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.

1. 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 described, a third partner can also be integrated: epi and/or endophytic fungi as well as bacteriobiont or associated bacterial communities [1] which are important constituents of many of them. The production of various unique extracellular secondary metabolites known as lichen substances is the result of this symbiosis. The specific condition in which lichens live is the reason for the production of many metabolites that provide good protection against negative physical and biological influences. [2] The majority of lichens-forming fungi belong to Ascomycetes Lecanoromycetes [3]. Due to the vast genetic diversity and interactions with various environmental factors, lichens have unique profiles of primary and secondary metabolites (i.e., lichen substances) with interesting physiochemical properties [4].

In this paper, we describe our studies on the chemical composition of the extracts of Parmotrema hypoleucinum, Lecanorales Order, family Parmeliaceae, which, with 2765 species all over the world, is the largest family [5].

In Algeria, the Parmeliaceae family is very present compared to other families [6]; four Parmotrema have been identified so far: P. perlatum, P. reticulatum, P. robustrum and P. hypoleucinum. The last one is very common in the Mediterranean area [7]; it belongs to the Lecanorales Order and to the Parmeliaceae Family. It corresponds to a foliaceous lichen that can be up to 12 cm in diameter. In general, the lobes are very irregular, wide and raised, often forming tufts on small branches and can appear like curly lettuce. The upper side has a grayish appearance, and the lower side is largely white, which allows it to be easily distinguished from other similar species that have a dark back side. This species has black cilia and marginal Soralies.

Several activities of these lichen molecules, mainly those resulting from the polymalonate acetate pathway, are of interest for cosmetics: photoabsorbing, antioxidant and inducing melanogenesis. These properties have been studied for a still limited number of secondary metabolites (Mitrović, 2011) (Figure 1).

Figure 1

Biosynthetic pathways of lichen secondary metabolites (Elix, 1996; Stocker wörgötter, 2008).

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 B4 (LTB4) [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 activity, among others, against microorganisms of the Mycobacterium genus. In the 1950s, Shibata and Miura [14] made modifications and derivatizations of the functional groups of usnic acid to carry out the structure–activity correlation study to enhance the biological activity profile.

Usnic and polyporic acids have a good growth inhibition activity of L1210 leukemic cells; in subsequent research [15,16], several derivatives of these acids have been prepared to enhance antitumor activity, but none of these derivatives have exceeded the activities presented by usnic and polyporic acids.

Kumar and Muller have prepared a series of analogs of barbatic, diffractaic and obtusatic acids isolated from lichens to evaluate the effects of inhibition of the biosynthesis of LTB4 and as antiproliferative agents. Some of these derivatives show good potential as LTB4 biosynthesis inhibitors [11,13].

Lichens can also have xanthones, that shown enzyme modulation that are therapeutic targets, such as protein kinase C [17], topoisomerase II [18,19], acetylcholinesterase [20] and monoamine oxidases [21]; antiretrovirals [22,23], antimalarials [24,25], antihypertensives [26], anti-inflammatory, cytotoxics [27] and antitumors [28,29]. For this reason, the use of the base skeleton of xanthone is justified to prepare derivatives with bioactive potential.

Many depsidones isolated from lichens and higher plants have important activities, including the inhibition of enzymatic activity [30], antimycobacterial, anti-inflammatory, analgesic, antitumor, cytotoxic and antiviral activity [31,32,33]. In the work published in 2015 by James C Lendemer and collaborators [34], they studied and delineated the Parmotrema species in eastern North America. Using morphological, chemical, reproductive and ecological characters, they define four species for this group: P. hypoleucinum, P. hypotropum, P. perforatum and P. subrigidum.

This group has found P. hypoleucinum and P. subrigidum to be momophyletic, the latter comprising two chemotypes that differ in the presence or absence of norstictic acid in addition to alectoronic acid.

Due to the pharmacological potential presented by compounds isolated in lichens, it was decided to study the chemical composition of the lichen Parmotrema hypoleucinum (J. Steiner) Hale, collected on two different supports in the area of Lac Tonga in Algeria.

2. 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.

The different extracts obtained were:

From P hypoleucinum (J. Steiner) Hale on Olea europaea

1-Hexane, 1-Cires, 1-C 1-Ethanol

From Parmotrema hypoleucinum (J. Steiner) Hale on Quercus coccifera.

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.

Table 1

Sample Parmotrema hypoleucinum (in Olea europaea), Hexane extract part soluble in MeOH esterified whit diazomethane, 1-Hexane.

Nº	RT	Identified Product	Mass	%	Natural Compound, Structure
1	19:84	Methyl 4-hydroxy-2-methoxy-3,5,6-trimethylbenzoate	224	0.5
2	20:00	Methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate	210	62.4
3	20:34	Methyl 2,4-dihydroxy-3,6-dimethylbenzoate	196	8.6
3	22:36	Methylhexadecanoate	270	5.3	C16H32O2 palmitic acid
4	23:53	13-methyl-17-norkaur-15-ene (hibaene)	272	10.1
5	24:19	Methyl 9,12-octadecadienoate	294	4.2	C18H32O2 linoleic acid
6	24:25	Methyl (Z)-9-octadecenoate	296	2.1	C18H34O2 oleic acid
7	24:48	Methyloctadecanoate	298	3.6	C18H34O2 stearic acid
8	25:39	Unidentified	288	0.8	Unidentified

Table 2

Sample Parmotrema hypoleucinum (in Quercus coccifera), Hexane extract part soluble in MeOH esterified whit diazometane, 2-Hexane.

Nº	RT	Identified Product	Mass	%	Natural Compound, Structure
1	20:01	Methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate	210	2.5
2	22:55	Methylhexadecanoate	270	2.8	C16H32O2 palmitic acid
3	23:54	13-Methyl-17-norkaur-15-ene Probably the natural product will be (-)-ent-Kauran-16α-ol	272	36.7
4	24:19	Methyl (Z,Z)-9,12-octadecadienoate	294	1.2	C18H32O2 linoleic acid
5	24:23	Methyl (Z)-9-octadecenoate	296	1.0	C18H34O2 oleic acid
6	24:38	Methyl octadecanoate	298	1.4	C18H34O2 stearic acid

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.

