Combined Analysis by GC(RI), GC-MS and ¹³C NMR of Leaf and Wood Essential Oils from Vietnamese Glyptostrobus pensilis (Staunton ex D. Don) K. Koch

Abstract

Glyptostrobus pensilis (Staunton ex D. Don) K. Koch is a critically endangered species, native to southeastern China and also very locally found in Dak Lak Province, Vietnam. Essential oil isolated from leaves is a monoterpene-rich oil containing mainly limonene (33.3%), α-pinene (23.4%) and bornyl acetate (9.2%). The composition of G. pensilis wood oil is rather complex and the identification of individual components needed fractionation over column chromatography. The main components, identified by GC(RI), GC-MS and ¹³C NMR, were cedrol (29.3%), occidentalol (6.6%) and occidentalol isomer (5.9%).

1. Introduction

Glyptostrobus pensilis (Staunton ex D. Don) K. Koch, also known as Chinese swamp cypress, is the only species in the genus Glyptostrobus (Cupressaceae). It is inventoried under various synonyms such as Glyptostrobus aquaticus, G. heterophyllus, G. sinensis, Sabina aquatica, Taxodium japonicum ssp. heterophyllum, T. sinense, Thuja pensilis. It is native to southeastern China and also very locally found in Dak Lak Province, Vietnam (two natural populations, with over 200 individuals) [1,2].

G. pensilis is a medium-sized tree, reaching 20–25 m tall and with a trunk diameter up to 1–1.6 m. Its brown bark is cracked into long, irregular strips. The main branches spread horizontally. The leaves are deciduous, spirally arranged, 5–20 mm long and 1–2 mm broad. The 2–3 cm long and 1–1.5 cm diameter cones are green, going yellow-brown during maturation. They open when mature to release winged seeds, 5–20 mm long. It typically grows in river banks, ponds and swamps [3].

G. pensilis is a critically endangered species. Indeed, the species is nearly extinct in the wild due to overcutting for its valuable wood. However, a few specimens are found in several botanical gardens around the world. Genetic variation within and between Chinese populations was investigated using inter-simple sequence repeats (ISSRs). The results show that genetic diversity of G. pensilis is rather low [4].

Studies of the last remnants of G. pensilis native populations in Vietnam have been carried out including standard taxonomical treatment, ecology, population structure and natural conditions of its habitats [2].

Concerning phytochemicals, a new abietane diterpene, glypensin A, and four known compounds, 12-acetoxy-ent-labda-8(17),13E-dien-15-oic acid, quercetin 3-O-α-L-arabino-furanoside, quercetin 3-O-β-D-galactopyranoside, β-sitosterol, were isolated from the branches and leaves of G. pensilis [5]. More recently, it has been reported that five abietane diterpenes isolated from the branches of G. pensilis are potential candidates for the prevention and treatment of SARS-CoV-2 [6]. Six spirobiflavonoid stereoisomers including two new ones, spiropensilisols A and B, were isolated from trunk bark [7]. The composition of leaf waxes has been investigated [8]. The resin of G. pensilis consisted mainly of diterpenoids: ferruginol, pisiferol, 6,7-dehydropisiferol, abeo-pisiferol, abeo-carnosol, sugiol, salvinolone and 6-hydroxy-salvinolone. The epicuticular wax extracted from shoots of G. pensilis comprised lipids, mainly n-nonacosan-10-ol, n-nonacosan-10-one and n-alkanes ranging from C27 to C33, with minor diterpenoids and triterpenoids. No sesquiterpenoids were detected [9].

Concerning volatiles, the composition of G. pensilis essential oil was reported [10]. The composition is dominated by α-pinene (18.9%) and limonene (23.9%). However, this oil sample was advisedly mentioned as “wood oil” instead of “leaf oil” (personal communication of the authors). This error was corroborated by the recent paper of Schmidt et al. (2016) [11] who reported on sesquiterpene-rich wood oil from G. pensilis harvested in Vietnam. Indeed, the composition of that wood oil was dominated by cedrol (16.4%), occidentalol (13.2%) and β-elemol (8.9%) besides the major component assumed as “dihydro-eudesmol isomer” (18.3%). The odor of this oil sample was defined as “soft woody, slightly terpeny top with fresh and green connotation, later soft woody, fine cedar note, tender warm woody notes reminding of cedar and cypress, later balsamic with slight burning note”.

