We report for the first time the chemical profiling of volatile organic compounds (VOCs) of gametophyte and sporophyte life stages of Leiosporoceros dussii, from Panama by using headspace-solid phase microextraction-gas chromatography-mass spectrometry in order to assess distinguishing chemical markers between the male and female gametophytes, and sporophytes of this hornwort.

A total of 27 VOCs were identified in L. dussii. Furthermore, the gametophyte and sporophyte showed clear differences in the type and amount of VOCs. The main constituents of L. dussii female thalli were menthacamphor (17.8%), hexanol (12.3%), and menthyl acetate (12.3%), while the major compounds of the male thalli were hexanol (25.3%), β-ionone (21.1%), benzeneacetaldehyde (17.6%), and β-cyclocitral (14.0%). The main VOCs of the sporophytes were hexanal (19.3%), β-cyclocitral (17.6%), 2-nonenal (15.8%), hexanol (12.5%), and β-ionone (10.2%). Unique compounds found in the female thalli were 3-pentanone, 3-octenol, nonanol, estragole, and menthyl acetate, and in the male thalli were methyl heptenone, nonanal, neoisomenthol, and bornyl acetate. Isomenthol, thymol, isomenthol acetate, and β-methylnaphthalene were only found in the sporophyte. The characteristic VOCs

identified in L. dussii suggest a difference between the chemical constituents of L. dussii and other hornworts species. The presence of simple VOCs when compared with compounds previously characterized in another hornwort genera may support the distinct genetic nature of this species

Durant-Archibold, Armando A.

Garrido, Anette

Gudiño Ledezma, Jose

Gupta, Mahabir P.

