breast cancer cell lines and genotoxic effect on human lymphocytes

Volatile constituents of selected Parmeliaceae lichens

Isolation of Atraric Acid, Synthesis of Atraric Acid Derivatives, and Use of Atraric Acid and the Derivatives thereofe for the Treatment of Benigne Prostatic Hyperplasia

A natural androgen receptor antagonist induces cellular senescence in prostate cancer cells

Biological activities of Pseudevernia furfuracea (L.) Zopf extracts and isolation of the active compounds

Nematocidal principles in "Oakmoss Absolute" and nematocidal activity of 2, 4-Dihydroxybenzoates

Fatty acid their composition of lichens and their phyco-and mycobionts

Seasonal variation in concentrations of carbohydrates and lipids in two epiphytic lichens with contrasting, snow-depth related distribution on subarctic birch trees

Fatty acids of lichen species from Tian Shan Mountains

Gas chromatographic analysis to compare the fatty acid composition of fifteen lichen species, with a focus on Stereocaulon

Biogenetic relationships of the lichen substances in the framework of systematics

Large genetic divergence of new, morphologically similar species of sterile lichens from

A Monograph of the Lichen Genus Parmelia Acharius sensu Strict (Ascomycotina: Pameliaceae)

A phylogenetic analysis of the boreal lichen Mycoblastus sanguinarius (Mycoblastaceae, lichenised Ascomycota) reveals cryptic clades correlated with fatty acid profiles

The Use of Metabolic Data in Lichenology at the Species and Subspecific Levels

Lichenology: Progress and Problems; Proceedings of an international Symposium

Metabolic diversity of lichen-forming ascomycetous fungi: Culturing, polyketide and shikimate metabolite production, and PKS genes

Role of fatty acids in signal transduction: Modulators and messengers

Study of biochemical aspects of lichen symbiosis. I. Lipids and fatty acids in cultured lichen symbionts

Characterization of surface n-alkanes and fatty acids of the epiphytic lichen Xanthoria parietina, its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills

Differences in the composition of phenolics and fatty acids of cultured mycobiont and thallus of Physconia distorta

Seasonal variation of lipids and fatty acids from tree-growing lichens of the genus Physcia

A Seasonal variability of lipids and fatty acids in the tree-growing lichen Xanthoria parientina L

Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts

Lipids in Algae, Lichens and Mosses

Chemical Constituents of the Epiphytic and Lithophilic Lichens of the Genus Collema

Fumaric acid: Production and separation

Bioproduction of fumaric acid: An insight into microbial strain improvement strategies

Fumarsäure in der Therapie der Multiplen Sklerose

Fumaric acid production using renewable resources from biodiesel and cane sugar production processes

Traumatic Acid Reduces Oxidative Stress and Enhances Collagen Biosynthesis in Cultured Human Skin Fibroblasts

Traumatic acid toxicity mechanisms in human breast cancer MCF-7cells

Esculetin Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Mice Via Modulation of the AKT/ERK/NF-κB and RORγt/IL-17 Pathways

A natural coumarin derivative esculetin offers neuroprotection on cerebral ischemia/reperfusion injury in mice

Esculetin Attenuates Th2 and Th17 Responses in an Ovalbumin-Induced Asthmatic Mouse Model

Cytoprotective effects of esculetin against oxidative stress are associated with the upregulation of Nrf2-mediated NQO1 expression via the activation of the ERK pathway

Coumarin: A Natural, Privileged and Versatile Scaffold for Bioactive Compounds

A Review of Coumarin Derivatives in Pharmacotherapy of Breast Cancer

Atranorin-an interesting lichen secondary metabolite. Mini-Rev

Chemie von Cladonia furcata und Cladonia rangiformis

Lichen compounds Restrain lichen feeding by bank voles (Myodes glareolus)

Chemical and Botanical Guide to Lichen Products

A Catalogue of Standardized Chromatographic Data and Biosynthetic Relationships for Lichen Substances

The antimicrobial activity of substances derived from the lichens Physcia aipolia, Umbilicaria polyphylla, Parmelia caperata and Hypogymnia physodes

Light screening in lichen cortices can be quantified by chlorophyll fluorescence techniques for both reflecting and absorbing pigments

Notes on the Lichens Physcia aipolia and P. alnophila in North America

Presence of atranorin in Physcia sorediosa

Cladonia lichens and their major metabolites as possible natural antioxidant, antimicrobial and anticancer agents

Antimicrobial and antibiofilm activity of secondary metabolites of lichens against methicillin-resistant Staphylococcus aureus strains from cystic fibrosis patients

Anti-inflammatory and toxicity studies of atranorin extracted from Cladina kalbii Ahti in rodents. Braz

Purification, physicochemical properties, thermal analysis and antinociceptive effect of atranorin extracted from Cladina kalbii

Evaluation of wound healing activity of atranorin, a lichen secondary metabolite, on rodents

Atranorin, an antimicrobial metabolite from lichen Parmotrema rampoddense exhibited in vitro anti-breast cancer activity through interaction with Akt activity

Usnic acid and atranorin exert selective cytostatic and anti-invasive effects on human prostate and melanoma cancer cells

Stereostructure of Asebotoxin I and II, Toxins of Pieris japonica

Bakkenolide A inhibits leukemia by regulation of HDAC3 and PI3K/Akt-related signaling pathways

Chemical constituents of the lichen species Cetraria islandica

Phenolic compounds in Chilean Mistletoe (quintral, Tristerix tetrandus) analysed by UHPLC-Q/Orbitrap/MS/MS and its antioxidant properties

New Results on the Chemistry of Lichen Substances. In Progress in the Chemistry of Organic Natural Products

Ursolic acid in health and disease

chemical constituents of the lichen Ramalina hierrensis

Lichen metabolites active against hepatitis C virus

Secondary products from Pannaria tavaresii (Lichens)

Chemical Constituents from Ouratea floribunda: Complete 1H and 13C NMR Assignments of Atranorin and its New Acetyl Derivative

Chemotype variations among lichen ecotypes of Umbilicaria aprina as revealed by LC-ESI-MS/MS: A survey of antioxidant phenolics

The optimal extraction and stability of atranorin from lichens, in relation to solvent and pH

Lichenic extracts and metabolites as UV filters

Seasonal trends in usnic acid concentrations of Arctic, alpine and Patagonian populations of the lichen Flavocetraria nivalis

Identification and quantitation of allelochemicals from the lichen Lethariella canariensis: Phytotoxicity and antioxidative activity

Possible functional roles of cortical depsides and medullary depsidones in the foliose lichen Hypogymnia physodes

Comparative Perspectives on Extraction Methods for Organic Metabolites and Pollutants from Lichens

Atmospheric Dioxin and Furan Deposition in Relation to Land-Use and Other Pollutants: A Survey with Lichens

Comparison of extraction techniques for polycyclic aromatic hydrocarbons from lichen biomonitors

Lichens as biomonitors for organic air pollutants

Methyl β-Orcinolcarboxylate and Depsides from Parmelia Furfuracea

Isolement et identification de l'acide furfurique, nouvelle depsidone du lichen Pseudevernia furfuracea (L.) Ach

Pseudevernia furfuracea-Olivetorina Relationships: Chemistry and Ecology

Lichen extracts as raw materials in perfumery

We thank the Ministry of Higher Education and Scientific Research (MESRS) of Algeria for the financial support to this study, and we would like to warmly thank Grira Abdeslem, Principal Forestry Inspector, El Kala National Park, for his presence and his invaluable assistance in the field.

The authors declare no conflict of interest.Sample Availability: Samples of all compounds used in the study are available from the authors.