Figure 2

Chromatogram of Hexane extract part soluble in MeOH esterified with diazomethane, 1-Hexane.

Figure 3

Chromatogram of Hexane extract part soluble in MeOH esterified whit diazomethane, 2-Hexane.

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.

P hypoleucinum (J. Steiner) Hale, on Quercus coccifera, the 2-Hexane sample, it has a lower number of components, and all were identified in the 1-Hexane sample. The most important fatty acids in the extracts were identified as palmitic, linoleic, oleic and stearic acids. 2,4-Dihydroxy-3,5,6-trimethylbenzoic acid and (-)-ent-Kauran-16α-ol alcohol is also identified in the two samples, 1-Hexane and 2-Hexane. Only in the 1-Hexane sample 4-hydroxy-2-methoxy-3,5,6-trimethylbenzoic acid and 2,4-dihydroxy-3,6-dimethylbenzoic acid are also identified.

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.

Figure 4

Chromatogram of Hexane extract part insoluble in MeOH esterified whit diazomethane, 1-Cires.

Table 3

Sample Parmotrema hypoleucinum (in Olea europaea), Hexane extract part insoluble in MeOH esterified whit diazomethane, 1-Cires.

Nº	RT	Identified Product	Mass	%	Natural Compound, Structure
1	20:02	Methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate	210	1.1
2	20:21	Methyl 4-hydroxy-2-methoxy-3,6-dimethylbenzoate	210	26.6
3	20:33	Methyl 2,4-dihydroxy-3,6-dimethylbenzoate	196 C9H10O4	38.9
4	22:51	Unidentified	312	0.9	Unidentified
5	22:51	13-Methyl-17-nor-8β,13β-kaur-15-ene Probably the natural product will be (-)-ent-Kauran-16α-ol	272 C20H32 Hibaene	1.8

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.

Figure 5

Chromatogram of Hexane extract part insoluble in MeOH esterified whit diazometane, 2-Cires.

Table 4

Sample Parmotrema hypoleucinum (in Quercus coccifera), Hexane extract part insoluble in MeOH esterified whit diazomethane, 2-Cires.

Nº	RT	Identified Product	Mass	%	Natural Compound, Structure
1	20:24	Methyl 2,4-dihydroxy-3,5,6-trimethylbenzoate	210	0.9
2	20:35	Methyl 2,4-dimethoxy-6-methylbenzoate	210	41.4
3	20:38	Methyl 2,4-dihydroxy-3,6-dimethylbenzoate	196	48.9

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 1-Cires sample, 4-hydroxy-2-methoxy-3,6-dimethylbenzoic acid is also identified, and this product does not appear in the 2-Cires sample; instead, the 2,4-dimethoxy-6-methylbenzoic acid appears in 2-Cires.

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).

Figure 6 and Figure 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.

Figure 6

Chromatogram of Hexane extract part soluble in MeOH, 1-Hexane.

Figure 7

Chromatogram of Hexane extract part soluble in MeOH esterified whit diazomethane, 2-Hexane.

Table 5

Samples Parmotrema hypoleucinum (in Olea europea), Hexane extract part soluble in MeOH 1-Hexane and Parmotrema hypoleucinum (in Quercus coccifera), Hexane extract part soluble in MeOH, 2-Hexane.

