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.

1. 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 1H- and 13C-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].

On the other hand, chemical constituents from Herbertus subdentatus (Steph.) Fulford and H. acanthelius Spruce have been detected by 1H- and 13C-NMR, UV, IR, thin-layer chromatography (TLC), and GC/MS [13]. In the family Lophocoleaceae, chemical constituents from Chiloscyphus pallidus (Mitt.) Engel & Schuster [14], Heteroscyphus planus [15], and Heteroscyphus billardierei (Schwägr.) Schiffn. have been isolated and studied by GC/MS and NMR techniques [16]. Finally, secondary metabolites in the liverwort Syzygiella rubricaulis (Nees) Stephani has been analyzed by GC/MS [17].

In the Ecuador, liverworts are plants that produce a wide array of biologically active compounds [13], however, less than 3% of these compounds have been investigated. As it is recorded in the bibliography of the large number of existing liverworts species in Ecuador, there is a record of the study of the compounds from Frullania brasiliensis, Herbertus acanthelius, Herbertus subdentatus, Plagiochila alternans Lindenb. & Gottsche, Plagiochila micropterys Gottsche, Macrolejeunea pallescens (Mitt.) Schiffn., Marchantia plicata Nees & Mont. [13], Marchesinia brachiate (Sw.) Schiffn. [18], Noteroclada confluens Taylor, Symphyogyna brasiliensis Nees & Mont. [19], and Syzygiella rubricaulis (Nees) Stephani [17].

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.

2. Results

2.1. 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.

2.2. Volatile Compounds Identification

The identification of volatile compounds present in liverworts of Ecuador was carried out by means of gas chromatography equipped with a flame ionization detector (GC/FID) and gas chromatography coupled to a mass spectrometer detector (GC/MS) using capillary nonpolar column DB-5ms. To our knowledge, this is the first report on the chemical composition of volatile compounds from Herbertus juniperinus, Leptoscyphus hexagonus, and Syzygiella anomala (Table 1). In total, sixty-seven chemical constituents were identified in essential oil samples, representing 80.12–90.17% of the total composition. These constituents were grouped into monoterpene hydrocarbons (0.14%), oxygenated monoterpenes (0.79–1.16%), sesquiterpene hydrocarbons (19.61–65.39%), oxygenated sesquiterpenes (23.48–66.24%), diterpene hydrocarbons (0.23–0.87%), and other compounds (0.76–1.16%).

Table 1

Volatile metabolites of Frullania brasiliensis (Fb), Herbertus juniperinus (Hj), Leptoscyphus hexagonus (Lh), and Syzygiella anomala (Sa).

