Phytochemical Composition of Lichen Parmotrema hypoleucinum (J. Steiner) Hale from Algeria

In this work, we carried out studies of the chemical composition of hexane, chloroform and ethanol extracts from two samples of the lichen Parmotrema hypoleucinum collected in Algeria. Each sample of the lichen P. hypoleucinum was collected on two different supports: Olea europaea and Quercus coccifera. Hexane extracts were prepared, in Soxhlet; each hexane extract was fractionated by its solubility in methanol; the products soluble in methanol were separated (cold): 1-Hexane, 2-Hexane; and the products insoluble in methanol (cold): 1-Cires, 2-Cires. A diazomethane esterified sample of 1-Hexane, 2-Hexane, 1-Cires and 2-Cires was analyzed by GC-MS, and the components were identified as methyl esters. In the 1-Hexane and 2-Hexane fractions, the methyl esters of the predominant fatty acids in the lichen were identified: palmitic acid, linoleic acid, oleic acid and stearic acid; a hydrocarbon was also identified: 13-methyl-17-norkaur-15-ene and several derivatives of orsellinic acid. In the 1-Cires and 2-Cires fractions, the previous fatty acids were no longer observed, and only the derivatives of orsellinic acid were found. The analysis of the 1-Hexane, 2-Hexane fractions by HPLC-MS/MS allows us to identify different chemical components, and the most characteristic products of the lichen were identified, such as Atranol, Chloroatranol, Atranorin and Chloroatranorin. In the fractions of 1-Cires and 2-Cires, the HPLC-MS/MS analysis reveals that they are very similar in their chemical components; the characteristic products of this lichen in this fraction are Atranorin and Chloroatranorin. In the extracts of chloroform, 1-Chloroform and 2-Chloroform, the analysis carried out by HPLC-MS/MS shows small differences in their chemical composition at the level of secondary products; among the products to be highlighted for this work, we have chloroatranorin, the stictic acid, norstictic acid and other derivatives. In the analysis of the most polar extracts carried out in ethanol: 1-Ethanol and 2-Ethanol, HPLC-MS/MS analysis shows very similar chemical compositions in these two extracts with small differences. In these extracts, the following acids were identified as characteristic compounds of this lichen: constictic acid, stictic acid, substictic acid and methylstictic acid. In the HPLC–MS/MS analysis of all these extracts, alectoronic acid was not found.


Introduction
Lichens live in symbiotic associations between fungi and algae and/or cyanobacteria, and in addition to these two symbiotic partners (photobiont and mycobiont) classically The natural products isolated from different lichens (such as Usnic acid, Lobaric acid, Atranorin, Protolichesterinic acid and Salazinic acid) have good antibiotic activities against Gram-positive bacteria and are also active against pathogenic dermatophyte fungi [8]. Other products found in lichens, such as anthraquinones derivatives, bianthrones and hypericin, have an inhibitory action on the activity of viral enzymes, such as the integrase of HIV-1 and HSV-1 [9,10] and also on enzymes such as lipooxygenases, histidine decarboxylase and tyrosinase; other derivatives inhibit the biosynthesis of Leukotriene B 4 (LTB 4 ) [11][12][13].
Polyphenolic products isolated from lichens have limitations, low solubility and, above all, toxicity. Usnic acid is a polyphenolic compound very common in lichens that has good