Nº	RT	[M-H]−	Mass Calc	Formula	Formula	Compounds
				1-Hexane	2-Hexane
1	2.67	181.0502	182.0574	C9H10O4	C9H10O4	3-Methylorsellinic acid
2	2.72	187.0970	188.1043	C9H16O4	C9H16O4	3,5-Dimethoxyciclohexanecarboxilic acid
3	2.75	293.0669	294.0741	C14H14O7	C14H14O7	6-(Hydroxymethyl)-3,5-bis(methoxycarbonyl)-2,4-dimethylcyclohex-1-ene-1-carboxylic acid
4	3.08	243.1239	244.1311	C12H20O5	C12H20O5	3,5,6-Hydroxymethyl-2,4-dimethylcyclohex-1-ene-1-carboxylic acid
5	3.15	151.0393	152.0465	C8H8O3	C8H8O3	Atranol
6	3.20	225.1129	226.1201	C12H18O4	C12H18O4	5-Formyl-3-hydroxymethyl-2,4,6-trimethylcyclohex-1-ene-1-carboxylic acid
7	3.47	199.0973	200.1046	C10H16O4	C10H16O4	3,5-Dihydroxy-2,4,6-trimethylciclohexenecarboxilic acid
8	3.55	195.0660	196.0730	-	C10H12O4	3,5-Dimethylorsellinic acid
9	3.58	149.0237	150.0310	C8H6O3	C8H6O3	4-Formylbenzoic acid
10	3.82	241.1081	242.1153	C12H18O5	C12H18O5	5-Formyl-3,6-dihydroxymethyl-2,4-dimethylcyclohex-1-enecarboxylic acid
11	4.10	201.1129	202.1202	C10H18O4	C10H18O4	2,4-Dihydroxy-3,5,6-trimethylcyclohexane-1-carboxylic acid
12	4.95	199.1337	200.1409	C11H20O3	C11H20O3	2-Hydroxy-10-undecenoic acid
13	5.00	185.0006	186.0079	C8H7ClO3	C8H7ClO3	Chloroatranol
14	5.10	213.1130	214.1203	C11H18O4	C11H18O4	2-Undecenedioic acid
15	5.21	169.0863	170.0936	C9H14O3	C9H14O3	4-Hydroxy-2,5-dimethylcyclohex-1-ene-1-carboxylic acid
16	5.49	163.0392	164.0470	C9H8O3	C9H8O3	p-Coumaric acid
17	5.60	209.1181	210.1253	C12H18O3	C12H18O3	6-(1-Oxopentyl)-1-cyclohexene-1-carboxylic acid
18	5.87	215.1286	216.1359	C11H20O4	C11H20O4	Undecanedioic acid
19	6.57	227.1288	228.1360	C12H20O4	C12H20O4	trans-Dodec-2-enedioic acid
20	7.44	282.2078	283.2150	C16H29NO3	C16H29NO3	N-Dodecanoyl-L-Homoserine lactone
21	7.67	329.2336	330.2252	-	C18H34O5	9,10-Dihydroxy-8-oxo-12-octadecenoic acid
22	7.77	209.0817	210.0889	C11H14O4	C11H14O4	3,4-Dimethoxyhydrocinnamic acid
23	8.70	373.1294	374.1366	C20H22O7	C20H22O7	4-O-demethyldivaricatic acid
24	9.01	253.1809	254.1882	C15H26O3	C15H26O3	9-Hydroxy-10,12-pentadecadienoic acid
25	9.53	243.1601	244.1675	C13H24O4	C13H24O4	Tridecanedioic acid
26	10.13	359.1139	360.1211	C19H20O7	-	Barbatic acid
27	10.30	375.1086	376.1159	C19H20O8	C19H20O8	8-Hydroxybarbatic acid
28	10.35	373.0931	374.1001	C19H18O8	C19H18O8	Baeomycesic acid
29	10.59	311.2230	312.2302	C18H32O4	C18H32O4	9Z-Octadecenedioic acid
30	10.77	351.2178	352.2256	C20H32O5	C20H32O5	structure proposed
31	10.87	233.1547	234.1619	C15H22O2	C15H22O2	Fukinanolide
32	10.89	323.2230	324.2303	C19H32O4	C19H32O4	allo-Protolichestrinic acid
33	10.91	411.0045	412.0118	C18H14Cl2O7	C18H14Cl2O7	Leoidin
34	10.91	396.9888	397.9961	C17H12Cl2O7	C17H12Cl2O7	Lecideoidin
35	11.49	403.1399	404.1473	C21H24O8	C21H24O8	4′-O-demethylsekikaic acid
36	12.28	313.2388	314.2266	-	C18H34O4	Octadecanedioic acid
37	13.00	269.2124	270.2195	C16H30O3	C16H30O3	2-Oxopalmitic acid
38	13.04	389.1245	390.1315	C20H22O8	C20H22O8	8-Hydroxydiffractaic acid
39	13.34	293.2124	294.2202	C18H30O3	C18H30O3	2-Hydroxylinolenic acid
40	13.58	291.1968	292.2041	C18H28O3	C18H28O3	α-Licanic acid
41	14.38	295.2279	296.2351	C18H32O3	C18H32O3	2-Hydroxylinoleic acid isomer
42	14.39	455.1711	456.1783	C25H28O8	C25H28O8	Glomelliferonic acid
43	14.40	295.2278	296.2352	-	C18H32O3	18-Hydroxylinoleic acid
44	14.68	321.2437	322.2509	C20H34O3	C20H34O3	Hydroxyeicosatrienoic acid
45	14.95	305.2125	306.2197	C19H30O3	C19H30O3	14-Oxo-7,10,12-nonadecatrienoic acid