Therefore, the aim of the present study was to investigate the composition of leaf and wood oil samples isolated from the same G. pensilis tree from Vietnam.

2. Materials and Methods

2.1. Plant Materials

Leaves and wood from G. pensilis were harvested at Trap Kso, Ea Ho Commune, Krong Nang District, Dak Lak Province, central highlands, Vietnam, in March 2020; geographical coordinates: 12 59′25″ N; 108 17′07″ E, at 713 m above sea level (Figure 1). Plant material was authenticated by Dr. Tran Huy Thai. A voucher specimen was deposited at the herbarium of the Institute of Ecology and Biological Resources (IEBR), Vietnam Academy of Science and Technology (Hanoi) with the reference HN-TN125.

2.2. Essential Oil Isolation

Leaves and wood were dried in the shade and hydrodistillated separately, 4–5 days after harvest using a Clevenger apparatus for 3 h for leaves and 4 h for wood, leading to colorless essential oils. Mass of material used for hydrodistillation/volume of collected essential oil: leaves: 3050 g/4.3 mL; wood: 2520 g/5.5 mL. Both oil samples were submitted to GC(RI), GC-MS and ¹³C NMR analyses.

2.3. GC-FID Analysis

GC-FID analyses were carried out using a Perkin Elmer Clarus 500 (Perkin Elmer, Courtaboeuf, France) system equipped with two FID and two fused-silica capillary columns (50 m length, 0.22 mm i.d., film thickness 0.25 μm), with polydimethylsiloxane (BP-1) and polyethylene glycol (BP-20). The oven temperature was programmed from 60 °C to 220 °C at 2 °C/min and then held isothermal at 220 °C for 20 min; injector temperature: 250 °C; detector temperature: 250 °C; carrier gas: H₂ (0.8 mL/min); split: 1/60; injected volume: 0.5 μL. The relative proportions of the essential oil constituents were expressed as percentages obtained by peak-area normalization, without using correcting factors. Retention indices (RIs) were determined relative to the retention times of a series of n-alkanes with linear interpolation (Target Compounds (V1.2019) software from Perkin Elmer).

2.4. GC/MS Analysis

GC/MS analyses were performed on a Perkin Elmer Clarus SQ8S TurboMass detector (quadrupole), directly coupled to a Perkin-Elmer Clarus 580 Autosystem XL, equipped with a polydimethylsiloxane (BP-1) fused-silica capillary column (50 m length, 0.22 mm i.d., film thickness 0.25 µm). The oven temperature was programmed from 60 to 220 °C at 2°/min and then held isothermal at 220° for 30 min; injector temp., 250 °C; ion source temp., 150 °C; carrier gas, He (1 mL/min); split ratio, 1:80; injection volume, 0.5 μL; ionization energy, 70 eV. The electron ionization (EI) mass spectra were acquired over the mass range 35–350 Da.

2.5. Nuclear Magnetic Resonance

¹³C NMR spectra were recorded on a Bruker AVANCE 400 Fourier transform spectrometer operating at 100.63 MHz for ¹³C, equipped with a 5 mm probe, in CDCl₃, with all shifts referred to internal TMS. The following parameters were used: pulse width = 4 μs (flip angle 45°); acquisition time = 2.7 s for 128 K data table with a spectral width of 25,000 Hz (250 ppm); CPD mode decoupling; digital resolution = 0.183 Hz/pt. The number of accumulated scans was 3000 for each sample (40 mg of essential oil in 0.5 mL of CDCl₃).