Results and Discussion
Parmotrema hypoleucinum (J. Steiner) Hale is an epiphytic lichen collected in Algeria and studied in order to determine its metabolic composition and chemical fingerprint. P hypoleucinum (J. Steiner) Hale was collected in two different supports, the first one in Lac Tonga (Sector Brabtia) on Olea europaea and the second one in Lac Tonga (Sector Brabtia) on Quercus coccifera. The metabolic compositions of each lichen sample were studied by sequential extraction, first of all, with hexane in Soxhlet for low polarity products. The remaining vegetable mass was placed with chloroform at room temperature to obtain the chloroform extract for the products of intermediate polarity, and finally, the vegetable mass was extracted with ethanol at room temperature for the products with higher polarity.
The hexane extract of each sample was dissolved in hot methanol and allowed to cool slowly to obtain the products insoluble in cold methanol. In this way, the products insoluble in methanol were separated: 1-Cires and 2-Cires; remaining soluble: 1-Hexane and 2-Hexane. Initially, an aliquot of the samples 1-Hexane, 2-Hexane, 1-Cires and 2-Cires were esterified with diazomethane to esterify the acid groups of the existing compounds. These esterified samples were analyzed by GC-MS to identify compounds of lower polarity.

2-Hexane, 2-Cires, 2-Choroform, 2-Ethanol
Chemical analysis of the hexane extract soluble in MeOH esterified with diazometane, 1-Hexan and 2-Hexane of Parmotrema hypoleucinum collected from two different phorophytes by GC/MS The 1-Hexane and 2-Hexane samples were esterified with diazomethane to esterify the existing acid groups to their methyl esters for GC-MS analysis, being the natural products of the acids indicated in Table 1 for 1-Hexane sample and Table 2 for 2-Hexane sample. The 1-Hexane sample was analyzed by GC-MS, and eight products were identified, among them palmitic, linoleic, oleic and stearic acids and an unidentified compound. Figure 2 and Table 1. The esterified 2-Hexane sample was also analyzed by GC-MS, identifying six products, palmitic, linoleic, oleic and stearic acid, a phenolic compound 2,4-dihydroxy-3,5,6-trimethylbenzoic acid and 13-methyl-17-norkaur-15-ene. Figure 3 and Table 2.   The compound identified as Hibaene (13-methyl-17-norkaur-15-ene) is the product with a retention time of 23:53 in GC-MS; its mass spectrum shows the molecular ion at 272 and comes from the dehydration of alcohol (-)-ent-Kauran-16α-ol in the ionization source of the mass spectrometer. The natural product should be the alcohol (-)-ent-Kauran-16α-ol.
For the fractions that have been obtained by crystallization from the crude hexane extract by solubilization in hot methanol, 1-Cires and 2-Cires are also esterified with diazomethane for GC-MS analysis.
In the 1-Cires analysis, five products are found, with four being identified. Figure 4 and Table 3. In the 2-Cires sample, three products have been identified from the methanol-insoluble part of the hexane extract of P. hypoleucinium (Quercus coccifera). Figure 5 and Table 4.    In the 1-Cires sample, there is a compound that could not be identified; this product does not appear in the 2-Cires sample. The kaurene-skeletal alcohol is found in 1-Cires and was not found in the 2-Cires sample. Of the other three compounds, two were identified in 1-Cires and 2-Cires: 2,4-dihydroxy-3,5,6-trimetylbenzoic acid and 2,4-dihydroxy-3,6dimethylbenzoic acid.
In the HPLC-MS/MS analysis of the 1-Hexane and 2-Hexane fractions of P. hypoleucinium, 95 products were detected in the 1-Hexane fraction, in which we proposed 78 structures (and 16 not identified). In total, 91 products were found in the 2-Hexane fraction, of which we proposed 78 structures (and 13 no identified ones). Figures 6 and 7 show the HPLC chromatograms that allowed this analysis and identification of the indicated compounds to be carried out; the complete result is shown in Table 5.
In the 1-Cires sample, there is a compound that could not be identified; this product does not appear in the 2-Cires sample. The kaurene-skeletal alcohol is found in 1-Cires and was not found in the 2-Cires sample. Of the other three compounds, two were identified in 1-Cires and 2-Cires: 2,4-dihydroxy-3,5,6-trimetylbenzoic acid and 2,4-dihydroxy-3,6-dimethylbenzoic acid.