46	15.19	297.2436	298.2508	C18H34O3	C18H34O3	9-Oxooctadecanoic acid
47	15.25	295.2280	296.2351	C18H32O3	C18H32O3	Coriolic acid
48	15.36	297.2435	298.2508	C18H34O3	C18H34O3	Ricinoleic acid
49	15.42	373.0925	374.0999	C19H18O8	C19H18O8	Atranorin
50	15.43	177.0187	178.0259	C9H6O4	C9H6O4	6,7-Dihydroxycoumarin
51	15.44	277.2536	278.2610	C19H34O	C19H34O	7,10,12-nonadecatrien-1-ol
52	15.46	365.2330	366.2403	C21H34O5	C21H34O5	Muronic acid
53	15.70	277.2538	278.2612	C18H30O2	C18H30O2	Linolenic acid
54	15.98	367.2488	368.2562	C21H36O5	C21H36O5	Murolic acid
55	16.16	311.2594	312.2667	C19H36O3	C19H36O3	Lichesterylic acid
56	16.29	461.2550	462.2619	C26H38O7	C26H38O7	Unidentified
57	16.37	471.3481	472.3553	C30H48O4	C30H48O4	Unidentified
58	16.45	381.2282	382.2356	C21H34O6	C21H34O6	Praesorediosic acid
59	16.47	210.9834	211.9873	C9H5ClO4	C9H5ClO4	7-Chloro-3-oxo-1,3-dihydroisobenzofuran-5- carboxylic acid.
60	16.47	381.2285	382.2355	C21H34O6	C21H34O6	19-Acetoxy-protolichesterinic acid
61	16.51	407.0540	408.0611	C19H17ClO8	C19H17ClO8	Chloroatranorin
62	16.90	421.9285	422.9363	C16H10Cl4O5	C16H10Cl4O5	Diploicin
63	16.97	201.1493	202.1564	C11H22O3	C11H22O3	11-Hydroxyundecanoic acid
64	16.97	385.2960	386.3032	C22H42O5	C22H42O5	Unidentified
65	17.59	387.2544	388.2616	C24H36O4	C24H36O4	Unidentified
66	17.68	389.1242	390.1314	C20H22O8	C20H22O8	Leprolomin
67	17.69	395.2442	396.2520	C22H36O6	-	structure proposed
68	17.80	357.0983	358.1057	C19H18O7	C19H18O7	structure proposed:
69	17.97	449.3276	450.3354	C35H38O7	C35H38O7	Unidentified
70	18.04	253.2173	254.2244	C16H30O2	C16H30O2	Palmitoleic acid
71	18.33	241.2172	242.2245	C15H30O2	C15H30O2	Pentadecanoic acid
72	18.35	455.3531	456.3605	C30H48O3	C30H48O3	Ursolic acid
73	18.50	279.2330	280.2403	C18H32O2	C18H32O2	Linoleic acid
74	18.66	299.2595	300.2667	C18H36O3	C18H36O3	2-Hydroxyoctadecanoic acid
75	18.78	279.2332	280.2403	C18H32O2	C18H32O2	Linoleic acid isomer, cis,trans
76	19.03	497.2548	498.2626	C29H38O7	-	Superpicrolichenic acid
77	19.16	255.2329	256.2401	C16H32O2	C16H32O2	Palmitic acid
78	19.49	281.2485	282.2559	C18H34O2	C18H34O2	Oleic acid
79	19.62	269.2488	270.2561	C17H34O2	C17H34O2	15-Methylhexadecanoic acid
80	19.89	269.2488	270.2561	C17H34O2	C17H34O2	Heptadecanoic acid
81	19.99	483.3481	484.3553	C31H48O4	C31H48O4	Unidentified
82	20.07	327.2543	328.2616	C20H40O3	C20H40O3	2-Hydroxyeicosanoic acid
83	20.17	473.2548	474.2626	C27H38O7	C27H38O7	Unidentified
84	20.54	283.2643	284.2716	C18H36O2	C18H36O2	Stearic acid (octadecanoic acid)
85	20.74	309.2801	310.2875	C20H38O2	C20H38O2	Eicosenoic acid
86	21.02	441.3377	442.3448	C29H46O3	C29H46O3	Unidentified
87	21.14	297.2801	298.2873	C19H38O2	C19H38O2	Nonadecanoic acid
88	21.70	311.2957	312.3029	C20H40O2	C20H40O2	(Eicosanoic acid) Arachidic acid
89	22.02	597.4164	598.4242	C37H58O6	-	Unidentified
90	22.02	669.4739	670.4817	C41 H66 O7	-	Unidentified
91	22.02	545.2758	546.2836	C30H42O9	C30H42O9	Structure proposed and confirmed by the ions
92	22.52	579.2368	580.2446	C36H36O7	-	Unidentified
93	22.66	753.4057	754.4135	C37H62O14	C37H62O14	Unidentified
94	22.68	637.4841	638.4908	C41H66O5	C41H66O5	Unidentified
95	22.69	751.4779	752.4857	C45H38O9	C45H38O9	Unidentified
96	23.78	603.3334	604.3412	C37H48O7	C37H48O7	Unidentified
97	23.81	633.3798	634.3876	C39H54O7	-	Unidentified
98	24.12	467.4109	468.4187	C30H56O4	-	12-Triacontenedioic acid
99	24.51	605.3483	606.3561	C37H50O7	C37H50O7	Unidentified

