Secondary Metabolite Profiling of Species of the Genus Usnea by UHPLC-ESI-OT-MS-MS

Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria, which are considered among the slowest growing organisms, with strong tolerance to adverse environmental conditions. There are about 400 genera and 1600 species of lichens and those belonging to the Usnea genus comprise about 360 of these species. Usnea lichens have been used since ancient times as dyes, cosmetics, preservatives, deodorants and folk medicines. The phytochemistry of the Usnea genus includes more than 60 compounds which belong to the following classes: depsides, depsidones, depsones, lactones, quinones, phenolics, polysaccharides, fatty acids and dibenzofurans. Due to scarce knowledge of metabolomic profiles of Usnea species (U. barbata, U. antarctica, U. rubicunda and U. subfloridana), a study based on UHPLC-ESI-OT-MS-MS was performed for a comprehensive characterization of their secondary metabolites. From the methanolic extracts of these species a total of 73 metabolites were identified for the first time using this hyphenated technique, including 34 compounds in U. barbata, 21 in U. antarctica, 38 in U. rubicunda and 37 in U. subfloridana. Besides, a total of 13 metabolites were not identified and reported so far, and could be new according to our data analysis. This study showed that this hyphenated technique is rapid, effective and accurate for phytochemical identification of lichen metabolites and the data collected could be useful for chemotaxonomic studies.


Introduction
Lichens are symbiotic associations of fungi with microalgae and/or cyanobacteria.Lichens are among the slowest growing organisms with strong tolerance to adverse environmental conditions ranging from plains to the highest mountains of tropical to Arctic regions under xeric to aquatic conditions.Lichens can grow on or within rocks, soil, trees, shrubs, trunks, on bricks, leather and wood.They have been used since ancient times as sources of color dyes, cosmetics, and as medicine for the treatment of bronchitis, asthma, leprosy, burning sensation, spleen enlargement, heart diseases, stomach disorders, liver pain, inflammation, and vomiting [1,2].
On the other hand, the genus Usnea contains more the 360 species, which are distributed in polar and tropical regions and their morphology is characterized by the presence of a cartilaginous central axis composed by dense fungal hyphae.Usnic acid is the most typical and abundant compound in the genus Usnea, which gives yellow color to thalli.Traditionally, the genus Usnea has been used to treat diarrhea, ulcers, urinary infections, tuberculosis, pneumonia, stomachache, antifungal, and cattle fungal diseases.The phytochemistry from Usnea species has revealed the presence of almost 60 compounds, distributed among depsidones, depsides, depsones, lactones, quinones, polyphenolics, polysaccharides, fatty acids, and dibenzofurans [2].
Continuing our research on lichens, we have analyzed the phytochemical profile of four Usnea species for the first time based on UHPLC-DAD coupled with high resolution electrospray ionization tandem mass spectrometry (ESI-MS-MS).

Results and Discussion
Four Usnea species were studied in order to determine their metabolomics profiles and chemical fingerprints: U. barbata from Longavi, Chile; U. antarctica from Antarctica, U. rubicunda and U. subfloridana from Colombia.

Metabolomics in Usnea barbata
Forty-four peaks (Figure 1) were detected for the first time in a methanolic extract (Table S1 in Supplementary Information) using UHPLC/ESI/MS/MS in negative mode.Thirty-four compounds identified in this species were mainly depsides, depsidones, lipids, diphenylether derivatives and dibenzofurans.

Metabolomics in Usnea antarctica
Twenty-one peaks (Figure 2) were identified for the first time in the methanolic extract (Table 1) using UHPLC/ESI/MS/MS in negative mode.Among the classes of compounds identified we can cite depsides, depsidones, lipids, and dibenzofurans.

Depsides
Five depsides were identified (peaks 48, 58, 63, 70 and 72) using UHPLC with DAD and HRMS and MS 2 analysis.Peak 48 was identified as gyrophoric acid.Peak 58 and 72 were identified as baeomycesic acid and 8-hydroxybarbatic acid, showing [M − H] − ions at m/z 373.0915 and 375.1070, respectively.Peak 63 and 70 were identified as methyl-8-hydroxy-4-O-demethylbarbatate and barbatic acid respectively.These findings are in good agreement with the studies of Castro et al. [7],

Metabolomics in Usnea antarctica
Twenty-one peaks (Figure 2) were identified for the first time in the methanolic extract (Table 1) using UHPLC/ESI/MS/MS in negative mode.Among the classes of compounds identified we can cite depsides, depsidones, lipids, and dibenzofurans.

