Volatile concentrate from the neotropical moss Neckeropsis undulata (Hedw.) Reichardt, existing in the brazilian Amazon

Background Many natural compounds have been identified and synthesized by the advancement of bryophytes phytochemistry studies. This work aimed to report the composition of Neckeropsis undulata (Hedw.) Reichardt moss volatiles, sampled in the Combú Island, Belém city, Pará state, Brazil. The volatile concentrate of N. undulata was obtained by a simultaneous distillation-extraction micro-system, analyzed by GC and GC-MS, and reported for the first time. Results Ten compounds were identified in the volatile concentrate, corresponding to 91.6% of the total, being 1-octen-3-ol (35.7%), α-muurolol (21.4%), naphthalene (11.3%), and n-hexanal (10.0 %) the main constituents. Most of the constituents of the N. undulata volatile concentrate have been previously identified in other mosses, and liverworts spread wide in the world. Conclusions 1-Octen-3-ol, n-hexanal, 2-ethylhexanol, isoamyl propionate, and octan-3-one are already known metabolic products obtained from enzymatic oxidation of polyunsaturated fatty acids, belonging to the large family of minor oxygenated compounds known as oxylipins. The knowledge of the composition of volatiles from moss N. undulata could contribute to the Neckeraceae species’ chemotaxonomy.


Background
Bryophytes are small terrestrial spore-forming green plants, phylogenetically placed between vascular plants and algae, and comprises about 22,000 species worldwide. They are most diverse in the tropics and fit in a general pattern of various groups of organisms, increasing species toward the equator line. Mosses (Bryophyta), liverworts (Marchantiophyta), and hornworts (Anthocerotophyta) compose the three lineages of bryophytes. The mosses are estimated to include about 12,700 species [1].

BMC Chemistry
Bryophytes have no physical defenses against any type of threat and, in general, are not attacked by insects and small animals. These plants emit scents such as sweetwoody, sweet-mossy, turpentine, carrot-like, mushroomy, and seaweed-like. However, bryophytes' volatiles chemistry is not well known because they are small species and challenging to collect. Besides, they are considered of little nutritional value to humans. However, some mosses have been widely used as medicinal plants in China to cure burns, bruises, external wounds, snake bite, fractures, and convulsions [6,7].
Few chemical and biological studies have been done with bryophytes in Brazil. Organic extracts of 25 bryophytes from the Brazilian Amazon, with solvents of different polarities, were tested against 13 pathogenic bacteria of a broad general spectrum [15]. The antibacterial activity of ethanol extract of the moss Octoblepharum albidum from Crato, Ceará state, Brazil, alone and in association with aminoglycosides, was determined against bacterial strains, resulting in significant and similar inhibitory activity for Escherichia coli ATCC 25,922 and Klebsiella pneumoniae ATCC 33,018. Also, phytochemical screening was performed with the same extract to identified the bioactive compounds [16]. Chemical diversity of the secondary metabolites of 12 populations of Syzygiella rubricaulis (Nees) Stephani (Jamesoniellaceae, Marchantiophyta) from the high tropical mountains of Mexico, Venezuela, Ecuador, and Brazil was performed. Sesquiterpenoids, diterpenoids, and long-chain hydrocarbons predominated in the CHCl 2 extracts of the 12 populations of S. rubricaulis [17]. To the best of our knowledge, no work with essential oils or volatile concentrates of bryophytes has been previously published in Brazil.
This work aimed to report the volatile composition from the moss Neckeropsis undulata (Hedw.) Reichardt, which occurs in the Brazilian Amazon, in order to extend the chemotaxonomic knowledge of this species.