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 fatty acid profile, we find the following acids common to the two samples: 2-hydroxy-10-undecenoic, 2-undecenedioic, undecanedioic, trans-2-dodecenedioic, 9-hydroxy-10,12-pentadecadienoic, tridecanedioic, 9Z-octadecenedioic, 10-acyl-9-formyl-13-hydroxyoctadeca-6,11-dienoic, octadecanedioic, 2-oxopalmitic, nonadecanoic, arachidic and 12-triacontenedioic acids.

Among the acids that have a cyclohexanecarboxylic base, the following derivatives were also found, as described in the publication of the lichen Physcia mediterranea Nimis [35], the 3,5-dimethoxycyclohexanecarboxylic acids, 6-(hydroxymethyl)-3,5-bis(methoxycarbonyl)-2,4-dimethylcyclohex-1-ene-1-carboxylic, 3,5,6-hydroxymethyl-2,4-dimethylcyclohex-1-ene-1-carboxylic, 5-formyl-3-hydroxymethyl-2,4,6-trimethylcyclohex-1-ene-1-carboxylic, 3,5-dihydroxy-2,4,6-trimethylcyclohex-1-ene-1-carboxylic, 5-formyl-3,6-dihydroxymethyl-2,4-dimethylcyclohex-1-ene-1-carboxylic, 2,4-dihydroxy-3,5,6-trimethylcyclohexane-1-carboxylic, 4-hydroxy-2,5-dimethylcyclohex-1-ene-1-carboxylic, 6-(1-oxopentyl)-cyclohex-1-ene-1-carboxylic acids.

Lactones were also identified: N-dodecanoyl-L-homoserine lactone and fukinanolide, other derivatives such as leoidin, lecideoidin, an alcohol such as 7,10,12-nonadecatrien-1-ol and a triterpenic acid identified as ursolic acid (found in the two extracts).

In the comparison of the characteristic polyphenolic compounds of the lichens in these two samples, 20 compounds were identified: 4-O-demethyldivaricatic, barbatic, 8-hydroxybarbatic, baeomycesic, allo-protolichesterinic, 4′-O-demethylsekikaic acids, 8-hydroxydiffractaic, glomelliferonic occurs in Xanthoparmelia subincerta [36], muronic is detected in P. praesorediosum [37], murolic, lichesterylic [35], praesorediosic, 19-acetoxyprotolichesterinic, diploicin, leprolomin, methyl 3-formyl-2-hydroxy-4-((4-methoxy-2-methylbenzoyl)oxy)-6-methylbenzoate, superpicrolichenic and 3-formyl-2-hydroxy-4-((2-(14-hydroxypentadecyl)-4-methyl-5-oxo-2,5-dihydrofuran-3-carbonyl)oxy)-6-methylbenzoic acids.

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-3-oxo-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 Figure 8 and Figure 9 and Table 6.

Figure 8

Chromatogram of Hexane extract part insoluble in MeOH, 1-Cires.

Figure 9

Chromatogram of Hexane extract part insoluble in MeOH, 2-Cires.

Table 6

Samples Parmotrema hypoleucinum (in Olea europea), Hexane extract part insoluble in MeOH, 1-Cires and Parmotrema hypoleucinum (in Quercus coccifera), Hexane extract part insoluble in MeOH, 2-Cires.