Salazar Allen, Noris

Villarreal A., Juan Carlos

UP-RID - Repositorio Institucional Digital de la Universidad de Panama

1Biochemistry and Molecular Medicine Research Unit, Center for Biodiversity and Drug Discovery, Institute for Scientific Research and High Technology Services (INDICASAT AIP), Panama, Panama2Smithsonian Tropical Research Institute (STRI), Ancon, Panama, Panama3Departamento de Bioquímica, Facultad de Ciencias Naturales, Exactas y Tecnología. Universidad de Panamá, Panamá4Department of Biology, Université Laval, Québec, Canada5Centro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Facultad de Farmacia, Universidad de Panamá, Panamá*The authors Armando A. Durant-Archibold and Noris Salazar Allen contributed equally to the work.Corresponding Author: Armando A. Durant-Archibold, Biochemistry and Molecular Medicine Research Unit, Center for Biodiversity and Drug Discovery, Institute for Scientific Research and High Technology Services (INDICASAT AIP), City of Knowledge, Panama, Panama.Email:  armando. durant@ gmail. comOriginal ArticleNatural Product CommunicationsAugust 2019: 1–4© The Author(s) 2019Article reuse guidelines: sagepub. com/ journals- permissionsDOI: 10.1177/1934578X19868875 journals. sagepub. com/ home/ npxCreative Commons Non Commercial CC BY-NC: This article is distributed under the terms of  the Creative Commons Attribution-NonCommercial 4.0 License (http://www. creativecommons. org/ licenses/ by- nc/ 4. 0/) which permits non-commercial use, reproduction and distribution of  the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https:// us. sagepub. com/ en- us/ nam/ open- access- at- sage).Chemical Profiling of Volatile Components of the Gametophyte and Sporophyte Stages of the Hornwort Leiosporoceros dussii (Leiosporocerotaceae) From Panama by HS-SPME-GC-MSAnette Garrido1, Jose Gudiño Ledezma2, Armando A. Durant-Archibold1,3*, Noris Salazar Allen2*, Juan Carlos Villarreal A2,4, and Mahabir P. Gupta5AbstractWe report for the first time the chemical profiling of volatile organic compounds (VOCs) of gametophyte and sporophyte life stages of Leiosporoceros dussii, from Panama by using headspace-solid phase microextraction-gas chromatography-mass spectrometry in order to assess distinguishing chemical markers between the male and female gametophytes, and sporophytes of this hornwort. A total of 27 VOCs were identified in L. dussii. Furthermore, the gametophyte and sporophyte showed clear differences in the type and amount of VOCs. The main constituents of L. dussii female thalli were menthacamphor (17.8%), hexanol (12.3%), and menthyl acetate (12.3%), while the major compounds of the male thalli were hexanol (25.3%), β-ionone (21.1%), benzeneacetaldehyde (17.6%), and β-cyclocitral (14.0%). The main VOCs of the sporophytes were hexanal (19.3%), β-cyclocitral (17.6%), 2-nonenal (15.8%), hexanol (12.5%), and β-ionone (10.2%). Unique compounds found in the female thalli were 3-pentanone, 3-octenol, nonanol, estragole, and menthyl acetate, and in the male thalli were methyl heptenone, nonanal, neoisomenthol, and bornyl acetate. Isomenthol, thymol, isomenthol acetate, and β-methylnaphthalene were only found in the sporophyte. The characteristic VOCs identified in L. dussii suggest a difference between the chemical constituents of L. dussii and other hornworts species. The presence of simple VOCs when compared with compounds previously characterized in another hornwort genera may support the distinct genetic nature of this species.Keywordsnatural products, hornwort, anthocerotophyta, Leiosporoceros dussii, gametophyte, sporophyte, chemosystematics, HS-SPME-GC-MSReceived: March 8th, 2019; Accepted: June 17th, 2019.Bryophytes (mosses, liverworts, and hornworts) are a group of  plants, which are taxonomically placed between the pterido-phytes and the algae. More than 20,000 species have been reported worldwide, among which the phylum Anthoceratophyta (named hornworts) contains around 300 species.1 A haploid-dominant life cycle characterizes all bryo-phytes with an alternance of  a multicellular gametophyte (n) and a multicellular sporophyte (2n). Bryophytes are known for being resilient organisms and resistant to the attack of  bacteria, fungi, insects, or snails.1 In contrast to flowering plants, the chemical characterization and pharmacological potential of  these plants have been underexplored, although many bryo-phytes have been used as medicinal plants to cure diseases such as tuberculosis, snake bite, neurasthenia, pneumonia, convul-sions and uropathy.2Natural Product Communications2The hornwort Leiosporoceros dussii (Steph.) Hässel is a dioicous species (separate male and female plants) native to the Americas. The species is found in Panama, Colombia, Costa Rica, the Greater Antilles, Ecuador, and Mexico.3 It is worth noting that L. dussii is the only species included within its own family Leiosporocerotaceae. Only 1 study has reported so far the chem-ical composition of  L. dussii species from the Neotropics.4 In this study, the chemical differences between the different stages of  the plant (gametophyte and sporophyte) were not evaluated, although this is valuable for supporting the biochemical knowl-edge, and potential pharmacological use of  the species.The study of  the volatile composition of  bryophytes has facilitated the differentiation between species, and resolved important taxonomic questions.5,6 Volatile organic compounds (VOCs) are remarkable secondary metabolites for protection of  plants against environmental pathogens and predators.7 Solid phase microextraction (SPME) has been widely used for research and identification of  volatile compounds present in plants, including bryophytes.6,8-10 SPME is a more sensitive and effective method, when compared with solvent