In the HPLC-MS/MS analysis of the 1-Hexane and 2-Hexane fractions of P. hypoleucinium, 95 products were detected in the 1-Hexane fraction, in which we proposed 78 structures (and 16 not identified). In total, 91 products were found in the 2-Hexane fraction, of which we proposed 78 structures (and 13 no identified ones). Figures 6 and 7 show the HPLC chromatograms that allowed this analysis and identification of the indicated compounds to be carried out; the complete result is shown in Table 5.     7-Chloro-3-oxo-1,3-dihydroisobenzofuran-5-carboxylic acid. Structure proposed and confirmed by the ions The most characteristic products of the 1-Hexane and 2-Hexane extracts of the lichens in the two samples are 3-methylorsellinic acid, atranol, 4-formylbenzoic acid, chloroatranol, p-coumaric acid, atranorin and chloroatranorin.
In the 1-Hexane sample, the following compounds were not identified: 3,5-dimethylorselinic acid, 9,10-dihydroxy-8-oxo-12-octadecenoic acid, octadecanedioic acid and 18-hydroxylinoleic acid, which were found in the 2-Hexane sample. The following compounds were not identified in the 2-Hexane sample: barbatic acid, tetraoxodocosanoic acid and superpicrolichenic acid; these products were identified in the 1-Hexane sample too. In the 1-Hexane sample, there are 17 products that could not be identified and in the 2-Hexane 13.
In the analysis of the 1-Cires and 2-Cires fractions, obtained by precipitation of the initial hexane extract by HPLC-MS/MS, it has been possible to detect in 1-Cires 56 products, of which 11 products were not identified. In 2-Cires, 53 products were detected, and 13 products could not be identified. For the acids and diacids identified in 1-Cires there are 23 products, and in 2-Cires, we have 27 products. As benzoic acids or derivatives we have p-coumaric acid and 6,7-dihydroxycoumarin. Among the polyphenolic compounds and esters, 1-Cires and 2-Cires were identified: allo-protolichestrinic acid, atranorin, 7-chloro-3oxo-1,3-dihydroisobenzofuran-5-carboxylic acid, chloroatranorin, 8-hydroxydiffractaic acid and 19-acetoxylichestrinic acid.
The chromatograms and the analysis of the 1-Cires and 2-Cires compounds are shown in Figures 8 and 9 and Table 6.    In the work carried out in 2016, in a general analysis of the chemical relationship in the group of Parmotrema perforatum (Parmeliaceae, Ascomycota), each sorediate species is descended from an apotheciate species with the same secondary chemicals [38]. The lichen Parmotrema hypoleucinum is derived from the ancestor Parmotrema perforatum represented by its secondary metabolites in stictic, constictic and norstictic acids. These acids have not been extracted in the Hexane extract, and they do not exist in the part insoluble in methanol, 1-Cires and 2-Cires nor in the part soluble in methanol: 1-Hexane and 2-Hexane.
The products identified in the 1-Chloroform and 2-Chloroform extracts are shown in the chromatograms in Figures 10 and 11 and the results of the identification of the compounds in Table 7.    In the analysis of 1-Chloroform and 2-Chloroform extracts, the acids of the secondary metabolites that define the Sorediate species are identified for Parmotrema hypoleucinum; these acids are as follows. Constictic acid is also detected in Parmotrema tinctorum; Norstictic acid, according to mycologia 2015, this compound appeared to be present in variable concentrations throughout the thallus. Often the medulla of a lobe tested negative while the medulla adjacent to the apothecia tested positive or vice versa. Stictic acid and other derivatives identified as Substictic acid, there are depsidone were detected in Parmotrema tinctorum, P. grayanum, also in P. robustum and P. andinum [39].
In sample 1-Chloroform, 60 products were detected, of which 52 products were identified, and 8 products were not identified, and in the sample 2-Chloroform, 59 compounds were detected, of which 52 products were identified and 7 unidentified.
In the extracts of the more polar products made with ethanol, in the 1-Ethanol sample 57 products were found, of which 54 products were identified, and 3 compounds were not identified. Figure 12 and Table 8. In the sample 2-Ethanol analyzed, 54 compounds were detected, of which 52 products were identified, and 2 compounds were not identified. Figure 13 and Table 8.    Praesorediosic acid or Protopraesorediosic acid [41][42][43] Among the compounds identified in the 1-Ethanol and 2-Ethanol samples were also found the acids that define this lichen Parmotrema hypoleucinum, the Constictic and Stictic acids and a derivative such as methylstictic acid.
The products analyzed by GC-MS were identified by their mass spectra and compared with the mass spectra of the NIST and Wiley databases.