Nº	RT	[M-H]−	MW Calc	Formula	Formula	Compounds
				1-Cires	2-Cires
1	2.67	146.9397	147.9475	C4H4O6	-	Dihydroxyfumaric acid
2	0.04	190.9281	191.9359	C5H4O8	-	Methanetetracarboxylic acid
3	1.80	116.9276	117.9354	C4H6O4	-	Butendioic acid
4	2.19	112.9845	113.9932	C4H2O4	C4H2O4	2,3-Dioxobuten-1,4-dial
5	2.72	187.0971	188.1049	C9H16O4	C9H16O4	3,5-Dimethoxyciclohexanecarboxilic acid
6	4.12	201.1129	202.1207	C10H18O4	C10H18O4	2,4-Dihydroxy-3,5,6-trimethylcyclohexane-1-carboxylic acid
7	5.51	163.0395	164.0473	C9H8O3	-	p-Coumaric acid
8	5.85	268.1919	269.1997	C12H28O6	C12H28O6	Unidentified
9	6.57	227.1286	228.1364	-	C12H20O4	trans-Dodec-2-enedioic acid
10	7.46	282.2077	283.2155	C16H29NO3	C16H29NO3	N-Dodecanoyl-L-homoserine lactone
11	8.09	174.9556	175.9634	C5H4O7	-	2-Hydroxy-3,4-dioxopentanedioc acid
12	8.15	293.1762	294.1840	C17H26O4	-	gingerol
13	8.39	323.2230	324.2308	-	C19H32O4	allo-protolichestrinic acid
14	9.50	350.2337	351.2415	C17H34O7	C17H34O7	Xylitollaurate
15	10.88	233.1546	234.1624	-	C15H22O2	Fukinanolide
16	11.43	334.2389	335.2467	C17H34O6	C17H34O6	Unidentified
17	11.75	311.2231	312.2309	C18H32O4	C18H32O4	9Z-octadecenedioic acid
18	13.16	293.2126	294.2204	-	C18H30O3	2-Hydroxylinolenic acid
19	13.95	319.2280	320.2358	-	C20H32O3	5-Hydroxyeicosatetraenoic acid
20	14.37	295.2280	296.2358	C18H32O3	C18H32O3	18-Hydroxylinoleic acid
21	14.67	321.2436	322.2514	C20H34O3	-	Hydroxyeicosatrienoic acid
22	14.90	346.2390	347.2468	C18H34O6	C18H34O6	9,10,14-trihydroxy-12-oxooctadecanoic acid
23	14.98	297.2434	298.2512	C18H34O3	C18H34O3	9-oxooctadecanoic acid
24	15.36	177.0186	178.0264	C9H6O4	C9H6O4	6,7-Dihydroxycoumarin
25	15.44	373.0929	374.1007	C19H18O8	C19H18O8	Atranorin
26	16.42	210.9801	211.9879	C9H5O4Cl	C9H5O4Cl	7-chloro-3-oxo-1,3-dihydroisobenzofuran-5- carboxylic acid.
27	16.47	407.0539	408.0617	C19H17O8Cl	C19H17O8Cl	Chloroatranorin
28	17.57	277.2175	278.2203	-	C18H30O2	Octadeca-9,12,15-trienoic acid
29	17.65	389.1246	390.1324	C20H22O8	-	8-Hydroxydiffractaic acid,
30	17.77	265.1480	266.1558	C15H22O4	C15H22O4	(4E,6E,9E)-Pentadeca-4,6,9-trienedioic acid
31	18.03	253.2331	254.2249	C16H30O2	C16H30O2	Palmitoleic acid
32	18.28	402.3016	403.3094	-	C22H42O6	Unidentified
33	18.34	241.2173	242.2251	C15H30O2	C15H30O2	Pentadecanoic acid
34	18.35	455.3534	456.3612	C30H48O3	-	Oleanolic acid
35	18.50	279.0936	280.2409	C18H32O2	C18H32O2	Linoleic acid
36	18.66	489.3375	490.3453	-	C26H50O8	Unidentified
37	18.86	403.2645	404.2723	-	C28H36O2	Unidentified
38	18.91	267.2331	268.2409	C17H32O2	C17H32O2	cis-9-Heptadecenoic acid
39	19.15	255.2329	256.2407	C16H32O2	C16H32O2	Palmitic acid
40	19.35	459.3271	460.3349	C25H48O7	C25H48O7	Unidentified
41	19.51	281.2487	282.2565	C18H34O2	C18H34O2	Oleic acid
42	19.75	459.3272	460.3350	C25H48O7	-	Methyl glucose isostearate
43	19.89	269.2488	270.2566	C17H34O2	C17H34O2	Heptadecanoic acid
44	19.99	307.2645	308.2723	C20H36O2	C20H36O2	11,14-Eicosadienoic acid
45	20.07	457.3722	458.3800	C27H54O5	C27H54O5	Unidentified
46	20.18	295.2645	296.2723	C19H36O2	C19H36O2	10E-nonadecenoic acid
47	20.54	283.2643	284.2721	C18H36O2	C18H36O2	Stearic acid (Octadecanoic acid)
48	20.82	309.2800	310.2878	C20H38O2	C20H38O2	Eicosenoic acid
49	20.86	505.3326	506.3404	-	C26H50O9	Unidentified
50	21.05	457.3722	458.3800	C27H54O5	-	Unidentified
51	21.71	311.2957	312.3035	-	C20H40O2	(Eicosanoic acid) arachidic acid
52	22.62	297.1532	298.1610	C12H26O8	C12H26O8	Unidentified
53	22.67	637.4836	638.4914	C24H60O12N7	-	Unidentified
54	22.77	339.3268	340.3346	C22H44O2	C22H44O2	Docosanoic acid
55	23.08	309.1743	310.1821	C17H26O5	C17H26O5	Portentol
56	23.58	353.2003	354.2081	C19H30O6	C19H30O6	Unidentified
57	23.81	311.1689	312.1767	C13H30O8	C13H30O8	Unidentified
58	23.89	367.3579	368.3657	C24H48O2	C24H48O2	Lignoceric acid
59	24.01	397.2266	398.2344	C21H34O7	C21H34O7	Stephanol
60	24.60	293.1793	294.1871	C17H26O4	C17H26O4	Heptadecatrienedioic acid
61	24.94	325.1844	326.1922	C14H30O8	C14H30O8	Unidentified
62	25.33	395.3895	396.3973	C26H52O2	-	Hexacosanoic acid or cerotic acid
63	25.89	337.2055	338.2133	C19H30O5	C19H30O5	6-Oxononadeca-8,11-dienedioic acid
64	26.10	339.2000	340.2078	C15H32O8	C15H32O8	Unidentified
65	26.24	381.2317	382.2395	C21H34O6	C21H34O6	19-Acetoxylichesterinic acid
66	26.75	425.2581	426.2659	C23H38O7	-	Asebotoxin I
67	27.53	321.2106	322.2184	C19H30O4	C19H30O4	Nonadecatrienedioic Acid
68	27.85	304.9143	305.9221	Noformula	-	Unidentified

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 Figure 10 and Figure 11 and the results of the identification of the compounds in Table 7.

