Phytochemical Investigation of New Algerian Lichen Species: Physcia Mediterranea Nimis

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.


Introduction
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.

GC-MS 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.

GC-MS 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.
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.

GC-MS 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].
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].

ME-hex Extract Analysis of Physcia mediterranea
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].

NMR Analysis
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].

NMR Analysis
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].

Lichen Material
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].

Lichen Material
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 •

Sample Preparation
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.

Preparation of Physcia mediterranea Extracts
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.

Instrumentation and Analysis Parameters
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.

GC-MS Analysis
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.

NMR Analysis
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.

Sample Preparation
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.

Preparation of Physcia mediterranea Extracts
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.

Instrumentation and Analysis Parameters
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.

GC-MS Analysis
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.

NMR Analysis
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 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.

Conclusions
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.