Integrated Analysis of the Wood Oil from Xanthocyparis vietnamensis Farjon & Hiep. by Chromatographic and Spectroscopic Techniques

In order to get better knowledge about the volatiles produced by Xanthocyparis vietnamensis, a species recently discovered in Vietnam, its wood oil has been analyzed by a combination of chromatographic (GC, CC) and spectroscopic (GC-MS, 13C-NMR) techniques. Forty components that accounted for 87.9% of the oil composition have been identified. The composition is dominated by nootkatene (20.7%), 11,12,13-tri-nor-eremophil-1(10)-en-7-one (17.2%), γ-eudesmol (5.1%), nootkatone (4.7%), valencene (3.5%) and 13-nor-eremophil-1(10)-en-11-one (2.6%). The structure of two new compounds—10-epi-nor-γ-eudesmen-11-one and 12-hydroxy-isodihydroagarofuran—has been elucidated, while 11,12,13-tri-nor-eremophil-1(10)-en-7-ol is reported as a natural product for the first time. The composition of X. vietnamensis wood oil varied drastically from those of leaf oils, dominated by hedycaryol (34.4%), phyllocladene (37.8%) or by pimara-6(14)-15-diene (19.4%).


Introduction
Xanthocyparis vietnamensis Farjon & Hiep (Cupressaceae), also known as Vietnamese gold cypress (Vietnamese name: Bách vàng), was discovered in 1999 in the calcareous mountains of Northern Vietnam (Quản Bạ, Hà Giang Province, Figure 1) [1]. Although found since that time in other localities (Hà Giang, Cao Bằng, Tuyên Quang provinces), it grows in a restricted area at an altitude of 1000-1600 m. The estimated number of adult trees is in the range 500-1000 individuals, therefore, X. vietnamensis remains among the endangered species [2]. This species has also been added to Group IA of the National List of Rare and Precious Flora and Fauna which prohibits any exploitation. An ex situ conservation program has been initiated and some restoration work undertaken [3]. This species grows associated with conifer species: Pseudotsuga sinensis, Nageia jneiryi, Podocarpus pilgeri, Calocedrus macrolepis, Taxils chinensis, Amentotaxus sp. The discovery of this new species led to the proposal of a new genus in 2002, the genus Xanthocyparis [4]. From the taxonomic point of view, analysis of 54 characteristics demonstrated the proximity of Chamaecyparis nootkatensis (D. Don) Spach with X. vietnamensis and therefore the former species has been transferred to the genus Xanthocyparis and it is nowadays inventoried as Xanthocyparis nootkatensis (D. Don) Farjon & Harder. This denomination was agreed in 2011 by the International Association for Plant Taxonomy [5,6].
X. vietnamensis is an evergreen, medium-sized tree, 10-15 m in height, 0.5 m in diameter. Bark is smooth, thin, red or brown-red and fibrous. The originality of this tree is the occurrence, at the adult stage, of two types of leaves (needles and tortoiseshells) [7]. The tree bears both male and female cones, solitary at the top of the branches, coming to maturity in two years [4]. Female cones are nearly globose, 9-11 mm × 10-12 mm, black or dark brown when ripe. Seeds occur in February. Male cones are oval, 2.5-3.5 mm × 2.0-2.5 mm. The wood is odoriferous and of excellent quality and it is used by local craftsmen for house building and house furniture as well as for wood-fired heating [1].
The aim of the present work was to get a better knowledge of the volatiles produced by X. vietnamensis by analyzing an essential oil sample isolated from the wood of this species. Due to the complexity of this essential oil, analysis was conducted by a combination of chromatographic and spectroscopic techniques.

Results
Wood from X. vietnamensis has been collected in Hà Giang Province ( Figure 1) and it produced by water-distillation a pale-yellow essential oil with a yield of 0.20% (v:m).

