New Volatile Constituents from Leaves of Stemodia trifoliata ( Link . ) Reichb . ( Schrophulariaceae )

Os óleos essenciais das folhas de Stemodia trifoliata (Scrophulariaceae), coletadas nos meses de agosto de 2005 e 2006, foram analisados por CG-EM e CG-DIC. Um total de 22 componentes voláteis, representados por sesquiterpenos e diterpenos, foi identificado. β-Cariofileno (9,4-15,4%) e óxido de cariofileno (6,2-9,0%) foram os principais componentes identificados na fração sesquiterpênica, enquanto os diterpenóides 6α-acetóxi-óxido de manoíla (13,9-23,2%) e 6α-hidróxi-óxido de manoíla (25,1-29,7%) foram os constituintes majoritários da fração diterpênica. Estes diterpenos, derivados do óxido de manoíla, são novos e suas estruturas foram determinadas por métodos espectroscópicos, particularmente RMN 1D e 2D. A investigação química dos componentes voláteis de S. trifoliata é descrita pela primeira vez neste trabalho.


Introduction
The genus Stemodia (Scrophulariaceae) comprises approximately 40 species represented by annual herbs and shrubs distributed in Asia, Africa, Australia and the Americas. 1][3][4][5] Secondary metabolites such as cucurbitacins, stearic acid derivatives and flavonoids have been isolated, but diterpenes containing an unusual tetracyclic skeleton, known as stemodane, are the most characteristic compounds elaborated by plants of this genus.7][8][9] S. trifoliata (Link) Reichb. is an annual plant, herbaceous and pubescent.Due to its preference by fertile and humid soils it is commonly found in flower beds, orchards and back yard gardens. 10n the present work, the chemical composition of the essential oil from the leaves of a wild population of S. trifoliata, as well as the isolation of two novel labdane diterpenes, are described.This is the first report on the phytochemical investigation of S. trifoliata and on the volatile chemical composition of a Stemodia species.

Results and Discussion
The essential oils of S. trifoliata, collected in August of two different years (2005 and 2006), but from the same site, were analyzed by GC-MS and GC-FID.The chemical composition of the oils, including the retention index and the percentage of each constituent is presented at Table 1.A total of 22 components was identified, representing 87.0 and 92.6% of the whole oils samples.Except by the presence of (E)-3-hexenol (2.6%), detected just in one of the analyzed oils, they were composed mainly by sesquiterpenes (33.2-48.0%)and diterpenes (44.6-51.2%),but the prevalent components were the same in both oils.In spite of this, marked qualitative differences were observed for the two oils, since a higher number of sesquiterpenes were identified for the sample B. Among the identified sesquiterpenes β-caryophyllene (9.4-15.4%)and caryophyllene oxide (6.2-9.0%) were the major ones.The diterpene fraction was dominated by manoyl oxide derivatives, which were identified as 6α-hydroxymanoyl oxide (25.1-29.7%)and 6α-acetoxymanoyl oxide (13.9-23.2%).In the GC-MS preliminary analysis of the oils, the peaks corresponding to 6α-hydroxymanoyl oxide (KI 1878) and 6α-acetoxymanoyl oxide (KI 1895), were not identified from their Kovats Indices and mass spectra.For this reason, an aliquot of the combined essential oils (samples A and B), was subjected to flash chromatography yielding both compounds, in the pure forms.Their structures were unequivocally elucidated by spectroscopy, especially NMR, including 1D and 2D experiments (Table 2).
Compound 1 was isolated as a colorless oil and its molecular formula C 22 H 36 O 3 was deduced by a combination of 13 C NMR spectral data and EIMS (m/z 333, [M] + -CH 3 ).Even though the molecular ion of 1 has not been detected it is plausible to speculate the loss of Me-16 to afford peak m/z 333 (5%) after the stability conferred by the conjugation of the positive charge with the vinyl group increased by the Compound 2 was also isolated as colorless oil and its molecular formula C 20 H 34 O 2 was deduced by a combination of NMR 13 C spectral data and EIMS (m/z 291, [M] + -CH 3 ).The same kind of explanation used for 1 should be used here to justify the peak at m/z 273 (12%) after dehydration from m/z 291 (24%).This molecular formula  along with the 1 H and 13 C NMR spectra similarities for both compound suggested that compound 2 could be the corresponding alcohol of 6α-acetoxymanoyl oxide, 1.This was reinforced by the absence of the typical signals of the acetyl group.Moreover, the oxymethine proton (H-6) was shifted highfield (d 3.88) in 2 compared with that at d 5.11 in 1.The expected deshielding effect on the chemical shifts of carbons C-5 (d 61.9) and C-7 (d 54.8) when compared with those of 1 (d 59.1, C-5; d 50.1, C-7) was in agreement with the presence of a hydroxyl group in C-6 after the disappearance of the γ-effect of the acetyl group.This was also supported by the HMBC data (Figure 1).The NOESY experiment (Figure 2) showed that the stereochemistry of diterpene 2 was consistent with that established for 1.It is worthy of notice that the isomer 6α-hydroxy-13-epimanoyl oxide has been previously isolated from southern pine (Pinus spp.) tall oil. 12

