Chemical constituents from Vellozia graminifolia ( Velloziaceae )

From the hexane and ethyl acetate extracts obtained from stems, roots and leaf sheaths ofVellozia graminifolia, a mixture of linear hydrocarbons, a mixture of pentacyclic triterpenes, five monoisoprenylated flavonoids and one labdane diterpene, (-)-ent-3β-hydroxy-8(17)-labden-15-oic acid, were isolated. The linear hydrocarbons and minor triterpenes were identified in their corresponding mixtures by High Resolution Gas Chromatography (HRGC) and HRGC coupled to mass spectrometry. The major pentacyclic triterpenes and the diterpene were characterized by spectral data, including 2D NMR experiments, and chemical transformations.


INTRODUCTION
Velloziaceae is a family of tropical monocotyledonous plants containing about 270 species, most of which occur in Brazilian tropical scrub on rock outcrops (''campos rupestres'') (Stannard 1995).The Vellozia genus presents the largest number of species in the Velloziaceae family.Previous phytochemical studies showed that the main constituents of the Vellozia genus are flavonoids (Harborne et al. 1994, Williams et al. 1994), diterpenoids (Pinto et al. 1983(Pinto et al. , 1991(Pinto et al. , 1996a) ) and triterpenoids.
The triterpenoids reported in the literature as constituents of Vellozia species are distributed into the following classes: dammarane, lupane, taraxerane, oleanane and euphane (Barnes et al. 1984, Pinto et al. 1996b, Peixoto et al. 1979).The application of High Resolution Gas Chromatography (HRGC) and HRGC coupled to mass spectrometer (MS) can be used to distinguish its triterpenoids (Shiojima et al. 1992, Ogunkoya 1981) by retention time and fragmentation patterns, respectively.Thus, the identification of these compounds can be accomplished by co-injection in the HRGC using authentic samples isolated from other plants (Pinto et al. 1994, Patitucci et al. 1995).On the other hand, HRGC requires special attention in Velloziaceae because the presence of peaks corresponding to sterols (Peixoto et al. 1979) and dimmeric diterpenes (Pinto et al. 1997) can be observed in the same region where the triterpenes appear.

General Experimental Procedures
Mps were uncorr.NMR spectra were recorded in CDCl 3 soln at 300 MHz for 1 H and 75 MHz for 13 C on a Bruker AC 300 spectrometer, using TMS as int.standard or by reference to the solvent signals: CHCl 3 at δ H 7.25 and CHCl 3 at δ C 77.00.HRGC analyses were recorded on a Hewlett Packard model 5790A gas chromatograph.Glass capillary column (11 m × 0.25µm) coated with SE-54 (df = 0.25µm).Analytical GC conditions: Hydrogen as carrier gas; flow rate of 2 ml/min; injector port was set at 280 • C and flame ionization detector (FID) at 290 • C. The temperature program for the analysis of mixtures ranged from 260 • C to 280 • C. The data were collected on an HP 3396-II integrator.HRGC-MS analyses were performed on an HP 5987A spectrometer (Hewlett Packard, Palo Alto, USA), with electron impact ionization (70eV).MS scan range was 40 to 700 Da.The GC-MS interface was at 350 • C and ion source temperature at 300 • C. Column temperature program and injection mode were as for chromatographic analysis.CC: silica gel (0.063 to 0.2 mm).TLC: silica gel (Merk, Kieselgel 60), spots visualized by UV (254 and 360 nm) and exposure to I 2 vapor.TLC was used to analyze frs collected from CC.

Plant Material
The Vellozia graminifolia was collected in Chapada da Diamantina, Minas Gerais State, Brazil and identified by Prof. Nanuza L. de Menezes of the Universidade de São Paulo, São Paulo-SP, Brazil.A voucher of this plant is deposited in the herbarium of the Instituto de Botânica at the same university.