Analysis of X. vietnamensis Wood Oil by GC(RI), GC-MS and 13 C-NMR
The oil sample has been analyzed by GC(RI), GC-MS and 13 C-NMR without isolation of individual components following a computerized method developed at the University of Corsica The discovery of this new species led to the proposal of a new genus in 2002, the genus Xanthocyparis [4]. From the taxonomic point of view, analysis of 54 characteristics demonstrated the proximity of Chamaecyparis nootkatensis (D. Don) Spach with X. vietnamensis and therefore the former species has been transferred to the genus Xanthocyparis and it is nowadays inventoried as Xanthocyparis nootkatensis (D. Don) Farjon & Harder. This denomination was agreed in 2011 by the International Association for Plant Taxonomy [5,6].
X. vietnamensis is an evergreen, medium-sized tree, 10-15 m in height, 0.5 m in diameter. Bark is smooth, thin, red or brown-red and fibrous. The originality of this tree is the occurrence, at the adult stage, of two types of leaves (needles and tortoiseshells) [7]. The tree bears both male and female cones, solitary at the top of the branches, coming to maturity in two years [4]. Female cones are nearly globose, 9-11 mmˆ10-12 mm, black or dark brown when ripe. Seeds occur in February. Male cones are oval, 2.5-3.5 mmˆ2.0-2.5 mm. The wood is odoriferous and of excellent quality and it is used by local craftsmen for house building and house furniture as well as for wood-fired heating [1].
The aim of the present work was to get a better knowledge of the volatiles produced by X. vietnamensis by analyzing an essential oil sample isolated from the wood of this species. Due to the complexity of this essential oil, analysis was conducted by a combination of chromatographic and spectroscopic techniques.

Results
Wood from X. vietnamensis has been collected in Hà Giang Province ( Figure 1) and it produced by water-distillation a pale-yellow essential oil with a yield of 0.20% (v:m).
The chromatographic profile as well as the 13 C-NMR spectrum of fractions F2, F8 and F9 demonstrated that every fraction contained an unidentified major component. Therefore, it was
The chromatographic profile as well as the 13 C-NMR spectrum of fractions F2, F8 and F9 demonstrated that every fraction contained an unidentified major component. Therefore, it was decided to combine column chromatography (CC on silica gel using a gradient of solvents pentane/diethyl ether) and the concept of "extraction" NMR [18,19] to identify the major components of every fraction. Therefore, A is a bicyclic, mono unsaturated tri-nor-sesquiterpene alcohol. Otherwise, 2D NMR experiments, particularly long range proton-carbon connectivities in the HMBC spectrum, suggested the 11,12,13-tri-nor-eremophil-1(10)-en-7-ol structure for A (component 16 in Table 1, Figure 3). It could be pointed out that the chemical shifts of seven out of twelve carbons appeared very close to those of 11,12,13-tri-nor-eremophil-1(10)-en-7-one (15), one of the major components of the EO. The axial stereochemistry of H7 was ensured by the coupling constant values of its signal (H7, triplet of triplet, J = 11.3 and 4. 2 Hz). A search in the computerized data library SciFinder revealed that the molecule has been already obtained by Revial et al. [20] in the course of their synthesis of valencenol. It appears that this compound is reported from natural sources for the first time. decided to combine column chromatography (CC on silica gel using a gradient of solvents pentane/diethyl ether) and the concept of "extraction" NMR [18,19] to identify the major components of every fraction.

Structure Elucidation of New Natural Compounds
2.3.1. Identification of 11,12,13-Tri-nor-eremophil-1(10)-en-7-ol (16) Fraction F9 was subjected to CC on silica gel using a gradient of solvents (pentane/Et2O). Subfraction F9-7 contained a major component A (52.5%, 0.4% in EO; RIa/RIp = 1458/2173). The 13 C-NMR spectrum of this fraction exhibited 12 signals with high intensities. Combination of the information provided by DEPT spectrum (2 C, 3 CH, 5 CH2 and 2 CH3), and by 1 H and 13 C chemical shift values (occurrence of one double bond C=CH and an alcohol function CH-OH), suggested the formula C12H20O, in agreement with the molecular ion in the MS (M + = 180). Therefore, A is a bicyclic, mono unsaturated tri-nor-sesquiterpene alcohol. Otherwise, 2D NMR experiments, particularly long range proton-carbon connectivities in the HMBC spectrum, suggested the 11,12,13-tri-nor-eremophil-1(10)en-7-ol structure for A (component 16 in Table 1, Figure 3). It could be pointed out that the chemical shifts of seven out of twelve carbons appeared very close to those of 11,12,13-tri-nor-eremophil-1(10)en-7-one (15), one of the major components of the EO. The axial stereochemistry of H7 was ensured by the coupling constant values of its signal (H7, triplet of triplet, J = 11.3 and 4. 2 Hz). A search in the computerized data library SciFinder revealed that the molecule has been already obtained by Revial et al. [20] in the course of their synthesis of valencenol. It appears that this compound is reported from natural sources for the first time.