extraction techniques for the analysis of  VOCs in plants and fruits.6,11 SPME is an analytical method that favors the isolation of  vol-atile and semivolatile compounds present in small amounts in the sample, allowing for the identification of  compounds oth-erwise undetectable by using other methods. In this paper, iso-lation and identification of  chemical compounds from the gametophyte and sporophyte stages of  L. dussii from Panama by headspace-solid phase microextraction-gas chromatogra-phy-mass spectrometry (HS-SPME-GC-MS) are reported for the first time. Furthermore, 27 compounds not previously reported in L. dussii were also identified.In general, the chemical compositions of  hornworts species have not been investigated thoroughly. Among VOCs, only a small number of  volatile mono-, phytane-type di-, and sesquit-erpenoids have been identified in few hornworts.1 Regarding the chemical composition of  L. dussii the only study reported so far was done in species from Panama, by using solvent extraction technique. Two compounds were detected, 16-kaurene and 4-hydroxy-3,3’,4-trimethoxystilbene.4 To undertake a more in-depth study on the chemical constituents of  L. dussii, both the gametophyte (female and male thalli) and the sporophyte stages were investigated for the identification of  the VOCs present in this plant, by using SPME. The iso-lated compounds were separated by gas chromatography (GC) and detected by mass spectrometry (MS). The main VOCs identified in the female and male thalli, and sporophyte of  L. dussii, and their Kovat’s retention indices and percentages are presented in Table  1. In total, HS-SPME-GC-MS permitted the identification of  27 VOCs in L. dussii. Eighteen compounds were identified in the female thalli and in the sporophyte, and 14 compounds in the male thalli. Alcohols (42.3%) were the main components identified in the female thalli, while alde-hydes were the most abundant both in male thalli (41.2%) and sporophyte (56.9%). In the female thalli, aldehydes (25.7%) were the second main compounds followed by monoter-penoids (18.9%), ketones (6.7%), esters (3.2%), and aromatic compounds (2.9%). On the other hand, alcohols (30.0%), ketones (22.0%), aromatic compounds (4.6%), and monoter-penoids (2.2%) were identified in the male thallus, while alco-hols (16.4%), ketones and monoterpenoids (10.2%), esters (4.1%), and aromatic compounds (2.3%) were found in the sporophytes. None of  the compounds identified in this study have been reported for L. dussii. Our results revealed that the main constituents of  L. dussii female thalli were menthacam-phor (17.8%), hexanol (12.3%), and menthyl acetate (12.3%) (Table  1). Furthermore, the major compounds of  the male thalli were hexanol (25.3%), β-ionone (21.1%), benzeneacetal-dehyde (17.6%), and β-cyclocitral (14.0%), while the main VOCs of  the sporophytes were hexanal (19.3%), β-cyclocitral (17.6%), 2-nonenal (15.8%), hexanol (12.5%), and β-ionone (10.2%). The presence of  the diterpenoid 16-kaurene was reported recently in L. dussii from Panama;4 however, this com-pound was not detected in this study.Anthocerotophyta phylum is known to produce small amounts of  terpenoids due to the absence of  oil bodies in their biological structure.12 Furthermore, L. dussii is the most simple of  the Anthocerotophyta.3 These 2 facts might explain why terpenoids such as di- and sesquiterpenoids were not identified in this hornwort in our study, and why there was a huge pres-ence of  alcohols and aldehydes in the plant. Diterpenoids and sesquiterpenoids have been found in more biologically evolved hornwort species such as Anthoceros caucasicus and Megaceros fla-gellaris.13,14 To the best of  our knowledge none of  the VOCs found in this investigation for L. dussii have been identified before in other Anthocerotophyta.HS-SPME-GC-MS allowed for chemical differentiation between gametophyte and sporophyte of  L. dussii. Overall, there were important differences in the type and percentage of  compounds found between gametophyte and sporophyte. Unique compounds found in the female thallus were 3-penta-none, 3-octenol, nonanol, estragole, and menthyl acetate, and in the male thallus were methyl heptenone, nonanal, neoisomen-thol, and bornyl acetate. Isomenthol, thymol, isomenthol ace-tate and β-methylnaphthalene were only found in the sporophyte.Our results suggest a difference between the chemical con-stituents of  L. dussii and other hornworts species. The pres-ence of  simple VOCs in L. dussii when compared with compounds previously characterized in other Anthocerotophyta confirmed the simple nature of  this species. Due to the fact that L. dussii do not possess oil bodies, is morphologically very small, and that it is difficult to collect enough plant material, the investigation on the volatile composition has been limited. Nevertheless, HS-SPME-GC-MS allowed for the profiling of  the VOCs from L. dussii with success, and also in Garrido et al. 3differentiating between the chemical nature of  the different stages of  the plantExperimentalPlant MaterialFresh samples of  L. dussii, female and male thallus, were col-lected at Monumento Natural Cerro Gaital, El Valle de Anton, Province of  Coclé (8°37′34.6″ N, 80°08′13.7″ O), between August and December. Samples were initially identified by Jose Gudiño, and identification was finally confirmed by the bryol-ogist Noris Salazar Allen. The plants were cleaned of  debris, protected from light and humidity, and stored at a temperature of  −20°C until analysis. Voucher specimens of  L. dussii were deposited at the Herbarium of  the University of  Panama, Panama, Panama. Sporophytes were separated from the thallus for further analyses. Two samples of  each type of  thalli and sporophytes were analyzed in duplicate