Peak N°	Compound a,b	RI	RI ref	Fb	Hj	Lh	Sa	Type	CF	MM (Da)
1	β-Phellandrene	1023	1025	-	-	-	0.14	MH	C10H16	136.13
2	1-Octen-3-ol, acetate	1107	1110	0.45	0.79	-	1.16	OM	C10H18O2	170.13
3	Thymol methyl ether	1217	1232	1.16	-	-	-	OT	C11H16O	164.12
4	Bicycloelemene	1321	1331	-	0.33	-	0.23	SH	C15H24	204.19
5	δ-EIemene	1324	1335	0.10	-	-	-	SH	C15H24	204.19
6	α-Cubebene	1335	1345	0.42	0.15	-	-	SH	C15H24	204.19
7	Silphiperfola-5,7(14)-diene	1355	1363	0.25	-	-	25.22	SH	C15H22	202.17
8	Isoledene	1358	1374	-	-	-	0.10	SH	C15H24	204.19
9	α-Copaene	1363	1374	0.44	0.27	0.68	-	SH	C15H24	204.19
10	β-Bourbonene	1370	1387	0.74	0.32	-	-	SH	C15H24	204.19
11	β-Elemene	1383	1389	3.03	0.78	-	-	SH	C15H24	204.19
12	Longifolene	1392	1407	1.77	2.58	0.33	-	SH	C15H24	204.19
13	β-Cubebene	1393	1387	0,63	-	-	-	SH	C15H24	204.19
14	α-Longipinene	1399	1350	-	-	3.62	-	SH	C15H24	204.19
15	α-Gurjunene	1412	1409	-	1.15	-	0.73	SH	C15H24	204.19
16	Caryophyllene	1412	1417	-	-	0.22	0.10	SH	C15H24	204.19
17	Aromandendrene	1423	1439	1.07	1.61	0.34	-	SH	C15H24	204.19
18	β-Barbatene	1431	1440	-	-	-	3.99	SH	C15H24	204.19
19	Aristolediene	1436	1435	-	-	1.49	-	SH	C15H22	202.17
20	cis-Thujopsadiene	1437	1465	-	-	-	7.00	SH	C15H22	202.17
21	cis-Thujopsene	1443	1429	-	-	0.18	-	SH	C15H24	204.19
22	Dehydroaromadendrene	1445	1460	-	-	1.29	1.61	SH	C15H24	204.19
23	(E)-β-Farnesene	1447	1454	--	3.20	-	1.95	SH	C15H24	204.19
24	β-Gurjunene	1448	1431	1.54	-	-	-	SH	C15H24	204.19
25	Germacrene-D	1466	1484	11.98	4.67	-	-	SH	C15H24	204.19
26	α-Patchoulene	1470	1454	-	-	1.16	-	SH	C15H24	204.19
27	Eremophilene	1473	1489	-	-	0.39	-	SH	C15H24	204.19
28	Alloaromadendrene	1474	1458	-	3.30	-	-	SH	C15H24	204.19
29	Viridiflorene	1476	1496	2.17	3.69	1.33	6.51	SH	C15H24	204.19
30	Bicyclogermacrene	1480	1500	tr	18.23	6.70	8.42	SH	C15H24	204.19
31	α-Selinene	1485	1498	-	-	1.30	-	SH	C15H24	204.19
32	Cuparene	1488	1504	-	-	-	0.55	SH	C15H22	202.17
33	Cabreuva oxide D	1492	1479	-	-	33.77	-	OS	C15H24O	220.18
34	Trichodiene	1501	1533	-	-	-	0.18	SH	C15H24	204.19
35	δ-Cadinene	1505	1522	-	3.38	-	-	SH	C15H24	204.19
36	Valencene	1508	1496	-	-	0.58	-	SH	C15H24	204.19
37	Sesquiphellandrene	1513	1521	-	3.05	-	-	SH	C15H24	204.19
38	trans-Cycloisolongifol-5-ol	1515	1513	-	-	2.57	-	OS	C15H24O	220.18
39	β-Vetispirene	1517	1493	-	-	-	8.01	SH	C15H22	202.17
40	α-Calacorene	1527	1544	0.80	-	-	-	SH	C15H20	200.16
41	γ-Selinene	1528	1522	2.53	-	-	-	SH	C15H24	204.19
42	γ-Dehydro-Ar-himachalene	1545	1530	-	-	-	0.79	SH	C15H20	200.16
43	Maaliol	1553	1566	-	-	-	0.84	OS	C15H26O	222.20
44	Caryophyllene oxide	1562	1582	-	15.29	-	8.98	OS	C15H24O	220.18
45	Spathulenol	1562	1577	-	11.90	0.57	-	OS	C15H24O	220.18
46	β-Oplopenone	1564	1575	-	-	-	6.4	OS	C15H24O	220.18
47	Elemol	1570	1548	4.19	-	18.55	-	OS	C15H26O	222.20
48	Globulol	1571	1590	-	-	-	1.83	OS	C15H26O	222.20
49	Viridiflorol	1579	1592	3.17	8.93	8.03	1.01	OS	C15H26O	222.20
50	Cubeban-11-ol	1582	1595	-	-	-	0.38	OS	C15H26O	222.20
51	Ledol	1591	1602	-	-	2.57	-	OS	C15H26O	222.20
52	Rosifoliol	1592	1600	5.08	-	-	1.04	OS	C15H26O	222.20
53	Muurola-4,10(14)-dien-1β-ol	1613	1630	-	-	-	0.36	OS	C15H24O	220.18
54	τ-muurolol	1642	1640	32.14	2.14	-	-	OS	C15H26O	222.20
55	τ-cadinol	1648	1638	4.71	-	-	-	OS	C15H26O	222.20
56	Valerenal	1650	1668	-	-	-	0.18	OS	C15H22O	218.17
57	Torreyol	1657	1656	0.66	-	-	-	OS	C15H26O	222.20
58	Selin-11-en-4-α-ol	1667	1658		0.82	-	-	OS	C15H26O	222.20
59	Acorenone	1673	1692	0.86	-	-	-	OS	C15H24O	220.18
60	3-Oxo-7,8-dihydro-β-ionol	1711	1695	-	0.48	-	-	OT	C13H20O2	208.15
61	Drimenol	1752	1757	-	-	0.18	-	OS	C15H26O	222.20
62	Aristolone	1757	1762	-	-	-	2.46	OS	C15H22O	218.17
63	Hexahydrofarnesyl acetone	1833	1843	-	0.28	-	--	OT	C18H36O	268.28
64	5,15-Rosadiene	1904	1896	-	0.39	-	-	DH	C20H32	272.25
65	Sandaracopimaradiene	1935	1935	0.23	0.17	-	-	DH	C20H32	272.25
66	Sclarene	1985	1974	-	0.16	-	-	DH	C20H32	272.25
67	Kaurene	2060	2042	-	0.15	-	-	DH	C20H32	272.25
Monoterpene hydrocarbons (MH)				-	-	-	0.14
Oxygenated monoterpenes (OM)				0.45	0.79	-	1.16
Sesquiterpene hydrocarbons (SH)				27.47	46.71	19.61	65.39
Oxygenated sesquiterpenes (OS)				50.81	39.08	66.24	23.48
Diterpene hydrocarbons (DH)				0.23	0.87	-	-
Other compounds (OT)				1.16	0.76	-
Total identified				80.12	88.21	85.85	90.17

a Compounds ordered according to the elution order in the column DB5-Ms. b All compounds were identified by MS and RI: MS, by comparison of the mass spectrum with those of the computer mass libraries Wiley 7, Adams 21 and NIST 05 22; RI, by comparison of RI with those reported in the literature; 21, 22, and 23. tr, trace (<0.05%); -, not detected; RI, retention indices in the a-polar column (DB5-MS); RIref, references: 21, 22, and 23. CF, Chemical Formula; MM, Monoisotopic mass.