Identification of 10-epi-Nor-γ-eudesmen-11-one (24)
Sub-fraction F2.3, obtained by fractionation of F2 on silica gel, contained a main compound (B, 40.4%, 0.9% in EO; RIa/RIp = 1553/2039). The 13 C-NMR spectrum of that fraction displayed 14 signals with high intensities that obviously belonged to B (Table 2). DEPT spectra differentiated four quaternary carbons (including a carbonyl carbon and two olefinic carbons), one CH, six CH2 and three CH3. Therefore, the formula C14H22O was deduced in agreement with the molecular ion M + = 206 in the MS. Beside the carbonyl function, that belonged to an acetyl group according to chemical shift data, and one double bond, the structure of B contained two rings and therefore it formed a bicyclodecane framework. Two possibilities have been considered: the bicyclo[4.4.0]decane and bicyclo[5.3.0]decane skeletons. Although the 1 H-NMR spectrum of the fraction was complex, the HSQC spectrum allowed the identification of protons that belonged to B. Then the long range heteronuclear connectivities reported in the Table 2 conducted to the structure of a nor-γ-eudesmenone ( Figure 4). The dimethylbicyclo[4.4.0]decane framework was constructed starting from signals of hydrogens of methyl groups located on C4 and C10, respectively, and from the H7 (methine) hydrogen. The location of the acetyl group on C7 was confirmed by the connectivities of H12 with C7. Other heteronuclear connectivities were in agreement with the proposed structure. Moreover, the structure was confirmed by the proximity of the chemical shift values with those of γ-eudesmol and 10-epi-γ-eudesmol [21,22]. Unfortunately, the relative cis or trans stereochemistry of C10-Me and the acetyl group bore by C7 cannot be deduced from the through

Identification of 10-epi-Nor-γ-eudesmen-11-one (24)
Sub-fraction F2.3, obtained by fractionation of F2 on silica gel, contained a main compound (B, 40.4%, 0.9% in EO; RIa/RIp = 1553/2039). The 13 C-NMR spectrum of that fraction displayed 14 signals with high intensities that obviously belonged to B (Table 2). DEPT spectra differentiated four quaternary carbons (including a carbonyl carbon and two olefinic carbons), one CH, six CH 2 and three CH 3 . Therefore, the formula C 14 H 22 O was deduced in agreement with the molecular ion M + = 206 in the MS. Beside the carbonyl function, that belonged to an acetyl group according to chemical shift data, and one double bond, the structure of B contained two rings and therefore it formed a bicyclodecane framework. Two possibilities have been considered: the bicyclo[4.4.0]decane and bicyclo[5.3.0]decane skeletons. Although the 1 H-NMR spectrum of the fraction was complex, the HSQC spectrum allowed the identification of protons that belonged to B. Then the long range heteronuclear connectivities reported in the Table 2 conducted to the structure of a nor-γ-eudesmenone (Figure 4). The dimethylbicyclo [4.4.0]decane framework was constructed starting from signals of hydrogens of methyl groups located on C4 and C10, respectively, and from the H7 (methine) hydrogen. The location of the acetyl group on C7 was confirmed by the connectivities of H12 with C7. Other heteronuclear connectivities were in agreement with the proposed structure. Moreover, the structure was confirmed by the proximity