2.6. Identification of Individual Components

Identification of the individual components was carried out: (i) by comparison of their GC retention indices (RIs) on non-polar and polar columns with those of reference compounds compiled in a laboratory-built library and with literature data [12,13,14]; (ii) by computer matching against commercial mass spectral libraries [14,15,16]; (iii) by comparison of the signals in the ¹³C NMR spectra of the samples with those of reference spectra compiled in the laboratory spectral library, with the help of laboratory-made software [17,18]. The usefulness of this technique has been highlighted [19,20], including the identification of epimers and stereoisomers [21,22].

2.7. Column Chromatography of the Essential Oil

G. pensilis wood oil (1.6803 g) was subjected to column chromatography over SiO₂ (250–500 µm, 40 g) and 13 fractions (F1–F13) were eluted using a gradient of solvents (pentane/Et₂O, from 0/100 to 100/0) (

Table 1. Components identified by GC(RI) and ¹³C NMR in the 13 fractions of CC (F1–F13), with their relative percentages.

F1 a 100:0 b 296 c	%	F2 95:5 36	%	F3 90:10 67	%	F4 90:10 141	%	F5 90:10 139	%	F6 90:10 144	%	F7 90:10 131	%
20	0.5	33	3.1	52	18.1	53	10.7	56	85.9	56	88.7	56	87.9
22	0.4	34	5.7	53	14.2	56	50.3	62	2.3
23	0.9	38	17.6	54	18.8	62	4.4	73	3.2
25	0.8	39	1.7	61	7.6	73	6.6	74	0.4
26	1.7	47	2.8	65	8.6	74	0.7
27	5.9	70	17.5	66	3.9	78	5.6
28	16.8	77	1.0	70	2.0
29	1.7			71	3.1
30	14.4			78	1.8
31	17.2
32	1.4
35	1.0
36	1.3
37	0.7
41	5.6
43	1.0
44	1.6
45	8.5
77	0.9
79	4.8
F8 90:10 162	%	F9 90:10 111	%	F10 75/25 149	%	F11 75:25 107	%	F12 75:25 99	%	F13 0:100 95	%
56	79.1	49	5.3	49	13.8	49	17.1	49	14.2	49	4.8
60	1.7	50	4.2	50	15.8	50	26.6	50	32.6	50	15.3
66	0.8	55	9.0	55	18.4	55	16.5	55	10.6	55	4.8
68	0.7	56	47.0	63	4.1	63	2.9	63	1.2	56	15.9
		63	3.5	66	9.5	66	10.3	66	7.0	67	2.1
		66	3.9	68	6.9	68	6.9	67	2.0
		68	3.0					68	4.5

^a Fraction number; ^b gradient of pentane:Et₂O; ^c mass fraction in mg.

). All the fractions of CC have been analyzed by GC(RI) and by ¹³C NMR.

Compound 50, occidentalol: 139.85 (C, C4), 133.20 (CH, C1), 123.53 (CH, C2), 116.84 (CH, C3), 72.91 (C, C11), 47.66 (CH, C5), 47.26 (CH, C7), 39.09 (CH₂, C9), 35.65 (C, C10), 27,30 (CH₂, C6), 27.10 (CH₃, C12/C13), 26.82 (CH₃, C12/13), 26.03 (CH₃, C14), 24.80 (CH₂, C8), 22.23 (CH₃, C15).

Compound 55, occidentalol isomer: 137.58 (C, C4), 136.62 (CH, C1), 121.28 (CH, C2/C3), 120.77 (CH, C2/C3), 72.92 (C, C11), 45.00 (CH, C5), 44.15 (CH, C7), 34.22 (C, C10), 32.70 (CH₂, C9), 27.03 × 2 (CH₃, C12/C13), 25.75 (CH₃, C14), 24.06 (CH₂, C6), 22.18 (CH₂, C8), 20.95 (CH₃, C15).

3. Results and Discussion

Leaf oil and wood oil have been separately isolated using a Clevenger-type apparatus. Yields were 0.143% and 0.220% (v/w vs. dry material), respectively.

3.1. G. pensilis Leaf Oil

Leaf oil was subjected to GC(RI), GC-MS and ¹³C NMR analyses. In total, 28 components have been identified, that accounted for 96.8% of the whole composition (

Table 2. Chemical composition of leaf and wood essential oils from Glyptostrobus pensilis.