Depsidones
Four depsidones corresponding to the peaks 24, 36, 40, and 73 were identified using UHPLC with DAD and HRMS-MS analysis.Peak 24 was identified as connorstictic acid.Peak 36 was identified as fumarprotocetraric acid (C 22 H 16 O 12 ), which showed an [M − H] − ion at m/z 471.0547.The fragmentation of this peak 36 produced ions at m/z 355.0441, 311.0545 and 115.0023.Peak 40 with a [M − H] − pseudomolecular ion at m/z 387.0705 was identified as hypoconstictic acid, which showed diagnostic daughter ions at m/z 343.0808 and 299.0923.Finally, peak 73 was identified as lobaric acid.These findings are in good agreement with the reports of Le Pogam et al. [8], Musharraf et al. [9] and Parrot et al. [10].

Other Compounds
Finally, an aromatic compound (peak 77) and two dibenzofurans (peak 64 and 78) corresponding to ethyl-4-O-methylolivetolcarboxylate, usnic acid and placodiolic acid were identified in this extract, respectively.Placodiolic acid showed a molecular anion at m/z 375.

Other Compounds
Finally, an aromatic compound (peak 77) and two dibenzofurans (peak 64 and 78) corresponding to ethyl-4-O-methylolivetolcarboxylate, usnic acid and placodiolic acid were identified in this extract, respectively.Placodiolic acid showed a molecular anion at m/z 375.

Metabolomics in Usnea rubicunda
Forty-five peaks (Figure 3) were detected for the first time in the methanolic extract of U. rubicunda using UHPLC/ESI/MS/MS in negative mode (Table 1).Thirty-eight compounds were identified in U. rubicunda and among them are depsides, depsidones, lipids, diphenylether and dibenzofurans.These findings are in good agreement with the reports of Cornejo et al. [6], Castro et al. [7], Le Pogam et al. [8], Musharraf et al. [9] and Parrot et al. [10].

Metabolomics in Usnea rubicunda
Forty-five peaks (Figure 3) were detected for the first time in the methanolic extract of U. rubicunda using UHPLC/ESI/MS/MS in negative mode (Table 1).Thirty-eight compounds were identified in U. rubicunda and among them are depsides, depsidones, lipids, diphenylether and dibenzofurans.These findings are in good agreement with the reports of Cornejo et al. [6], Castro et al. [7], Le Pogam et al. [8], Musharraf et al. [9] and Parrot et al. [10].

Unknown Compounds
Seven compounds were detected in this extract by the peaks 2, 3, 5, 12, 17, 27 and peak 45 but none of them were identified.

Other Compounds
Finally, a dibenzofuran (peak 78) corresponding to usnic acid was detected and identified in this extract.

Unknown Compounds
Seven compounds were detected in this extract by the peaks 2, 3, 5, 12, 17, 27 and peak 45 but none of them were identified.

Other Compounds
Finally, a dibenzofuran (peak 78) corresponding to usnic acid was detected and identified in this extract.

Metabolomics in Usnea subfloridana
Forty-five (Figure 4) peaks were detected for the first time in the methanolic extract of U. subfloridana using UHPLC/ESI/MS/MS in negative mode (Table 1).Thirty-seven compounds were identified in this species which can be arranged as depsides, depsidones, lipids and dibenzofurans.These findings are in good agreement with the reports of Cornejo et al. [6], Castro et al. [7], Le Pogam [8], Musharraf et al. [9] and Parrot et al. [10].

Metabolomics in Usnea subfloridana
Forty-five (Figure 4) peaks were detected for the first time in the methanolic extract of U. subfloridana using UHPLC/ESI/MS/MS in negative mode (Table 1).Thirty-seven compounds were identified in this species which can be arranged as depsides, depsidones, lipids and dibenzofurans.These findings are in good agreement with the reports of Cornejo et al. [6], Castro et al. [7], Le Pogam [8], Musharraf et al. [9] and Parrot et al. [10].