of the chemical shift values with those of γ-eudesmol and 10-epi-γ-eudesmol [21,22]. Unfortunately, the relative cis or trans stereochemistry of C10-Me and the acetyl group bore by C7 cannot be deduced from the through space H-H connectivities in the NOESY spectrum that appeared really poor. In both cases, the acetyl group should adopt an equatorial (or pseudo-equatorial) position. This point is confirmed by the multiplicity (doublet of triplets) and the coupling constant values (14.8 and 2.2 Hz) of equatorial H6 ( 2 J H6eq-H6ax ; 3 J H6-H7 and 4 J H6eq-H8eq , W stereochemistry) and it is in agreement with the occurrence of a correlation plot between H 6eq and H13 in the NOESY spectrum.  Comparison of the chemical shift values of B with those of the γ-eudesmol epimers reported in the literature could be informative. Indeed, the chemical shift of carbon C7 that differs drastically in γ-eudesmol (50.56 ppm) and epi-γ-eudesmol (44.21 ppm) [21,22] cannot be taken into consideration since compound 24 bore an acetyl group instead of the "isopropyl alcohol" group. However, a significant difference was observed on the chemical shift value of olefinic quaternary carbon C4, 124.48 ppm for γ-eudesmol and 125.98 ppm for 10-epi-γ-eudesmol, the latter being close to the C7 of  1.60, 1.01 ppm). Finally, the multiplicity (dt) and coupling constants (14.8 and 2.2 Hz) of H6eq of 24 are in agreement with those of epi-γ-eudesmol (dt, 14.8 and 2.8 Hz) [21,22].
It should be mentioned that the C10 epimeric ketone has been reported by Marshall and Pike in the course of the stereoselective total synthesis of γ-eudesmol [23]. The relative cis stereochemistry of acetyl and methyl groups was deduced from mechanistic considerations and confirmed by MeLi addition to the keto group leading to γ-eudesmol. Although 1 H-NMR data were not reported in detail, the chemical shifts of C4-Me and C10-Me (1.61 and 1.04 ppm) fit perfectly with those of γ-  Comparison of the chemical shift values of B with those of the γ-eudesmol epimers reported in the literature could be informative. Indeed, the chemical shift of carbon C7 that differs drastically in γ-eudesmol (50.56 ppm) and epi-γ-eudesmol (44.21 ppm) [21,22] cannot be taken into consideration since compound 24 bore an acetyl group instead of the "isopropyl alcohol" group. However, a significant difference was observed on the chemical shift value of olefinic quaternary carbon C4, 124.48 ppm for γ-eudesmol and 125.98 ppm for 10-epi-γ-eudesmol, the latter being close to the  1.60, 1.01 ppm). Finally, the multiplicity (dt) and coupling constants (14.8 and 2.2 Hz) of H 6eq of 24 are in agreement with those of epi-γ-eudesmol (dt, 14.8 and 2.8 Hz) [21,22].
It should be mentioned that the C10 epimeric ketone has been reported by Marshall and Pike in the course of the stereoselective total synthesis of γ-eudesmol [23]. The relative cis stereochemistry of acetyl and methyl groups was deduced from mechanistic considerations and confirmed by MeLi addition to the keto group leading to γ-eudesmol. Although 1 H-NMR data were not reported in detail, the chemical shifts of C4-Me and C10-Me (1.61 and 1.04 ppm) fit perfectly with those of γ-eudesmol (1.60 and 1.01 ppm), and therefore confirm the trans stereochemistry of C10-Me and acetyl group in 24.