General experimental procedures
The qualitative analysis was carried out on a Shimadzu GC-17A/QP5050 using a non-polar OV-5 fused silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness); carrier gas helium, flow rate 1 mL min -1 and with split mode (ratio 1:48).The injector temperature and detector temperature were 250 and 280 ºC, respectively.The column temperature was programmed from 40 to 180 ºC at 4 ºC min -1 and then 180 to 250 ºC at 20 ºC min -1 and held isothermal for 7 min.Mass spectra were recorded from 30-450 m/z.NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for 1 H and 125 MHz for 13 C) spectrometer equipped with 5 mm inverse detection z-gradient probe.The chemical shifts, given on the d scale, were referenced to the residual undeuterated CHCl 3 portion (d H 7.27) and, the center peak of the deuterated CDCl 3 (d C 77.23).

Plant material
The leaves of S. trifoliata were collected during the flowering stage, in August 2005 and 2006, from a population growing on the road margins at Pico Alto, Guaramiranga ridge (Ceará State, Brazil), at an elevation of approximately 1000 m.A voucher specimen (No. 39550) has been deposited at the Herbário Prisco Bezerra (EAC) of the Departamento de Biologia, Universidade Federal do Ceará.

Essential oil distillation
The fresh leaves of S. trifoliata were hydrodistilled in a Clevenger-type apparatus for a period of 2 h to afford yellowish oils, which were dried over Na 2 SO 4 , stored in sealed glass vials and preserved under refrigeration before analysis.The yield of the oils (0.02%, m/m), were calculated on fresh weight of the plant materials.

Isolation of compounds 1 and 2
An aliquot of the leaf essential oil (86 mg) of S. trifoliata, was subjected to silica gel column flash chromatography using a gradient of n-hexane:CH 2 Cl 2 4:6 to give 72 fractions of approximately 8 mL each.After TLC analysis, fractions 65 to 67 were combined (48 mg) and further submitted to silica gel flash chromatography using CH 2 Cl 2 as eluent to yield the pure compounds 1 (17 mg) and 2 (10 mg).

Compound identification
The individual components of the oil were identified on the basis of their GC retention indices (RI) with reference to a homologous series of C 8 -C 26 n-alkanes, and by matching their 70 eV mass spectra with those of the spectrometer data base using the Wiley Class-5000 library and, by comparison of the fragmentation patterns of the mass spectra with those reported in the literature. 13The structures of the new volatile diterpenes (1 and 2) were elucidated based on spectroscopic analysis and comparison with published data for known manoyl oxide diterpenes.

Table 1 .
11emical composition of the leaf oils of Stemodia trifoliateThe above data are consistent with a manoyl oxide diterpene with an acetoxy substituent.The presence of the acetylated hydroxyl group at the C-6 position was supported by the observed 3 J C-H long-range correlations displayed for H-6 (d 5.11, td, J = 4.0 and 11.4 Hz) and the carbon signals at d 170.3 (C-21) and 74.5 (C-8).In the HMBC spectrum the correlation of H-14 (d 5.87, dd, J = 10.7 and 17.3 Hz) with the carbon signal at d 29.4 was sufficient to assure the correct assignment of CH 3 -16.11The4-axial orientation inferred to this methyl group was in agreement with the chemical shift at d 29.4.The proposed relative stereochemistry was further supported by the NOESY experiment, which clearly revealed dipolar interaction between CH 3 -16/CH 3 -17 and CH 3 -16/H-12eq.Similarly, the α-equatorial orientation suggested to the acetyl group was defined based on nOe correlations of H-6 with the methyl groups 17, 19 and 20.
a Constituents listed in order of elution on DB-5 column; b RI = Kovats retention index according to n-alkanes (C 8 -C 26 ); c A = Essential oil percentage of S. trifoliata collected in August 2005; d B = Essential oil percentage of S. trifoliata collected in August 2006; e Methods used in the identification of the volatile constituents.dd, J = 17.3 and 1.4 Hz, H-15a) and 4.94 (1H, dd, J = 10.7,1.4 Hz, H-15b).The 13 C NMR (CPD) spectrum showed the presence of 22 carbon signals assigned to five methyls, six methylenes, three methines, a vinyl moiety, one acetyl group, as well as to four non-hydrogenated carbon atoms after comparison with both DEPT 135 and HMQC spectra.

Table 2 .
13 and13C NMR spectral data for compounds 1 and 2, in CDCl 3 a All hydrogen and carbon assignments were made based on DEPT 135°, 1 H, 1 H-COSY, HMQC and HMBC experiments; Chemical shifts (d) in ppm; Coupling constants (J) in Hz; Several multiplicities were not determined due to bond complexity or superimposition.