Lichen Material
Parmotrema hypoleucinum is a foliose epiphytic lichen, which was collected on Quercus coccifera and on Olea europea at Lake Tonga (Sector Brabtia), at an altitude of 2.20 m above sea level, coordinate 36 • 51 38 N; 08 • 28 46 E in June 2017. The area of lake tonga is 2600 ha communicating with the sea through the artificial channel of the Messida. This station is located in the national park of el kala (80,000 ha) Figure 14, classified as a biosphere reserve by UNESCO in 1990, located in the extreme northeast of Algeria.

Sample Preparation
Hexane extraction was carried out for each lichen sample; for Parmotrema hypoleucinium (J. Steiner), Hale was made from 40 g of powder material for the two samples. The extraction was carried out in Soxhlet apparatus with hot n-Hexane for 24 h; after this time, the solvent was evaporated to obtain the Parmotrema hypoleucinium (Olea europaea) n-Hexane extract 0.66 g, which represents (1.65%) and Parmotrema hypoleucinium (Quercus coccifera)) n-Hexane extract 0.27 g, which represent (0.68%). The n-Hexane extracts are then dissolved in hot methanol and allowed to cool to room temperature so that insoluble products crystallize. With this treatment, the methanol insoluble part (cires) and the cold methanol soluble part (Hexane) are obtained for each n-Hexane extract. For P hypoleucinium, the part insoluble in methanol produced 1-Cires of 0.379 g and 2-Cires of 0.058 g; the part soluble in methanol produced 1-Hexane of 0.281 g and 2-Hexane of 0.219 g.
The vegetable mass recovered and dried from the extractions with Hexane was placed to extract with chloroform at room temperature for 5 days. After this time, the chloroform extract was filtered, and the solvent was evaporated, obtaining the 1-Chloroform extract with a weight of 0.226 g, which represents (5.65%) and the 2-Chloroform extract with a weight of 0.196 g, which represents (4.90%).
After being extracted the vegetable mass with chloroform was placed with ethanol for 5 days to prepare the Ethanol extracts. Evaporation of the solvent gave the ethanol extracts: for 1-Ethanol, a mass of 2385 g was recovered, which represented 5.96%, and for the 2-Ethanol extract, it presented a mass of 2.417 g, which represented 6.04%.

Instruments
For the GCMS analysis, an Agilent MS220 mass spectrometer coupled to a 7890A GC was used.
HPLC analyses were carried out on an orbitrap Thermo q-Exactive mass spectrometer coupled to a Vanquish HPLC.

GCMS Parameters
The oven temperature was initially set to 50 • C, held for 5 min and then a ramp of 30 • C/min was applied up to 270 • C that was held for 5 additional mins. A VF-5 ms columns was used, with a length of 30 m, inner diameter 0.25 mm and layer width of 0.25 micron.
MS spectra were acquired in EI mode with a mass range from 50 uma to 600 uma.