N°	Component	RI Apol Lit	RI Apol	RI Pol	RI Pol Lit	Leaf Oil %	Wood Oil %	Identification
1	Tricyclene	922	920	1016	1012	1.1	-	RI, MS, NMR
2	α-Pinene	934	930	1016	1025	23.4	-	RI, MS, NMR
3	Camphene	947	943	1063	1068	5.6	-	RI, MS, NMR
4	Sabinene	968	964	1120	1122	0.1	-	RI, MS
5	β-Pinene	973	969	1109	1110	1.6	-	RI, MS, NMR
6	Myrcene	983	980	1159	1161	4.9	-	RI, MS, NMR
7	α-Phellandrene	999	996	1163	1168	0.1	-	RI, MS
8	α-Terpinene	1011	1008	1178	1178	0.1	-	RI, MS
9	p-Cymene	1015	1011	1269	1270	0.1	-	RI, MS
10	Limonene *	1024	1022	1201	1198	33.3	-	RI, MS, NMR
11	β-Phellandrene *	1021	1022 *	1209	1209	0.8	-	RI, MS, NMR
12	γ-Terpinene	1050	1047	1243	1245	0.1	-	RI, MS
13	Terpinolene	1079	1077	1280	1282	0.2	-	RI, MS
14	Linalool	1086	1082	1544	1543	0.1	-	RI, MS
15	Borneol	1153	1147	1696	1700	0.1	-	RI, MS
16	Terpinen-4-ol	1164	1160	1598	1601	0.1	-	RI, MS
17	α-Terpineol	1176	1170	1693	1694	0.4	-	RI, MS, NMR
18	Bornyl acetate *	1270	1268	1577	1579	9.2	-	RI, MS, NMR
19	iso-Bornyl acetate *	1271	1268	1582	1573	1.8	-	RI, MS, NMR
20	α-Cubebene	1352	1345	1451	1460	-	0.1	RI, MS, NMR
21	Geranyl acetate	1361	1358	1753	1751	0.6	-	RI, MS, NMR
22	α-Copaene	1375	1372	1485	1491	-	0.1	RI, MS
23	α-Funebrene	1385 a	1377	1498	1500 a	-	0.2	RI, MS, NMR
24	α-Duprezianene	1388 b	1381	1516	1524 b	-	0.2	RI, MS
25	β-Elemene	1388	1383	1584	1591	-	0.2	RI, MS, NMR
26	Sibirene	1392 c	1396	1543	1528 c		0.4	RI, MS
27	α-Cedrene *	1417 a	1408	1560	1562 a	-	1.4	RI, MS, NMR
28	β-Funebrene *	1415 a	1408	1564	1570 a	-	3.5	RI, MS, NMR
29	β-Cedrene **	1425 a	1414	1590	1594 a	-	2.3 **	RI, MS, NMR
30	(E)-β-Caryophyllene **	1419	1414	1590	1598	6.5	2.3 **	RI, MS, NMR
31	Thujopsene	1435 d	1424	1614	1618 d	-	4.4	RI, MS, NMR
32	Prezizaene	1452 d	1440	1652	1632 d	-	0.4	RI, MS, NMR
33	(E)-β-Farnesene	1449	1445	1665	1664	-	0.3	RI, MS, NMR
34	α-Humulene	1449	1446	1662	1667	0.7	0.3	RI, MS, NMR
35	α-Acoradiene	1462 e	1455	nd	nd	-	0.2	RI, MS, NMR
36	β-Acoradiene	1462	1458	1684	1688	-	0.3	RI, MS
37	trans-Cadina-1(6),4-diene	1472 e	1464	nd	nd	-	0.4	RI, MS
38	ar-Curcumene	1471	1466	1773	1773	-	0.6	RI, MS, NMR
39	γ-Curcumene	1473	1468	1684	1692	-	0.5	RI, MS, NMR
40	Germacrene D	1476	1481	1706	1708	-	0.2	RI, MS, NMR
41	Cuparene	1505	1489	1815	1816	-	1.4	RI, NMR