Twenty-seven components were determined in essential oil of H. juniperinus, representing 88.21% of the total oil, the principal constituents are found to be sesquiterpene hydrocarbons: bicyclogermacrene (18.23%) and germacrene-D (4.67%) and oxygenated sesquiterpene: caryophyllene oxide (15.29%), spathulenol (11.90%) and viridiflorol (8.93%). Twenty-one components were identified for the essential oil of L. hexagonus, representing 85.85% of the total oil, the principal group was oxygenated sesquiterpenes such as cabreuva oxide D (33.77%), Elemol (18.55%) and Viridiflorol (8.03%) and sesquiterpene hydrocarbons like bicyclogermacrene (6.70%). Τ-muurolol (32.14%), germacrene-D (11.98%), Rosifoliol (5.08%), τ-cadinol (4.71%) and elemol (4.19%) were the main compounds of F. brasiliensis out of Twenty-five components, representing 80.12% of the total essential oil.

In essential oils of F. brasiliensis the principal groups were oxygenated sesquiterpenes (50.81%) and sesquiterpene hydrocarbons (27.47%). Other compounds such as thymol methyl ether, 3-Oxo-7,8-dihydro-β-ionol, and Hexahydrofarnesyl acetone were detected from the essential oils of H. juniperinus and F. brasiliensis, representing 0.76% and 1.16%, respectively. A monoterpene hydrocarbon β-Phellandrene was detected in essential oils of S. anomala and an oxygenated monoterpenes 1-octen-3-ol acetate was detected in the oils of S. anomala and F. brasiliensis.

3. 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 C15H26O.

Between 2006 and 2001 Bardón et al. [12] studied the nonvolatile compounds from Frullania brasiliensis of Argentina, achieving isolate and identify two eremophilanolides, 5-epidilatanolides A and B, as well as a new natural bibenzyl. In this same study, also were identified the nonvolatile compounds eudesmane-type sesquiterpene lactones nepalensolide A, nepalensolide B, (+)-frullanolide, and (+)-dihydrofrullanolide, hopanoid zeorin, four sterols stigmasta-4,22-dien-3,6-dione, stigmasta-4,22-dien-3-one, stigmasterol, and sitosterol, and a trace amount of atraric acid.

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.

In studies related with Ecuadorian liverwort Nagashima et al. [13,18] report that the compounds 3,4-Dimethoxy-1-vinylbenzene, 2,4,5-trimethoxy-1-vinylbenzene and apigenin-7,4′-dimethylether were identified in Marchesinia brachiate. 1,4-Dimethylazulene was isolated from Plagiochila micropterys and Macrolejeunea pallescens. The major component of Marchantia plicata is marchantin A and pinguisane-type sesquiterpenoid is the major component in Plagiochila alternans.

In the Ecuadorian species, Noteroclada confluens Taylor Ludwiczuk et al. [19] determined that the major component with 49% is an unknown sesquiterpene alcohol (molecular ion M+ 222), and that species also produced a large amount of bicyclogermacrene and an unidentified bibenzyl derivative (molecular ion M+ 344]. According to Ludwiczuk et al. the main compounds occurring in Symphyogyna brasiliensis Nees from Ecuador are Dihydroagarofurane (36.3%) and δ-selinene (20.6%), these species also produced cascarilladiene, selina-4,7-diene, eudesma-5,7(11)-diene, Bicyclogermacrene, thujopsene, calarene, γ- and δ-cuprenene, β-cubebene, bourbon-7(11)-ene and trans-dauca-4(11),7-diene [19]. On the other hand, Costa et al. [17] determined that chemical profiles of lipophilic extracts of Syzygiella rubricaulis (Nees) Stephani are rich in sesquiterpenes. Regarding the species Leptoscyphus hexagonus and Syzygiella anomala, no previous studies have been carried out.

4. Materials and Methods

4.1. 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.

4.2. 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.

4.3. 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].

4.4. 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.

4.5. 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.

5. Conclusions

Volatile metabolites of Herbertus juniperoideus (Sw.) Grolle, Leptoscyphus hexagonus (Nees) Grolle, and Syzygiella anomala (Lindenb. & Gottsche) Steph were qualitatively and quantitatively characterized for the first time, in addition to these three species of liverworts, volatile secondary metabolites of Frullania brasiliensis Raddi were studied. A total of sixty-seven compounds were identified, which were mainly grouped into sesquiterpene hydrocarbons and oxygenated sesquiterpenes. The major components were τ-muurolol in F. brasiliensis, bicyclogermacrene in H. juniperoideus, Cabreuva oxide D in L. hexagonus and silphiperfola-5,7(14)-diene in S. anomala.