Saponins and Sapogenins from Brachiaria decumbens Stapf

Quatro saponinas esteroidais e três sapogeninas foram identificadas das partes aéreas de Brachiaria decumbens. As estruturas desses compostos foram estabelecidas através de métodos químicos e espectroscópicos (RMN de H e C, HMBC, HMQC) como 3 -metóxi-lanost-9(11)-eno (1), diosgenina (2a), iamogenina (2b), 3-O-D-glicopiranosil-24(S)-etil-22E-deidrocolesterol (3a), 3O-D-glicopiranosil-24(R)-etil-22E-deidrocolesterol (3b), dioscina (4a) e 3-O-{ -Lramnopiranosil-(1 4)-[ -L-ramnopiranosil-(1 2)]-D-glicopiranosil}-25(S)-espirost-5-en-3 -ol (4b). Esses compostos foram isolados pela primeira vez em B. decumbens, sendo que o composto 4b não foi ainda descrito na natureza conforme revisão realizada.


Introduction
Brachiaria decumbens Stapf belongs to a group of plants capable of inducing hepatogenous photosensitizations [1][2][3][4] similar to those described for Panicum spp., [5][6][7][8] Tribulus terrestris, 8,9 Agave lecheguilla 10 and Narthecium ossifragum 8 .These species are all known to contain steroidal saponins which have been associated with deposition of crystalloid material within the biliary system and photosensitization.In our previous paper, 11 we reported the isolation and structural determination of steroidal sapogenins present in rumen contents of lambs intoxicated with B. decumbens.In this paper we describe the structural elucidation of triterpene and steroidal compounds isolated from the aerial parts of B. decumbens as free aglycones or as saponins.The latter compounds were isolated as epimeric pairs.

Plant material and extraction
Aerial parts from B. decumbens Stapf were collected in Teutônia, State of Rio Grande do Sul, Brazil, in January 2000.A herbarium specimen is on deposit in the Herbarium of the Botany Department of the Federal University of Rio Grande do Sul (Herbarium ICN, Porto Alegre, Brazil, voucher nº 121456).Air-dried powdered plant material (500 g) was extracted during 7 days at room temperature with ethanol.The solvent was evaporated and the residual alcoholic phase was suspended in water.This suspension was partitioned with dichloromethane and the main dichloromethane compounds were isolated using column chromatography.

Results
Solvent partition and chromatographic procedures allowed the isolation of the main compounds 1-4 (Figure 1) from the aerial parts of B. decumbens.
Acid hydrolysis of 3 yielded only one sugar identified as glucose (glc), and acid hydrolysis of 4 yielded two sugars identified as rhamnose (rha) and glucose (glc).In both cases, the sugars were identified by co-TLC using authentic samples.
The 13 C NMR spectrum of compound 1 displayed 31 signals, whereas the DEPT spectrum revealed 9 methyl, 10 methylene, 7 methine and 5 quartenary carbon atoms.The presence of an insaturation can be observed in the 13   The IR spectrum showed an hydroxy group (3445 cm -1 ).The 13 21 The 13 C NMR spectrum displayed 57 signals, determined from the DEPT spectrum as 8 methyl, 15 methylene, 28 methine and 5 quaternary carbon atoms.Careful comparison of 13 C NMR spectral data of 4 with that of 2 showed that 4 has same aglycone moieties (an epimeric mixture) and that 4 differs structurally from 2 only by the presence of sugar signals.This assumption was confirmed by HMBC, HMQC and 1 H, 1 H-COSY experiments.Comparing the chemical shifts of sugars with the literature data 7,17,22 it was possible to identify them as two rhamnoses and one glucose.Using the HMBC, HMQC and 1 H, 1 H-COSY spectra it was possible to identify the interglycosidic linkage.Through all these data we could identify 4 as dioscin (4a) and 3-O-{a-L-rhamnopyranosyl-(1®4)-[a-L-rhamnopyranosyl-(1®2)]-b-D-glucopyranosyl}-25(S)-spirost-5-en-3b-ol (4b).The latter compound was not described in the literature, as far as we know.

Discussion
Over the past years photosensitization in ruminants grazing B. decumbens had been associated with sporidesmin from Pithomyces chartarum.However, hepatogenous photosensitization was detected without the presence of P. chartarum, [1][2][3][4] proving the association of the disease with the ingestion of plant saponins as, demonstrated by Miles et al. 8 Such compounds are metabolized by microbial flora in ruminants to b-D-glucuronide insoluble salts of episarsasapogenin and epismilagenin.
C NMR spectrum by the signals at d c 149.0 (Cq) and d c 115.2 (CH) and, by one-proton absorbance at d H 5.25 (H-11) in the 1 H NMR spectrum.These data suggest a D 9(11) -lanostene skeleton.The 1 H NMR spectrum of 1 showed the presence of a singlet at d H 3.40 (3H) correlated with the carbon at d c 57.9 (OCH 3 ), demonstrating the presence of a methoxyl group.Long range correlation peaks were detected between the methoxyl signal and C-3 (d c 89.0).The others signals of 1 were established by HMBC, HMQC and 1 H, 1 H COSY experiments.Through detailed comparison of these data with those from literature, 12-14 1 was identified as 3bmethoxy-lanost-9(11)-ene.Compound 2 presented the same Rf value of an authentic sample of diosgenin on silica gel plates (TLC).