and mean reported in Table 1 as percent relative concentration of  each compound.Analysis of  the Volatile Chemical Composition of  L. Dussii by HS-SPME-GC-MSHS-SPME-GC-MS analysis of  the female thallus, male thallus and sporophytes was done following the procedure described by Durant et al,8 with some modifications. Briefly, divinylben-zene-carboxen-polydimethylsiloxane StableFlex SPME fiber (50/30 µm) (Supelco, Bellefonte, PA, USA) was conditioned in the GC following the manufacturer’s recommendation. A blank test was done before each analysis to verify for possible carry-over effect. For SPME analysis 1 g of  each sample and 1 mL of  NaCl solution were added in a 10 mL hermetically sealed vial. The fiber was then exposed to the headspace of  the sample and maintained there for 15 minutes in a thermostatic bath at 50°C. All experiments were done under constant stir-ring of  400 rpm. GC quadrupole MS analyses were performed Table 1. Volatile Compounds Identified in the Female Thallus, Male Thallus, and Sporophyte of the Panamanian Leiosporoceros Dussii by Headspace-Solid Phase Microextraction-Gas Chromatography-Mass spectrometry.PeakNo. KRI KRI* CompoundsRelative content (%)Ft Mt S1 652 647 3-Pentanone 0.4 - -2 808 800 Hexanal 7.7 5.1 19.33 871 865 Hexanol 12.3 25.3 12.54 1003 987 Methyl heptenone - 0.9 -5 1107 1102 Nonanal - 3.4 -6 1042 1036 3-Octenol 3.4 - -7 1057 1049 Benzeneacetaldehyde 2.6 17.6 4.08 1151 1147 2-Nonenal 5.1 - 15.89 1180 1173 Nonanol 5.6 - -10 1146 1141 Menthacamphor 17.8 1.6 3.911 1150 1146 Camphor 0.7 1.4 0.412 1189 1182 Isomenthol - - 3.113 1195 1188 Neoisomenthol - 3.1 -14 1197 1190 α-Terpineol 0.2 - 0.515 1200 1192 Dihydrocarveol 5.7 - 1.516 1205 1196 Estragole 3.5 - -17 1208 1200 Decanal 0.5 1.0 0.218 1227 1222 β-Cyclocitral 9.9 14.0 17.619 1285 1281 β-Methylnaphthalene - - 1.020 1288 1283 Bornyl acetate - 0.8 -21 1298 1294 Menthyl acetate 12.3 -22 1301 1297 Thymol - - 1.623 1307 1301 Anethole 2.9 4.2 1.224 1311 1305 Isomenthol acetate - - 3.025 1452 1446 Citronellyl propanoate 3.2 - 4.126 1494 1490 β-Ionone 6.3 21.1 10.227 1537 1532 Dihydroactinidiolide - 0.3 0.1Ft, female thallus; KRI: Kovat’s retention index;KRI*, reported Kovats retention index from reference databases; Mt, male thallus; S, sporophyte.Natural Product Communications4in a GC 6890N coupled to a mass spectrometer 5975C (Agilent Technologies, Palo Alto, CA, USA). After extraction of  the volatile compounds the SPME fiber was inserted in the GC injection port. Desorption of  the compounds was performed at 240°C in splitless mode for 2 minutes. Separation of  the extracted compounds was achieved on a HP-5MS capillary col-umn (30 m length, 0.25 mm id, 0.25 µm film thickness). Helium was used as a carrier gas at 1 mL/min. The oven temperature was programmed at 50°C for 2 minutes, then increased to 240°C at 6 °C/min and held for 5 minutes. The MS detector was operated in electron impact mode (EV = 70 eV); with an ion source temperature of  250°C; and operated in scan mode from 30 to 550 m/z. All experiments were performed in dupli-cate and the results obtained expressed as average. Identification of  VOCs was achieved by comparing the mass spectra with those provided by the Registry of  Mass Spectral Data with Structures library (Wiley 7th edition, USA), National Institute of  Standards and Technology library (NIST/11), and by using authentic standards when available. Further identification was performed by comparison of  the calculated Kovat’s retention index (KI) with those reported in the literature. KI was deter-mined by using an alkane standard solution C8-C20 (Sigma-Aldrich, Saint Louis, MO, USA). The relative quantities of  the volatile compounds are expressed as percent peak areas relative to the total peak area of  identified compounds. The percent-ages reported in Table 1 for each stage of  the plant are means of  2 samples of  each stage and each analysis carried out in duplicate.AcknowledgmentsMahabir P. Gupta and Armando Durant-Archibold gratefully acknowledge the National Secretariat for Science, Technology, and Innovation of  Panama (SENACYT) for support through its National System of  Research Awards (SNI). Armando Durant-Archibold wishes to acknowledge Dr. Jagannatha Rao, Director of  the Institute for Scientific Research and Technology Services (INDICASAT AIP) for support with the GC-MS. Noris Salazar Allen and José Gudiño acknowledge support provided by research funds from the Smithsonian Tropical Research Institute (STRI).Declaration of  Conflicting InterestsThe author(s) declared no potential conflicts of  interest with respect to the research, authorship, and/or publication of  this article.FundingThe author(s) disclosed receipt of  the following financial support for the research, authorship, and/or publication of  this article: This work was supported by National Secretariat for Science, Technology, and Innovation of  Panama (SENACYT) through its National System of  Research Awards (SNI).References 1. Asakawa Y, Ludwiczuk A. Chemical constituents of  bryophytes: structures and biological activity. J Nat Prod. 2018;81(3):641-660. 2. Asakawa Y, Ludwiczuk A. Bryophytes: liverworts, mosses, and hornworts: extraction and isolation procedures. Methods Mol Biol. 2013;1055:1-20. 3. Villarreal A JC, Renzaglia KS. 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Chemical profiling of volatile components of the gametophyte and sporophyte stages of the hornwort leiosporoceros dussii (Leiosporocerotaceae) from Panama by HSSPME-GC-MS

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Chemical profiling of volatile components of the gametophyte and sporophyte stages of the hornwort leiosporoceros dussii (Leiosporocerotaceae) from Panama by HSSPME-GC-MS

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