Volatile Metabolites in Liverworts of Ecuador

Volatile metabolites from Frullania brasiliensis Raddi, Herbertus juniperoideus (Sw.) Grolle, Leptoscyphus hexagonus (Nees) Grolle, and Syzygiella anomala (Lindenb. & Gottsche) Steph collected in the south of Ecuador were investigated. Volatile secondary metabolites were extracted by hydrodistillation and analyzed by gas chromatography/flame ionization detector (GC/FID) and Gas chromatography/Mass spectrometry (GC/MS). Sixty-seven volatile compounds were identified in the four species, which represent between 80.12–90.17% of the total chemical composition. The major components were τ-muurolol (32.14%) and Germacrene-D (11.98%) in the essential oil of F. brasiliensis, bicyclogermacrene (18.23%), and Caryophyllene oxide (15.29%) in the oil of H. juniperoideus, Cabreuva oxide D (33.77%) and Elemol (18.55%) in the oil of Leptoscyphus hexagonus, and Silphiperfola-5,7(14)-diene (25.22%) and Caryophyllene oxide (8.98%) in the oil of Syzygiella anomala. This is the first report on volatile compounds for the species Herbertus juniperoideus, Leptoscyphus hexagonus, and Syzygiella anomala.


Introduction
The Ecuador has a very rich liverworts flora over 770 species [1][2][3]. Liverworts produce secondary metabolites in abundance [4] with more than 1500 terpenoids and 350 aromatic compounds flavonoids [5][6][7]. In this context, liverworts produce terpenoids and aromatic compounds, many of which exhibit diverse, interesting biological properties related to antitumor, antimicrobial, antifungal, antioxidative, and insecticidal activities; cytotoxic and insect antifeedant [5]. Several studies have shown abundant secondary metabolites in foliose liverworts. For example, Asakawa et al. [8] studied by gas chromatography coupled to mass spectrometry (GC/MS) the volatile components of 25 taxa of the liverwort family Frullaniaceae from New Zealand, Australia and South America.
In addition, chemical constituents of Frullania serrata were isolated, and their structure was elucidated by nuclear magnetic resonance (1D-and 2D-NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and infrared spectroscopy (IR) [9]. The chemical composition of Frullania tamarisci essential oil was investigated using gas chromatography coupled to flame ionization detection (GC/FID), GC/MS, and NMR analyses [10]. Compounds of Frullania hamatiloba have been isolated and their structures were determined by a combination of the 1 H-and 13 C-NMR, MS, ultraviolet-visible spectroscopy (UV), IR, and X-ray crystallographic (X-ray) analysis [11]. In another species of this genus, the F. brasiliensis two eremophilanolides were established by NMR and X-ray analysis [12].
The purpose of the present investigation was the isolation and identification of volatile metabolites of four species of liverworts from Ecuador, three of which, Herbertus juniperoideus, Leptoscyphus hexagonus, and Syzygiella anomala, do not have previous chemical or phytochemical studies.

Volatile Compounds Isolation
By means of hydrodistillation in a Clevenger-type apparatus, 0.3 mL of essential oil was obtained from 1500 g of Frullania brasiliensis, what represents a yield of 0.02% (v/w). For the species Herbertus juniperinus, Leptoscyphus hexagonus, and Syzygiella anomala, yields close to 0.01% (v/w) were obtained.

Discussion
Many liverworts are endemic to the southern hemisphere, including Oceania and South America [7]. In the Ecuador (South America), there are around 770 species of liverworts [1][2][3], of which about eleven species have been previously studied. Liverworts (Hepaticae) are a rich source of terpenoids and aromatic compounds [7]. In this research, chemical constituents of studied species of liverworts were mainly grouped into sesquiterpene hydrocarbons (SH) and oxygenated sesquiterpenes (OS). Only one monoterpene hydrocarbon (MH) (β-phellandrene) with RI 1023 was determined; below RI 1000, no compound was identified in any of the four species studied. However, in Frullania tamarisci of France [10], three compounds (α-pinene, 1-Octen-3-ol, and β-pinene) with RI below 1000 were identified, which would indicate that it is possible to isolate this type (MH) of compounds from liverworts.
Around the year 1991, Nagashima et al. [13] investigated terpenoids and aromatic compounds of seven Ecuadorian liverworts. In the spieces Frullania brasiliensis found in total two compounds, the sesquiterpenoids Arbusculin B and α-Bisabolol, the relative abundance percentages are not mentioned. In our study, for species Frullania brasiliensis twenty-five compounds were identified, the main compound identified for this species was τ-muurolol (32.14%), that like the α-Bisabolol is an oxygenated sesquiterpenes of formula C 15 H 26 O.
The principal constituents in H. juniperinus are bicyclogermacrene (18.23%) and caryophyllene oxide (15.29%). For this species, no previous chemical or phytochemical studies have been carried out. However, two Ecuadorian species of the genus Herbertus, H. acanthelius, and H. subdentatus, were previously studied by Nagashima et al. [13] in which identified isocuparene-type sesquiterpenoids as the major components.