Other Compounds
Finally, a dibenzofuran corresponding to usnic acid (peak 78) was detected and identified in this extract.
So far, twenty-three depsides have been reported from Usnea genus according to Huneck et al. 1996 [3] and Singh et al. 2016 [2].In this study, we found twenty depsides distributed as: thirteen in U. barbata, five in U. antarctica, nine in U. rubicunda and seven in U. subfloridana.We identified barbatolic acid (peak 8), thamnolic acid (peak 10), squamatic acid (peak 15), baeomycesic acid (peak 58), diffractaic acid (peak 60), barbatic acid (peak 70), and atranorin (peak 79), which were previously reported in the genus Usnea.The others depsides identified by us are reported for the first time in the genus.
Regarding depsidones, thirteen compounds have been reported in the genus Usnea [2,3], while in this study we could identify seventeen.Among the depsidones reported in Usnea genus and identified by us were: protocetraric acid (peak 16), salazinic acid (peak 18), constictic acid (peak 21), menegazziaic acid (peak 25), galbinic acid (peak 32), norstictic acid (peak 35), fumarprotocetraric acid (peak 36), hypoconstictic acid (40), stictic acid (peak 50), and lobaric acid (peak 73).The other depsidones identified in this study are reported for the first time in the genus.Some eight dibenzofurans have been published in the Usnea genus [2,3] so far, but we identified two: placodiolic acid (peak 64) and usnic acid (peak 78).It is necessary to mention that usnic acid has been identified in all the species studied.Thirteen compounds have not been identified, which could be new according to our data.Therefore, these compounds should be worthy of further research (isolation and NMR identification).A pioneering work to determine the components in crude extracts of lichens using MS/MS was done by Leuckert and Holzmann [11].At that study its authors identified usnic acid, diffractaic acid, gyrophoric acid, lecanoric acid, orsellinic acid, ovoic acid, thamnolic acid, hypothamnolic acid, divaricatic acid, fumarprotocetraric acid, protocetraric acid, homosekikaic acid and sekikaic acid by their specific fragmentation patterns, without an isolation methodology, in the following lichens: Alectoria ochroleuca, Umbilicaria torrefacta, Thamnolia vermicularis, Ophioparma ventosa, Cladonia cryptochlorophaea and Cladonia rei.The Tomasi group [10] has reported a chemical study of eight chemotypes of Ramalina siliquosa using LC-ESI-MS/MS and identified ten compounds-conhypoprotocetraric acid, salazinic acid, peristictic acid, cryptostictic acid, protocetraric acid, stictic acid, norstictic acid, hypoprotocetraric acid, 4-O-demethylbarbatic acid, usnic acid-and twenty-two more, which were detected but not identified.In another study, nine compounds were identified using a HPLC-MS/MS approach in nine lichens belonging to the genus Lichina, Collema and Roccella [12].Among them β-orcinol, orsenillic acid, choline sulphate, roccellic acid, montagnetol, lecanoric acid, erythrin, lepraric acid and acetylportentol were identified based on their fragmentation pathways.Choudhary et al. 2015 [9] studied the lichens Parmotrema grayana and Heterodermia obscurata using HPLC-ESI-QqTOF-MS/MS on negative ion mode.A total of fifteen compounds were detected and identified from the dichloromethane and methanolic extracts.Finally, Le Pogam et al. [8] proposed the rapid identification of lichen extracts using laser desorption/ionization time of flight mass spectrometry instead of electrospray ionization.The analyzed samples were Diploicia canescens, Evernia prunastri, Ophioparma ventosa, Pseudevernia furfuracea, Roccella fuciformis, Xanthoria parietina, Cladonia portentosa, flavocetraria nivalis, Lecidella asema, Ramalina siliquosa, Vulpicida pinastri and Usnea filipendula and, in general in each studied species 2-5 compounds were detected.For example in the lichen Usnea filipendula only salazinic acid and usnic acid were detected.

Instruments
A Thermo Scientific Dionex Ultimate 3000 UHPLC system equipped with a quaternary Series RS pump and a Thermo Scientific Dionex Ultimate 3000 Series TCC-3000RS column compartments with a Thermo Fisher Scientific Ultimate 3000 Series WPS-3000RS autosampler and a rapid separations PDA detector controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA and Dionex Softron GmbH, a part of Thermo Fisher Scientific, Bremen, Germany) hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo, Bremen, Germany) were used for analysis.The chromatographic system was coupled with the MS with a Heated Electrospray Ionization Source II (HESI II).Nitrogen (purity > 99.999%) obtained from a Genius NM32LA nitrogen generator (Peak Scientific, Billerica, MA, USA) was employed as both the collision and damping gas.Mass calibration for the Orbitrap™ was performed once a week, in both negative and positive modes, to ensure a working mass accuracy lower than or equal to 5 ppm.Cafeine and n-butylamine (Sigma Aldrich, St. Louis, MO, USA) were the calibration standards for positive ions and buspirone hydrochloride, sodium dodecyl sulfate, and taurocholic acid sodium salt (Sigma Aldrich) were used to calibrate the mass spectrometer.These compounds were dissolved in a mixture of acetic acid, acetonitrile, water and methanol (Merck, Darmstadt, Germany) and were infused using a Chemyx Fusion 100 syringe pump (Thermo Fisher Scientific, Bremen, Germany).XCalibur 2.3 software (Thermo Fisher Scientific, Bremen, Germany) and Trace Finder 3.2 (Thermo Fisher Scientific, San José, CA, USA) were used for UHPLC control and data processing, respectively.Q Exactive 2.0 SP 2 from Thermo Fisher Scientific was used to control the mass spectrometer.

Table 1 .
Identification of metabolites in Usnea species by UHPLC-ESI-MS-MS.