Identification of 12-Hydroxy-isodihydroagarofuran (39)
Sub-fraction F8.15, obtained by fractionation of F8 on silica gel, contained a main component (C, 60.1%; 2.3% in EO; RIa/RIp = 1747/2452). The 13 C-NMR spectrum of that fraction exhibited 15 signals with high intensities. DEPT spectra allowed the differentiation of three C, two CH, seven CH 2 and three CH 3 . Chemical shift values suggested the occurrence of an oxide function (2 C at δ = 88.21 ppm and 82.91 ppm) and a primary alcohol function CH 2 -OH (δ = 69.29 ppm) corroborated by the occurrence of two doublets at 3.40 and 3.24 ppm ( 2 J = 10.4 Hz) in the 1 H spectrum. The molecule contained also three methyl groups ( 1 H-NMR signals: a doublet and two singlets). The formula C 15 H 26 O 2 was deduced from these data in agreement with the molecular ion M + = 238 in the MS. Obviously, the molecule displays a tricyclic structure. Owing to the occurrence of a CH 2 -OH group, one of the three rings is oxygenated. This suggestion was confirmed by examination of long range correlations in the HMBC spectrum. The bicyclo [4.4.0]decane skeleton was constructed starting from methyls C14 and C15 and the dihydroagarofuran framework resulted inter alia from correlations of H7 and H13 with various carbons ( Table 3). The primary alcohol function was assigned to carbons C12 or C13 on the basis of the correlation plot of the methylenic hydrogens with C7. It was attributed to C12, the γ-shielding effect of the hydroxyl group on C13 being in the range 5-6 ppm, as expected. At this stage, component 39 should be a 12-hydroxydihydroagarofuran ( Figure 5). Two points should be specified: (i) the stereochemistry of the ring junction of the decalin substructure; (ii) the relative stereochemistry of methyls C14 and C15. Unfortunately, the NOESY spectrum was particularly poor and therefore no significant through space interaction between hydrogen atoms has been detected. The correct stereochemistry was obtained by comparison of the 13 C-NMR and 1 H-NMR chemical shifts of 39 with those of the four dihydroagarofuran isomers reported in the literature [24][25][26][27]. Indeed, the 13 C-NMR chemical shifts of C2 (21.33 ppm) and C4 (32.23 ppm) of 39 differ substantially from those of trans-dihydroagarofuran and 4-epi-cis-dihydroagarofuran (17.0/17.7 and 40.5/43.1, respectively) [25,27]. In contrast they are close to those of isodihydroagarofuran and cis-dihydroagarofuran (21.4/21.6 ppm and 32.2/32.6 ppm), respectively [24,26]. Differentiation between the last two isomers has been achieved by comparison of the 1 H chemical shifts of methyls C14 and C15, 1.01 and 0.85 for 39, much most closer to those of isodihydroagarofuran (1.00 and 0.88 ppm) than to those of cis-dihydroagarofuran (0.88 and 0.82 ppm). Therefore, compound 39 is 12-hydroxy-isodihydroagarofuran. Sub-fraction F8.15, obtained by fractionation of F8 on silica gel, contained a main component (C, 60.1%; 2.3% in EO; RIa/RIp = 1747/2452). The 13 C-NMR spectrum of that fraction exhibited 15 signals with high intensities. DEPT spectra allowed the differentiation of three C, two CH, seven CH2 and three CH3. Chemical shift values suggested the occurrence of an oxide function (2 C at δ = 88.21 ppm and 82.91 ppm) and a primary alcohol function CH2-OH (δ = 69.29 ppm) corroborated by the occurrence of two doublets at 3.40 and 3.24 ppm ( 2 J = 10.4 Hz) in the 1 H spectrum. The molecule contained also three methyl groups ( 1 H-NMR signals: a doublet and two singlets). The formula C15H26O2 was deduced from these data in agreement with the molecular ion M + = 238 in the MS. Obviously, the molecule displays a tricyclic structure. Owing to the occurrence of a CH2-OH group, one of the three rings is oxygenated. This suggestion was confirmed by examination of long range correlations in the HMBC spectrum. The bicyclo [4.4.0]decane skeleton was constructed starting from methyls C14 and C15 and the dihydroagarofuran framework resulted inter alia from correlations of H7 and H13 with various carbons ( Table 3). The primary alcohol function was assigned to carbons C12 or C13 on the basis of the correlation plot of the methylenic hydrogens with C7. It was attributed to C12, the γ-shielding effect of the hydroxyl group on C13 being in the range 5-6 ppm, as expected. At this stage, component 39 should be a 12-hydroxydihydroagarofuran ( Figure 5). Two points should be specified: (i) the stereochemistry of the ring junction of the decalin substructure; (ii) the relative stereochemistry of methyls C14 and C15. Unfortunately, the NOESY spectrum was particularly poor and therefore no significant through space interaction between hydrogen atoms has been detected. The correct stereochemistry was obtained by comparison of the 13 C-NMR and 1 H-NMR chemical shifts of 39 with those of the four dihydroagarofuran isomers reported in the literature [24][25][26][27]. Indeed, the 13 C-NMR chemical shifts of C2 (21.33 ppm) and C4 (32.23 ppm) of 39 differ substantially from those of trans-dihydroagarofuran and 4-epi-cis-dihydroagarofuran (17.0/17.7 and 40.5/43.1, respectively) [25,27]. In contrast they are close to those of isodihydroagarofuran and cisdihydroagarofuran (21.4/21.6 ppm and 32.2/32.6 ppm), respectively [24,26]. Differentiation between the last two isomers has been achieved by comparison of the 1 H chemical shifts of methyls C14 and C15, 1.01 and 0.85 for 39, much most closer to those of isodihydroagarofuran (1.00 and 0.88 ppm) than to those of cis-dihydroagarofuran (0.88 and 0.82 ppm). Therefore, compound 39 is 12-hydroxyisodihydroagarofuran.  Table 3. NMR data of 12-hydroxyisodihydroagarofurane (39).
linear interpolation (Target Compounds software, version 6.3.2, Perkin-Elmer). with those of authentic compounds or literature data; (b) on computer matching with laboratory-made and commercial mass spectral libraries [14,29,30]; and (c) on comparison of the signals in the 13 C-NMR spectra of essential oils with those of reference spectra compiled in the laboratory spectral library with the help of laboratory-developed software [10].