Figure 10

Chromatogram of 1-Chloroform extract from P. hypoleucinum.

Figure 11

Chromatogram of 2-Chloroform extract from P. hypoleucinum.

Table 7

Samples Parmotrema hypoleucinum (in Olea europea), Chloroform extract, 1-Chloroform and Parmotrema hypoleucinum (in Quercus coccifera), Chloroform extract, 2-Chloroform.

Nº	RT	[M-H]−	Mass Calc	Formula	Formula	Compounds
				1-Chloroform	2-Chloroform
1	0.98	174.9557	175.9635	C5H4O7	-	2-Hydroxy-3,4-dioxopentanedioic acid
2	1.01	145.0975	146.1053	-	C7H14O3	2-Hydroxyheptanoic acid
3	1.05	112.9845	113.9923	-	C4H2O4	Acetylenedicarboxylic acid (Squaric acid)
4	1.12	182.9882	183.9960	-	C7H4O6	Chelidonic acid
5	1.14	341.1091	342.1169	-	C12H22O11	Sucrose
6	1.52	215.0097	216.0175	C8H8O7	C8H8O7	D-diacetyltartaric anhydride
7	1.74	433.0778	434.0856	C20H18O11	C20H18O11	Avicularin
8	1.76	403.0675	404.0753	C19H16O10	C19H16O10	Euxanthic acid
9	1.92	417.0468	418.0546	C19H14O11	-	Shoyuflavone C
10	2.41	433.0780	434.0858	C20H18O11	C20H18O11	Morin 3-alpha-L-arabinopyranoside
11	2.57	401.0518	402.0596	C19H14O10	C19H14O10	Shoyuflavone B
12	2.64	403.0672	404.0750	C19H16O10	C19H16O10	Euroxanthone B
13	3.15	447.0934	448.1012	C21H20O11	C21H20O11	Quercitrin
14	3.23	401.0515	402.0593	C19H14O10	C19H14O10	Constictic acid
15	3.38	182.9882	183.9960	C8H5O3Cl	C8H5O3Cl	3-Chloro-4-formylbenzoic acid
16	3.48	187.0972	188.1050	C9H16O4	-	Azelaic acid
17	3.63	371.0412	372.0490	C18H12O9	-	Norstictic acid
18	3.90	313.0722	314.0800	-	C17H14O6	Cirsimaritin
19	3.91	442.1145	443.1223	C18H29O8Cl2	-	Unidentified
20	3.99	373.0567	374.0645	-	C18H14O9	Menegazziaic acid
21	4.02	399.0361	400.0439	C19H12O10	-	Kynapcin-28
22	4.13	357.0617	358.0695	C18H14O8	C18H14O8	Succinyldisalicylic acid
23	4.24	401.0515	402.0593	C19H14O10	-	Siphulellic acid
24	4.27	373.0568	374.0646	C18H14O9	-	Protocetraric acid
25	5.03	417.0830	418.0908	C20H18O10	C20H18O10	Conphysodalic acid
26	5.09	385.0568	386.0646	C19H14O9	C19H14O9	Stictic acid
27	5.18	387.0721	388.0799	C19H16O9	C19H16O9	Cryptostictic acid
28	6.02	385.0565	386.0643	C19H14O9	C19H14O9	3,3′-Carbonylbis [6-(methoxycarbonyl)-benzoic acid]
29	6.14	431.0984	432.1062	C21H20O10	C21H20O10	Genistein 7-glucoside (Genistin)
30	6.32	209.0849	210.0927	-	C11H14O4	Sinapyl alcohol
31	7.63	309.1017	310.1095	-	C15H18O7	1-O-cis-cinnamoyl-β-D-glucopyranose
32	8.11	373.0568	374.0646	-	C18H14O9	Menegazziaic acid isomer
33	8.67	371.0408	372.0486	C18H12O9	C18H12O9	Substictic acid
34	9.54	293.1763	294.1841	C17H26O4	C17H26O4	Nordihydrocapsiate
35	10.26	771.1205	772.1283	C38H28O18	C38H28O18	fucofuroeckol A hepta-acetate
36	10.61	426.9681	427.9759	-	C16H12O14	Unidentified
37	11.11	475.3278	476.3356	C25H48O8	C25H48O8	Tetrahydroxypentacosanedioic acid
38	12.21	345.0982	346.1060	C18H18O7	-	Isooptusatic acid (or 3′-Methylevernic acid)
39	13.12	265.1482	266.1560	C15H22O4	C15H22O4	EthyI 4-O-methylolivetolcarboxylate
40	13.34	467.0985	468.1063	C24H20O10	C24H20O10	Gyrophoric acid
41	13.36	317.0670	318.0748	C16H14O7	-	Lecanoric acid
42	13.80	503.3593	504.3671	C27H52O8	C27H52O8	Tetraglyceryl monooleate
43	14.47	517.3745	518.3823	C28H54O8	-	13-beta-D-glucosyloxy)docosanoic acid
44	14.83	359.0776	360.0854	-	C18H16O8	Ramalinaic acid
45	15.40	365.2334	366.2412	C21H34O5	C21H34O5	Muronic acid
46	15.87	265.1479	266.1557	C15H22O4	C15H22O4	Ivambrin
47	16.02	367.2491	368.2569	-	C21H36O5	Constipatic acid or Protoconstipatic acid
48	16.28	297.1532	298.1610	C12H26O8	C12H26O8	Unidentified
49	17.14	177.0187	178.0265	C9H6O4	C9H6O4	6,7-dihydroxycoumarin
50	17.37	309.1743	310.1821	C17H26O5	C17H26O5	Portentol
51	18.01	311.1690	312.1768	C13H28O8	C13H28O8	heptahydroxytridecanol
52	18.03	407.0541	408.0619	C19H17O8Cl	C19H17O8Cl	Chloroatranorin
53	18.05	210.9801	211.9879	C7H10O3Cl2	C7H10O3Cl2	2-Methoxy-3,4-dichloro-6-methyltetrahydropyran-5-one
54	18.32	353.2004	354.2082	C19H30O6	C19H30O6	tetraoxononadecanoic acid
55	18.36	421.2265	422.2343	-	C23H34O7	Sarmentologenin
56	18.85	397.2268	398.2346	C21H34O7	-	Stephanol
57	19.13	387.2544	388.2622	-	C24H36O4	Dehydrodeoxycholic acid
58	19.16	441.2530	442.2608	C23H38O8	-	Asebotoxin IV
59	19.32	325.1846	326.1924	C21H26O3	-	Linderanolide
60	19.55	253.2172	254.2250	C16H30O2	C16H30O2	palmitoleic acid (9-cis-hexadecenoic acid)
61	19.64	255.2331	256.2409	C16H32O2	-	palmitic acid
62	19.74	293.1796	294.1874	-	C17H26O4	Gingerol
63	19.92	279.2332	280.2410	-	C18H32O2	Linoleic acid
64	20.35	267.2332	268.241	C17H32O2	C17H32O2	2-Heptadecenoic acid
65	20.51	283.2645	284.2723	C18H36O2	C18H36O2	Stearic acid
66	20.88	281.2488	282.2566	C18H34O2	C18H34O2	Oleic acid
67	22.25	565.3784	566.3862	C32H54O8	-	Unidentified
68	23.08	679.4650	680.4728	C35H68O12	C35H68O12	Unidentified
69	23.11	761.5977	762.6055	C35H68O12	C35H68O12	Unidentified
70	23.14	395.3897	396.3975	-	C26H52O2	Hexacosanoic acid
71	23.84	337.2057	338.2135	C19H30O5	C19H30O5	Idebenone
72	23.91	367.3583	368.3661	C24H48O2	-	Lignoceric acid (tetracosanoic acid)
73	24.13	637.4844	638.4922	-	C34H70O10	Unidentified
74	24.25	639.3973	640.4051	C31H60O13	-	Unidentified
75	24.44	381.3741	382.3819	C25H50O2	-	Pentacosanoic acid
76	24.56	339.2000	340.2078	C15H32O8	C15H32O8	Heptahydroxypentadecanol
77	24.60	679.4650	680.4728	C35H68O12	C35H68O12	Unidentified
78	24.80	535.3132	536.3210	C26H48O11	C26H48O11	Unidentified