42	β-Curcumene	1503	1498	1734	1737	-	0.4	RI, MS
43	α-Alaskene	1512 e	1502	1760	1763 f	-	0.4	RI, MS, NMR
44	trans-Calamenene	1513	1505	1826	1823	-	0.3	RI, MS, NMR
45	δ-Cadinene	1514	1510	1750	1756	-	2.4	RI, MS, NMR
46	epi-Zonarene	1508 g	1512	nd	nd		0.3	RI, MS
47	Zonarene	1521 e	1519	nd	nd	-	0.6	RI, NMR
48	α-Calacorene	1530	1523	1933	1921	-	0.1	RI, MS
49	β-Elemol	1537	1530	2076	2088	-	4.2	RI, MS, NMR
50	Occidentalol	1548 h	1530	2098	2097 h	-	6.6	RI, MS, NMR
51	(E)-Nerolidol	1550	1545	2038	2036	-	0.2	RI, MS
52	Caryophyllene oxide	1570	1566	1974	1986	3.7	0.9	RI, MS, NMR
53	Juniper cedrol	1583 d	1573	2098	2102 d	-	2.1	RI, NMR
54	Sesquithuriferol *	1600 d	1582	2104	2113 d	-	0.9	RI, NMR
55	Occidentalol isomer *	nd	1582	2198	nd	-	5.9	RI, NMR
56	Cedrol	1597	1587	2112	2120	-	29.3	RI, MS, NMR
57	β-Biotol	1595 d	1589	nd	2149 d		0.3	RI, MS
58	Humulene oxide	1597	1589	2030	2047	0.4	-	RI MS
59	epi-Cedrol	1613 a	1596	2166	2163 a	-	0.3	RI, MS, NMR
60	Eudesm-6-en-4α-ol	1607 i	1606	2158	2170 i	-	0.7	RI, NMR
61	1-epi-Cubenol	1614	1610	2054	2088	-	1.1	RI, MS, NMR
62	α-Acorenol	1616 d	1613	2128	2124 d	-	0.8	RI, MS, NMR
63	γ-Eudesmol	1616	1613	2161	2166	-	1.2	RI, MS, NMR
62	α-Acorenol	1616 d	1613	2128	2124 d	-	0.8	RI, MS, NMR
64	τ-Muurolol	1631	1624	2186	2186	-	0.1	RI, MS, NMR
65	Cubenol	1620	1626	2050	2068	-	0.5	RI, MS, NMR
66	β-Eudesmol	1634	1630	2222	2238	-	2.6	RI, MS, NMR
67	Selin-11-en-4α-ol	1640 i	1632	2245	2249 i	-	0.4	RI, MS, NMR
68	α-Eudesmol	1641	1635	2213	2223	-	1.8	RI, MS, NMR
69	Cedr-8-en-15-ol	1646 e	1642	nd	nd	-	0.6	RI, MS
70	Caryophylla-3,8(15)-dien-5β-ol	1655	1649	2366	2392	0.6	-	RI, NMR
71	Prezizaan-15-al	1661 d	1650	2142	2155 d	-	1.5	RI, NMR
72	allo-Cedrol (Khusiol)	1680 e	1659	2311	nd	-	0.6	RI NMR
73	α-Bisabolol *	1668	1662	2210	2213	-	1.3	RI, MS, NMR
74	epi-α-Bisabolol *	1674	1662	2210	2214	-	0.2	RI, MS, NMR
75	β-Acoradienol	1769 j	1772	nd	nd	-	0.2	RI, MS
76	13-epi-Pimaradiene	1941 k	1954	2238	nd	0.7	-	RI, NMR
77	Abietatriene	2033	2027	2483	2506	0.5	0.2	RI, MS, NMR
78	Manool	2047	2034	2648	2628	-	0.8	RI, MS, NMR
79	Abieta-3,7-diene	2062	2063	2444	2450	-	1.8	RI, MS, NMR
80	Feruginol	2283 l	2273	nd	nd	0.8	0.2	RI, NMR
	Total identified					96.8	92.3

