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

: 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 13 C NMR, were cedrol (29.3%), occidentalol (6.6%) and occidentalol isomer (5.9%).


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 yellowbrown 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 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.

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.

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 13 C NMR analyses.

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: H 2 (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).

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 13 C NMR analyses.

Nuclear Magnetic Resonance
13 C NMR spectra were recorded on a Bruker AVANCE 400 Fourier transform spectrometer operating at 100.63 MHz for 13 C, equipped with a 5 mm probe, in CDCl 3 , 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 CDCl 3 ).

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 13 C NMR spectra of the samples with those of reference spectra com-piled 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].

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.

G. pensilis Wood Oil
G. pensilis wood oil has also been analyzed by GC(RI), GC-MS and 13 C 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 13 C 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 nonpolar 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 SiO 2 and 13 fractions were eluted using a gradient of solvents (pentane/diethyl oxide, 100/0 to 0/100) and analyzed by GC(RI) and 13 C NMR (Table 1).
Various major components have been identified by combination of the three techniques, GC(RI), GC-MS and 13 C NMR in the whole oil sample.Identification was confirmed by 13 C 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 13 C NMR in fractions of CC.Lastly, a few compounds have been identified in the fractions of CC by 13 C NMR and quantified in the EO through their RIs.
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 13 C 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 13 C NMR in a fraction of CC.Similarly, MS suggested αor β-alaskene for component 43, the α-isomer being identified by 13 C 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 13 C NMR in the F8 fraction of CC as eudesm-6-en-4αol 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 13 C 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 13 C 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 13 C 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 13 C 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 icy-clo[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].
EO and reached 26.6% in fraction F11.It could be noted that occidenta with β-elemol on the non-polar column used for GC-MS and with jun polar column.
Occidentalol 50 was identified as early as 1956 in the wood oil fr talis [34].It is a eudesmane derivative characterized by the cis ju clo[4.4.0]decane skeleton and conjugated double bonds.The relative the substituents has been reported [35,36] and later corrected ( Although co-eluted with sesquithuriferol on a non-polar column, also identified as occidentalol by MS and both components were differ lar column (RI = 2104 vs. 2198).It accounted for 5.9% in the whole o reached 18.4% in fraction B10.Unfortunately, our efforts to purify this c sive CC, preparative GC) in order to submit it to a full set of spectrosco to elucidate its structure remained unsuccessful, probably due to the g EO, of oxygenated sesquiterpenes bearing the bicyclo[4.4.0]decane ske alcohol function.Therefore, we attempted to assess the structure of 55 w disposal.Besides 55 (18.4%), fraction B10 contained occidentalol (16%) cedrol (9.8%), the 13 C NMR spectra of the three compounds being comp data library.It was possible to extract from the spectrum of B10 the 15 to 55, and to assess the number of hydrogens linked to every carbon spectrum (see Section 2).The molecule contained three quaternary car ethylenic carbon and a carbon linked to an oxygen atom), five CH (inc enic carbons), three CH2 and four CH3.These findings corroborated th that suggested "occidentalol", the MS spectra of 50 and 55 being nearl Compound 55 is probably an isomer of occidentalol.Indeed, 7-epi-occ reported [38] as well as trans-occidentalol, but 13 C NMR data were no paring the chemical shifts of occidentalol isomer with those of occident est difference is observed for carbon C9, 32.70 ppm vs. 39.09ppm.T ppm is probably due to a γ steric effect, observable, for instance, in a m 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 13 C 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 CH 2 and four CH 3 .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 13 C 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. 39.09ppm.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.
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 path-way".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].

Figure 1 .
Figure 1.Vietnam.Dak Lak Province in red.Krong Nang District in green.Sample collection site is the black dot.

Figure 1 .
Figure 1.Vietnam.Dak Lak Province in red.Krong Nang District in green.Sample collection site is the black dot.

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