Plant material and extraction procedure
Neckeropsis undulata (Hedw.) Reichardt (Neckeraceae) is a native and non-endemic moss species, with occurrence throughout all Brazil regions [5] and easily found in the Brazilian Amazon. The samples of N. undulata were collected in a floodplain area close to Belém, Pará state, Brazil, on the trunk of live cocoa trees, using techniques proposed by Yano (1984) [18]. Different bryophytes can occur together, and this species was separated using tweezers with magnifying glasses, considering its gametophyte and sporophyte phases. The botanical identification was carried out by Prof. Ana Cláudia Martins, a Bryophytes specialist from Departamento de Ciências Naturais, Universidade do Estado do Pará, Belém, Pará state, Brazil. The moss sample (100 g) was air-dried for three days, and its volatile concentrate was obtained by micro hydrodistillation-extraction in a Likens-Nickerson apparatus (25 g, 2 h, duplicate) [19], using n-pentane (3 mL) as the solvent.

Volatile concentrate analysis
The volatile concentrate of N. undulata was submitted to GC and GC-MS analysis. It was performed on a GCMS-QP2010 Ultra system (Shimadzu Corporation, Tokyo, Japan), equipped with an AOC-20 i autoinjector and the GCMS-Solution software containing the NIST and FFNSC 2 libraries [20,21]. . Individual components were identified by comparing their retention indices and mass spectra (molecular mass and fragmentation pattern) with those existing in the GCMS-Solution system libraries [20,21,23]. The quantitative data regarding the volatile constituents were obtained using a GC 2010 Series, operated under similar conditions to those of the GC-MS system. The relative amounts of individual components were calculated by peak-area normalization using a flame ionization detector (GC-FID). The analyses were performed in duplicate.
The addition of molecular oxygen to polyunsaturated fatty acids and the transformations of the resulting hydroperoxides leads to the production of a large family of minor oxygenated compounds known as oxylipins. The main constituent of the volatile concentrate on N. undulata was 1-octen-3-ol (35.7%). It is allyl alcohol known as mushroom alcohol, and an oxylipin produced by enzymatic oxidation and cleavage of the linoleic, arachidonic, and eicosapentaenoic acids. In nature, the 1-octen-3-ol is indicated as a signaling molecule in plant cellular responses, plant-herbivore interactions, and plant-plant interactions [11,13,24]. In the oil of the moss Rhodobryum ontariense Kindb. from Serbia, 1-octen-3-ol has been identified as the secondary constituent, following the diterpene phytol, which was the main component [10]. The thalli of the liverwort Marchantia polimorpha emitted high amounts of C8 volatiles, after mechanical wounding, mainly consisting of 1-octen-3-ol and octan-3-one, which were also isolated from the N. undulata volatile concentrate, together with another unidentified C8 alcohol, with a low percentage. The presence of arachidonic acid or eicosapentaenoic acid seemed essential for the formation of these volatile compounds of C8-type [25].
The second most abundant constituent of the volatile concentrate on N. undulata was the oxygenated sesquiterpene α-muurolol (21.4 %). Terpene compounds are present in little amounts in mosses when compared to liverworts [26]. α-Muurolol has a cadinane skeleton, and it is found in liverworts and mosses. Many sesquiterpene and diterpene compounds are also identified in higher plants [6]. For example, in the liverwort Frullania brasiliensis Raddi and moss Campylopus richardii Brid. oils, the stereoisomer epi-α-muurolol was the primary constituent, with 32.18 % and 15.1 %, respectively [9,27].

Conclusions
In this work, the volatile composition of Neckeropsis undulata moss was reported for the first time, and it is assumed that this knowledge can contribute to a better understanding of the Brazilian bryophytes. 1-Octen-3-ol, the primary constituent, is already a known metabolic product, obtained from enzymatic oxidation of polyunsaturated fatty acids. α-Muurolol is a oxygenated sesquiterpene, with a cadinane skeleton, found in other mosses and of chemotaxonomic importance. 1-Octen-3-ol, n-hexanal, 2-ethylhexanol, isoamyl propionate, and octan-3-one belong to the large family of minor oxygenated compounds oxylipins, generated from long-chain fatty acids.