Materials
Dichloromethane and sodium sulfate anhydrous were purchased from Sigma-Aldrich. The standard of aliphatic hydrocarbons was purchased from CHEM SERVICE under the name of Diesel Range Organics Mixture #2-GRO/DRO and with the code M-TPH6X4-1ML. Helium was purchased from INDURA, Ecuador. All chemicals were of analytical grade and used without further purifications.

Plant Material
The plant material of the four species was collected in "El Tiro", in the province of Loja (Southern Ecuador, latitude, 3 • 58 59" S; longitude, 79 • 08'05" W; the altitude ranged from 2800-3000 m a.s.l). The storage and transfer of the plant material were carried out in airtight plastic containers until they are hydrodistilled. The collection temperature was 14-16 • C (ambient temperature), and the transfer temperature was 16-18 • C, the pressure was approximately 80 KPa (ambient pressure). Voucher specimens were deposited in the Herbarium of the Universidad Técnica Particular de Loja (HUTPL)-Bryophytes and lichen collection under the acquisition numbers AB-1299 for Frullania brasiliensis, AB-1300 for Herbertus juniperoideus, AB-1264 for Leptoscyphus hexagonus, AB-1301 for Syzygiella anomala. The identity of the plant material was confirmed by the curator of lichens and bryophytes, the mentioned herbarium.

Volatile Compounds Isolation
The volatile compounds isolation was realized from 1500 g of vegetal material of Frullania brasiliensis, 4029 g of Herbertus juniperoideus, 6045 g of Leptoscyphus hexagonus and 4874 g of Syzygiella anomala. The material was processed fresh, immediately after arriving at the laboratory, between 8 and 12 h after being collected. The plant material of each species was hydrodistilled for four hours using a Clevenger-type apparatus. Subsequently, each extract sample (essential oil) was dried over sodium sulfate anhydrous and was stored in sealed vials, protecting them from light at 4 • C until being used in the analysis [20].

Gas Chromatography/Flame Ionization Detector (GC/FID)
The analyses of the chemical composition of the essential oils were performed on an Agilent gas chromatograph (model 6890N series) equipped with a flame ionization detector (FID). A nonpolar column DB-5ms (5%-phenyl-methylpolyxilosane) 30 m × 0.25 mm, thickness 0.25 µm was used. An automatic injector (series 7683) in split mode was used. The samples, 1 µL of solution (1/100, v/v, essential oil/dichloromethane), were injected with a split ratio of 1:50. Helium was used as a carrier gas at 0.9 mL/min in constant flow mode. The initial oven temperature was held at 50 • C for 3 min, and then it was heated to 210 • C with a ramp of 2.5 • C/min, and the temperature was maintained for 3 min until the end. The injector and detector temperatures were of 210 • C and 250 • C, respectively. The retention index (IR) of the compounds was determined based on the standard of aliphatic hydrocarbons, which were injected after the oils at the same conditions.

Gas Chromatography/Mass Spectrometry (GC/MS)
The GC/MS analyses were performed using an Agilent chromatograph coupled to a mass spectrometer (quadrupole) detector (model Agilent series 5973 inert). The spectrometer was operated at 70 eV, electron multiplier 1600 eV, scan rate: 2 scan/s, and mass range: 40-350 m/z. It was provided with a computerized system MSD-Chemstation D.01.00 SP1. The same columns described in GC/FID section were used. The ion source temperature was set at 250 • C. The identification of the oil components was based on a comparison of both mass spectrum data and relative retention indices with the published literature [21][22][23]. The relative amounts of individual components were calculated based on the GC peak area (FID response) without using a correction factor.