Extraction and Purification
Dried and powdered material (roots, stems and leaf sheaths) was successively extracted with n-hexane and ethyl acetate at room temp.and the solvents removed under vacuum to yield 12.9 and 13.9 g of oily residue, respectively.The n-hexane extract (12.9 g) was chromatographed on a column of silica gel (112 g) and eluted with hexane/EtOAc and EtOAc/MeOH mixtures in increasing polarity.A total of 29 fractions, each containing ca 120 mL, were collected and combined on the basis of TLC comparative analysis.Fraction 1, eluted with hexane, afforded a mixture of alkanes.Fractions 4 and 5, eluted with hexane/EtOAc (3:1), furnished a mixture of pentacyclic triterpenes (1-15) (36 mg, as red oil) after removal of aliphatic esters with acetone.Fractions 10 and 11, eluted with hexane/EtOAc (2:1), yielded a mixture of 20 and 21.Fraction 20 eluted with EtOAc/MeOH (5:1) afforded 16 (48 mg), after recrystallization from acetone.The EtOAc extract (13.90 g) was chromatographed using the same procedures described above, yielding 22-24.

RESULTS AND DISCUSSION
The crude hexane extract from V. graminifolia was initially analyzed by HRGC and HRGC-MS.The previous knowledge of the heat stable chemical constituents present has been established for retention time (R t ) and comparison of the mass spectral data with those of standards available in our laboratory.The complexity observed in the terpenoid eluting regions in the HRGC chromatogram led to fractionating on open silica gel column in the usual manner (see Experimental), yielding initially two fractions.
The presence of the n-alkanes (C 27 H 56 to C 30 H 62 ) in the more apolar fraction (eluted with hexane) of the hexane extract have been determined on the basis of mass fragmentograms (m/z 57 and 85) (Silverstein et al. 1995) obtained by HRGC-MS.

Minor Pentacyclic Triterpenes by HRGC-MS
The fraction containing 2 and 8 was submitted to recrystallization to remove components in major percentage to improve the analysis of the minor components by HRGC-MS.
Thus, the above triterpenoids 1-15 (Figure 3) were identified by their mass spectra (Table I and  Figure 4) and by comparison with standards previously isolated from species of Velloziaceae.
Obviously, pentacyclic triterpenoids of the ursane-type may be included as structural alternatives, since the occurrence of mixtures containing oleanane and ursane isomers is relatively common.Secondary biomodifications of triterpenes may involve the introduction of an additional hydroxyl group and double bond, oxidation of hydroxyl functions to carbonyl groups, reduction of double bond, Wagner-Meerwein rearrangement and alkylation by S-adenosyl methionine (Torssell 1997).
The presence of the signal corresponding to hydrogens of a hydroxymethylene group at δ H 3.62 (m).The molecular ion at m/z 308 observed in the mass spectrum was also used to characterize the structure of this diol.The negative optical rotation compared with the recently synthesized (+)-3β-hydroxylabd-8(17)-ene-15-ol (Pemp and Seifert 1997) allowed determination of 19 as an ent-labdane.Thus, the natural diterpene isolated from Vellozia graminifolia was characterized as (-)-3βhydroxylabd-8(17)-en-15-oic acid (16).To the best of our knowledge 16, is the first diterpene with a labdane skeleton from the Velloziaceae family; its enantiomer was isolated from Araucaria imbricata (Chandra et al. 1964) and from Moldenhawera nutans (David et al. 1998).

Monoisoprenylated Flavonols
The structures of the new prenylated flavonols (20-22) (Figure 6) have been identified on the basis of spectral analysis, including 2D NMR techniques   such as HMQC, HMBC and NOESY, together with chemical shift correlations (Branco et al. 1998, 2001, Branco 2001).The additional new flavonoids 23 and 24 were characterized by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry experiments (Branco et al. 2001, Branco 2001).

Fig. 4 -
Fig.4-Ionic fragments corresponding to principal peaks observed in the mass spectra.Obviously, the ionic fragments that appear with numbers and letters to indicate the triterpene correspondents can be used for other triterpenoids if compatible.