Components listed following their elution order on non-polar column (BP-1). Percentages of individual components on non-polar column, except those with *, % on polar column and those with **, % by combination of GC and NMR. NMR: compound identified by ¹³C NMR in at least one oil sample; NMR (italic) compound identified by ¹³C NMR in one or more fraction(s) of CC; RI lit apol and pol ref. [12], otherwise stated: ^a comparison with RIs of pure compounds, ^b [23], ^c [24], ^d [25], ^e [14], ^f [26], ^g [27], ^h [28], ⁱ [29], ^j [30], ^k [31], ^l [32].

). The composition is dominated by monoterpene hydrocarbons, limonene (33.3%) and α-pinene (23.4%), followed by camphene (5.6%) and myrcene (4.9%). Bornyl acetate (9.2%) is the major oxygenated monoterpene accompanied by iso-bornyl acetate (1.8%). Sesquiterpenes are represented by (E)-β-caryophyllene (6.5%) and α-humulene (0.7%) and their oxides, caryophyllene oxide (3.7%) and humulene oxide (0.4%). Abietatriene, (synonym dehydroabietane, 0.5%), 13-epi-pimaradiene (synonym sandaracopimaradiene, 0.7%) and feruginol (0.8%) are the identified diterpenes. These results confirmed that the essential oil previously described by Dai and Thai in 2012 [10] was a leaf oil sample instead of wood oil. Although limonene (23.9%), α-pinene (18.9%), bornyl acetate (5.2%) and (E)-β-caryophyllene (6.1%) were its main components, the previously reported oil sample differed from the present one by the content of various components: camphene (5.6 vs. 1.4%), myrcene (4.9 vs. 2.2%), bornyl acetate (9.2 vs. 5.2%). In contrast, the 2012 oil sample contained various sesquiterpenes not detected in the present sample, for instance, among sesquiterpene hydrocarbons, germacrene D (3.6%), β-elemene (2.5%) and β-selinene (2.1%) were found. A few diterpenes, such as ferruginol, were found in both oil samples.

3.2. G. pensilis Wood Oil

G. pensilis wood oil has also been analyzed by GC(RI), GC-MS and ¹³C NMR. In contrast with the leaf oil sample, it is a sesquiterpene-rich essential oil. Indeed, neither monoterpene hydrocarbon nor oxygenated monoterpene has been detected. Various major components have been identified by GC(RI), GC-MS and/or ¹³C NMR (Table 2). However, the composition of this oil sample appeared complex, first by the occurrence of uncommon compounds and second by the number of overlapped peaks on the chromatogram that induced, on the one hand, a lack of correspondence between various percentages on non-polar and polar columns, respectively, and, on the other hand, poor fits during GC-MS analysis. Therefore, the sample was subjected to column chromatography (CC) over SiO₂ and 13 fractions were eluted using a gradient of solvents (pentane/diethyl oxide, 100/0 to 0/100) and analyzed by GC(RI) and ¹³C NMR (Table 1).

Various major components have been identified by combination of the three techniques, GC(RI), GC-MS and ¹³C NMR in the whole oil sample. Identification was confirmed by ¹³C NMR in one or more fractions of CC. Some minor components have been identified by GC(RI) and GC-MS in the whole oil and by ¹³C NMR in fractions of CC. Lastly, a few compounds have been identified in the fractions of CC by ¹³C NMR and quantified in the EO through their RIs.

Cedrol (29.3%) significantly dominated the essential oil composition. Among other components bearing the tricyclo[5.3.1.0^1,5]undecane framework (cedrane), the following have been identified: α-cedrene (1.4%) and β-cedrene (2.3%), as well as β-funebrene (3.5%) and α-funebrene (0.2%, identification ensured by ¹³C NMR in fraction B1 of CC). Cedrene and funebrene (α- and β-isomers) differ only by the stereochemistry of the ring junction and ¹³C NMR was useful to ensure their identification. Two minor components bearing the cedrane skeleton have been identified, β-biotol (0.3%) and epi-cedrol (0.3%). It could be noted that β-cedrene and (E)-β-caryophyllene displayed very close retention indices on non-polar and polar columns and they were fortuitously overlapped on both columns in our oil sample. They have been quantified by combining GC (whole percentage) and ¹³C NMR (ratio of the mean intensities of the signals of each component).