LC Parameters
For the HPLC separation, a Kinetex XB-C18 (Phenomenex) with a particle size of 2.6 microns, 100 mm in length and a diameter of 2.1 mm was used as column. As solvent A, water with 0.1% of formic acid was used, and as solvent B, acetonitrile was chosen. The column flow war 0.200 mL/min. The following gradient was used (in Table 9):

MS Parameters
For the ionization electrospray in negative mode was used, with the following parameters: Electrospray voltage −3.8kV, Sheath gas 30, Aux gas 10, drying gas temperature 310 • C. Capillary temp. 320 and S-lens value of 55.0.
The acquisition was performed in a mass range from 100 to 1000 uma, and an auto MS2 program was used with a fragmentation voltage of 30.

Conclusions
Due to the biological importance of lichens isolated natural products, studies of the chemical composition of two samples of the lichen Parmotrema hypoleucinum, collected on two different supports: Olea europaea and Quercus coccifera, in Algeria, were carried out. For each sample, the extracts of hot n-Hexane, Chloroform at room temperature and Ethanol at room temperature were carried out.
The n-Hexane extract of each sample is dissolved in hot methanol and allowed to cool slowly so that the products insoluble in methanol precipitate. The parts soluble in methanol are obtained by filtration, the solvent is evaporated, and they are designated as 1-Hexane and 2-Hexane fractions for each sample, respectively. The product insoluble in methanol, washed and dried, are designated as the 1-Cires and 2-Cires fractions, respectively. An aliquot sample of the fractions: 1-Hexane, 2-Hexane, 1-Cires and 2-Cires, were esterified with diazomethane to produce the methyl esters of the existing acids.
The 1-Cires and 2-Cires esterified samples were analyzed by GC-MS to separate and identify the components of lower polarity. In the analysis carried out, 2,4-dihydroxy-3,5,6-trimethylbenzoic and 2,4-dihydroxy-3,6-dimethylbenzoic acids were found as existing products in the two samples. In the 2-Cires esterified sample, 2,4-dimethoxy-6-methylbenzoic acid, which does not exist in the esterified 1-Cires fraction, was also identified in this analysis. In the esterified fraction 1-Cires, the analysis also identified 4-hydroxy-2-methoxy-3,6-dimethylbenzoic acid, the hydrocarbon 13-methyl-17-norkaur-15-ene, and a product that was not identified with a mass of 312, which were not found in the esterified sample of 2-Cires.
The analysis, identification and comparison of the components of the original fractions of 1-Hexane and 2-Hexane by HPLC-MS/MS indicate that they are very similar and show almost no differences, except for some very minor components; in these fractions of low polarity, the most characteristic components P hypoleucinum will be: Atranol, Chloroatranol, Atranorin and Chloroatranorin; a triterpenic acid identified as Ursolic acid has also been found in both samples.
The analysis of the original 1-Cires and 2-Cires fractions by HPLC-MS/MS shows that they are very similar, presenting some small differences in some minor components. The predominant components identified are the fatty acids indicated in the Tables in addition to the predominant product Atranorin and Chloroatranorin, which will be characteristic for this lichen.
In the analysis carried out by HPLC-MS/MS of the original extracts of 1-Chloroform and 2-Chloroform of the two samples of P hypoleucinum and in the comparison of their components, small differences were found in some secondary compounds. In this analysis, the following acids can be considered as characteristic products of the lichen: Constictic acid, Norstictic acid (this acid was only found in the 1-Chloroform extract), Stictic acid, Substictic acid and Chloroatranorin.
The HPLC-MS/MS analysis of the original extracts of 1-Ethanol and 2-Ethanol did not suggest great differences, and most of their components were identified; the following compounds were found as more representative of the lichen: Constictic acid, Stictic acid, Substictic acid; Methylstictic acid was only identified in the extract of 1-Ethanol.
In this work, a more complete study of the components of the different extracts made from P. hypoleucinum was carried out due to the importance of the biological activities. In the most important identified products, in general, their biological activities were referred to derivatives of orsellinic acid, atranol, chloroatranol, atranorin, chloroatranorin, stictic acid and norstictic acid.
For these components, their antioxidant activities, apoptotic effects, cytotoxic, antimicrobial and antitumor activity are already known.