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.

As in the previous analyses, fatty acids, hydroxy acids, oxo acids, some xanthones and flavones have been found, in addition to menegazziaic, siphullelic, protocetraric, conphysodalic, cryptostictic, menegazziaic isomer, gyrophoric, lecanoric and muronic acids among others.

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.

Figure 12

Chromatogram of 1-Ethanol extract from P. hypoleucinum.

Table 8

Samples Parmotrema hypoleucinum (in Olea europea), Ethanol extract, 1-Ethanol and Parmotrema hypoleucinum (in Quercus coccifera), Ethanol extract, 2-Ethanol.

Nº	RT	[M-H]−	MW Calc	Formula	Formula	Compounds
				1-Ethanol	2-Ethanol
1	0.98	174.9556	175.9634	C5H4O7	C5H4O7	2-Hydroxy-3,4-dioxopentanedioic acid
2	1.05	112.9845	113.9923	C4H2O4	C4H2O4	Acetylenedicarboxylic acid (Squaric acid)
3	1.15	311.1156	312.1234	C15H20O7	C15H20O7	Neoanisatin
4	1.19	151.0603	152.0681	C5H12O5	C5H12O5	Arabitol
5	1.82	182.9882	183.9960	C7H4O6	C7H4O6	Chelidonic acid
6	2.99	401.0517	402.0595	C19H14O10	C19H14O10	Constictic acid
7	3.95	357.0617	358.0695	C18H14O8	C18H14O8	Hyposalazinic acid, Psoromic acid or Virensic acid
8	4.17	519.1147	520.1225	C24H24O13	-	Eujambolin
9	4.91	387.0722	388.0800	C19H16O9	C19H16O9	Cryptostictic acid
10	5.06	385.0568	386.0646	C19H14O9	C19H14O9	Stictic acid
11	5.19	417.0830	418.0908	C20H18O10	C20H18O10	Juglanin
12	5.71	373.0566	374.0644	C18H14O9	C18H14O9	Protocetraric acid
13	5.88	431.0985	432.1063	C21H20O10	-	Genistin
14	6.51	328.0597	329.0675	C20H11O4N	-	Unidentified
15	6.80	163.0393	164.0471	C9H8O3	C9H8O3	Coumaric acid
16	7.25	359.0775	360.0853	-	C18H16O8	Conhypoprotocetraric acid, [40]
17	7.83	399.0723	400.0801	C20H16O9	-	Methylstictic acid
18	8.44	371.0408	372.0486	C18H12O9	C18H12O9	Substictic acid
19	8.98	209.0453	210.0531	C10H10O5	-	5,6-Dihydroxy-7-methoxy-4-methyl-2-benzofuran-1(3H)-one
20	9.24	293.1763	294.1841	C17H26O4	C17H26O4	(+)-[6]-Gingerol
21	9.33	413.0881	414.0959	C21H18O9	C21H18O9	Vesuvianic acid
22	9.84	461.3123	462.3201	-	C24H46O8	Unidentified
23	10.28	389.2911	390.2989	-	C21H42O6	9,10,12,13-tetrahydroxyheneicosanoic acid
24	10.49	243.0065	244.0143	C10H9O5Cl	-	(4-Chloro-2-formyl-6-methoxyphenoxy)acetic Acid
25	10.97	475.3276	476.3354	C25H48O8	C25H48O8	Tetrahydroxypentacosanedioic acid
26	11.38	265.1481	266.1559	C15H22O4	C15H22O4	EthyI 4-O-methylolivetolcarboxylate
27	11.42	403.3068	404.3146	C22H44O6	C22H44O6	9,10,12,13-Tetrahydroxydocosanoic acid
28	11.62	447.3331	448.3409	-	C24H48O7	D-Glucitol monostearate
29	11.96	489.3434	490.3512	C26H50O8	C26H50O8	Icosanedioic acid bis(2,3-dihydroxypropyl) ester
30	12.05	345.0982	346.1060	C18H18O7	C18H18O7	Isooptusatic acid (or 3′-Methylevernic acid)
31	12.54	417.3224	418.33.02	C23H46O6	C23H46O6	Heptadecyl D-glucoside
32	13.20	343.0823	344.0901	C18H16O7	C18H16O7	Usnic acid
33	13.57	431.3379	432.3457	C24H48O6	C24H48O6	6-Ethyl-6-n-pentyl-pentadecan -4,5,7,8,15-pentol-I5-acetate
34	13.73	503.3593	504.3671	C27H52O8	C27H52O8	Tetraglyceryl monooleate
35	14.25	309.1746	310.1824	C17H26O5	C17H26O5	Portentol
36	14.38	293.2126	294.2204	C18H30O3	C18H30O3	17-Hydroxylinolenic acid
37	14.40	517.3750	518.3828	C28H54O8	C28H54O8	13-(beta-D-Glucosyloxy)docosanoic acid
38	15.34	365.2335	366.2413	C21H34O5	C21H34O5	Muronic acid
39	16.68	297.2438	298.2516	C18H34O3	C18H34O3	Ricinoleic acid
40	18.00	311.1691	312.1769	C13H28O8	C13H28O8	Heptahydroxytridecanol
41	18.20	353.2005	354.2083	C19H30O6	C19H30O6	Tetraoxononadecanoic acid
42	18.71	397.2267	398.2345	C21H34O7	C21H34O7	Stephanol
43	18.93	421.2269	422.2347	C23H34O7	C23H34O7	Sarmentologenin
44	19.09	441.2531	442.2609	C23H38O8	C23H38O8	Asebotoxin IV
45	19.41	485.2796	486.2874	C25H42O9	C25H42O9	2-(7Z,10Z,13Z)-hexadecatrienoyl-3-(β-D-galactosyl)-sn-glycerol
46	19.54	253.2173	254.2251	C16H30O2	C16H30O2	palmitoleic acid (9-cis-hexadecenoic acid)
47	19.83	241.2172	242.2250	-	C15H30O2	Pentadecanoic acid
48	19.93	279.2332	280.2410	C18H32O2	C18H32O2	Linoleic acid
49	20.03	293.1795	294.1873	C17H26O4	C17H26O4	Gingerol
50	20.50	325.1844	326.1922	C21H26O3	C21H26O3	Linderanolide
51	20.63	255.2329	256.2407	C16H32O2	C16H32O2	Palmitic acid
52	20.89	281.2488	282.2566	C18H34O2	C18H34O2	Oleic acid
53	21.18	325.1846	326.1924	-	C14H30O8	Hexahydroxytetradecan-1-ol
54	21.38	591.2616	592.2694	C34H40O9	-	Scortechinone F
55	22.01	283.2645	284.2723	C18H36O2	C18H36O2	Stearic acid
56	22.21	761.5975	762.6053	C46H82O8	C46H82O8	Unidentified
57	22.92	373.0932	374.1010	C19H18O8	-	Baeomycesic acid
58	22.94	535.3136	536.3214	C26H48O11	-	Unidentified
59	23.31	311.2958	312.3036	C20H40O2	C20H40O2	Arachidic acid
60	23.54	337.2057	338.2135	C19H30O5	C19H30O5	Idebenone
61	24.82	339.2000	340.2078	C15H32O8	C15H32O8	Heptahydroxypentadecanol
62	26.80	367.3584	368.3662	C24H48O2	C24H48O2	Tetracosanoic acid
63	27.03	381.2319	382.2397	C21H34O6	C21H34O6	Praesorediosic acid or Protopraesorediosic acid [41,42,43]

Figure 13

Chromatogram of 2-Ethanol extract from P. hypoleucinum.

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.

3. Materials and Methods

3.1. 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.

Figure 14

Location of El Kala National Park (P.N.E.K., 2010).

Parmotrema hypoleucinum (J. Steiner) Hale was identified by Professor Monia Ali Ahmed lichenologist and research director of the Pathology of Ecosystems team at the University of Badji-Mokhtar, Annaba, Algeria. This sample has been deposited in Badji-Mokhtar University, Annaba, code AAM-2.

3.2. HPLC Orbitrap

3.2.1. 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%.

3.2.2. 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.

3.2.3. 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.

3.2.4. 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):

Table 9

Solvents used, % of solvent A and % of solvent B.

Time (min)	%A	%B
0	50	50
20	0	100
25	0	100
26	50	50

Total analysis time was set to 30 min.

3.2.5. 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 ºC. 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.

4. 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.

Esterified samples of 1-Hexane and 2-Hexane were analyzed by GC-MS to identify components of lower polarity. In both samples, the methyl esters of 2,4-dihydroxy-3,5,6-trimethylbenzoic acids, palmitic acid, linoleic acid, oleic acid, stearic acid and the hydrocarbon 13-methyl-17-norkaur-15-ene (Probably the natural product will be (-)-ent-kauran-16α-ol). 4-Hydroxy-2-methoxy-3,5,6-trimethylbenzoic and 2,4-dihydroxy-3,6-dimethylbenzoic acid methyl esters and a product that does not it has been possible to identify mass 288; these products were not found in the esterified sample of 2-Hexane.

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.