Other sesquiterpene hydrocarbons present with appreciable contents were thujopsene (4.4%), δ-cadinene (2.4%) and cuparene (1.4%). Cuparene was identified by GC(RI) and ¹³C NMR directly in the essential oil and confirmed in a fraction of CC. Similar identification was performed for zonarene (0.6%). In parallel, calamenene was identified by GC-MS (cis/trans stereochemistry not determined), the trans isomer being differentiated by ¹³C NMR in a fraction of CC. Similarly, MS suggested α- or β-alaskene for component 43, the α-isomer being identified by ¹³C NMR in fraction F1 of CC.

In addition to the oxygenated cedrane derivatives already mentioned, various oxygenated cyclic or bicyclic sesquiterpenes have been identified in the EO: β-elemol (4.2%), β-eudesmol (2.6%), α-eudesmol (1.8%), γ-eudesmol (1.2%), as well as occidentalol (6.6%). Identification of minor sesquiterpenes demonstrated the utility of combining various techniques. For instance:

-

Compound 60 was identified by ¹³C NMR in the F8 fraction of CC as eudesm-6-en-4α-ol and quantified in the EO through its retention indices;

-

Compound 62 α-acorenol, although co-eluted with γ-eudesmol on the non-polar column, has been identified by MS and ¹³C NMR;

-

For component 67 (RI apol 1632), MS suggested various sesquiterpenes bearing the bicyclo[4.4.0]decane skeleton and a tertiary alcohol function, such as intermedeol and isomers as well as selina-11-en-4α-ol. The last compound was elected by observation of its chemical shifts in the ¹³C NMR spectrum of fractions F12 and F13 of CC.

-

Component 71, prezizaan-15-al, has been identified only by NMR in fraction B2 of CC and quantified in the EO through its retention indices;

-

Components 73 and 74 gave overlapped signals on non-polar and polar GC columns and were identified as α-bisabolol or its epimer by GC-MS. ¹³C NMR demonstrated the occurrence of both epimers and they were quantified through the ratio of the mean intensities of the corresponding signals.

Special attention should be devoted to components 50 (RIa/RIp = 1530/2098) and 55 (RIa/RIp = 1582/2198), both being identified as occidentalol by GC-MS. The 15 signals of occidentalol were observed in the ¹³C NMR spectra of the EO and various fractions of CC and chemical shift values agree with those reported [33]. Component 50 is occidentalol in accordance with RI values, MS spectrum and ¹³C NMR data. It accounted for 6.6% in the EO and reached 26.6% in fraction F11. It could be noted that occidentalol was overlapped with β-elemol on the non-polar column used for GC-MS and with juniper cedrol on the polar column.

Occidentalol 50 was identified as early as 1956 in the wood oil from Thuja occidentalis [34]. It is a eudesmane derivative characterized by the cis junction of the icyclo[4.4.0]decane skeleton and conjugated double bonds. The relative stereochemistry of the substituents has been reported [35,36] and later corrected (Figure 2) [37].

Although co-eluted with sesquithuriferol on a non-polar column, compound 55 was also identified as occidentalol by MS and both components were differentiated on the polar column (RI = 2104 vs. 2198). It accounted for 5.9% in the whole oil composition and reached 18.4% in fraction B10. Unfortunately, our efforts to purify this compound (successive CC, preparative GC) in order to submit it to a full set of spectroscopic techniques and to elucidate its structure remained unsuccessful, probably due to the great number, in the EO, of oxygenated sesquiterpenes bearing the bicyclo[4.4.0]decane skeleton and a tertiary alcohol function. Therefore, we attempted to assess the structure of 55 with the data at our disposal. Besides 55 (18.4%), fraction B10 contained occidentalol (16%), elemol (14%) and cedrol (9.8%), the ¹³C NMR spectra of the three compounds being compiled in our spectral data library. It was possible to extract from the spectrum of B10 the 15 signals belonging to 55, and to assess the number of hydrogens linked to every carbon through the DEPT spectrum (see Section 2). The molecule contained three quaternary carbons (including an ethylenic carbon and a carbon linked to an oxygen atom), five CH (including three ethylenic carbons), three CH₂ and four CH₃. These findings corroborated the results of GC-MS that suggested “occidentalol”, the MS spectra of 50 and 55 being nearly superimposable. Compound 55 is probably an isomer of occidentalol. Indeed, 7-epi-occidentalol has been reported [38] as well as trans-occidentalol, but ¹³C NMR data were not mentioned. Comparing the chemical shifts of occidentalol isomer with those of occidentalol itself, the largest difference is observed for carbon C9, 32.70 ppm vs. 39.09 ppm. The shielding of 6.4 ppm is probably due to a γ steric effect, observable, for instance, in a molecule bearing the cis junction of the bicyclo[4.4.0]decane skeleton and the cis stereochemistry of the methyl and isopropanol groups.

Although some main components of our sample of G. pensilis wood oil were previously reported by Schmidt et al. [11], the compositions varied substantially, qualitatively and quantitatively. Indeed, some components displayed higher content in our oil sample, particularly cedrol (29.3% vs. 16.4%). In contrast, other components were less abundant in our oil sample: occidentalol (6.6% vs. 13.2%), elemol (4.2% vs. 8.9%), α-cedrene (1.4% vs. 6.1%). The major component of the Schmidt et al. oil sample, dihydro-eudesmol isomer (18.3%), and dihydro-eudesmol itself (5.7%) were not detected in our sample and, conversely, occidentalol isomer was not suggested in the previous paper. The occurrence of a misprint that reported “dihydro-eudesmol isomer” instead of “dehydro-eudesmol isomer”, i.e., occidentalol isomer, could be hypothesized.

According to Hortman et al. [38], “the coincident presence of a rarely occurring cis ring junction and a 1,3-diene system in occidentalol suggests that a unique biosynthetic pathway”. Therefore, we compiled below the plants that produce occidentalol (or occcidentalol isomer) as a secondary metabolite.

Following the pioneering work of Nakatsuka and Hirose [34] who identified occidentalol for the first time (percent not mentioned), a few papers reported on the occurrence of this sesquiterpene in Thuja occidentalis wood oil with appreciable content, 19–51%, depending on the duration of hydrodistillation [28,39]. In contrast, occidentalol has never been reported in foliage nor in cone essential oils of this tree [40,41,42,43,44].

However, occidentalol has been identified, to a lesser extent, in essential oils isolated from other species, inter alia, essential oils from wood of Thuja koraiensis [45]; from branches of Taxus canadensis, 1.4% [46]; from aerial parts of Bupleurum candollii, 1.7% [47]; from leaves and fruits of Julocoton triqueter, 7.4% [48]; from bark of Duguetia lanceolata, 1.4% [49]. Occidentalol has also been observed in tobacco leaves in response to a virus infection [33].

4. Conclusions

Leaves and wood of G. pensilis, a species scarcely found in Vietnam, in Dak Lak, a province of the central highlands, produced by hydrodistillation essential oils, rich in monoterpenes and sesquiterpenes, respectively. Limonene (33.3%) and α-pinene (23.4%) were the main components of leaf essential oil, which contained also sesquiterpenes and diterpenes to a lesser extent. In contrast, G. pensilis wood produced a sesquiterpene-rich essential oil whose composition was significantly dominated by cedrol (29.3%), besides other compounds bearing the tricyclo[5.3.1.0^1,5]undecane framework (cedrane). Wood oil contained also appreciable contents of occidentalol (6.6%) and occidentalol isomer (5.9%). Yet, none of these compounds can be actually considered as chemotaxonomic markers since they all are widespread compounds in the plant kingdom.