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Journal of the Brazilian Chemical Society - Saponins and Sapogenins from Brachiaria decumbens Stapf

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Journal of the Brazilian Chemical Society

Print version ISSN 0103-5053

J. Braz. Chem. Soc. vol.13 no.2 São Paulo  2002

http://dx.doi.org/10.1590/S0103-50532002000200002 

Article

 

Saponins and Sapogenins from Brachiaria decumbens Stapf

 

Viviane S. Piresa, Alexandre T. C. Taketab, Grace Gosmanna and Eloir P. Schenkel*a

aFaculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga, 2752, 90610-000 Porto Alegre, RS - Brazil

bKekulé-Institut für Organische Chemie und Biochemie der Rheinischen Friedrich-Wilhelms-Universität, Bonn, Germany

 

 

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 1H e 13C, HMBC, HMQC) como 3b-metóxi-lanost-9(11)-eno (1), diosgenina (2a), iamogenina (2b), 3-O-b-D-glicopiranosil-24(S)-etil-22E -deidrocolesterol (3a), 3-O-b-D-glicopiranosil-24(R)-etil-22E -deidrocolesterol (3b), dioscina (4a) e 3-O-{a-L-ramnopiranosil-(1®4)-[a-L-ramnopiranosil-(1 ®2)]-b-D-glicopiranosil}-25(S)-espirost-5-en-3 b-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.

Four steroidal saponins and three sapogenins were identified from aerial parts of Brachiaria decumbens. Their structures were established by chemical and spectroscopic means (1H and 13C NMR, HMBC, HMQC) as 3b-methoxy-lanost-9(11)-ene (1), diosgenin (2a), yamogenin (2b), 3-O-b-D-glucopyranosyl-24(S)-ethyl-22E -dihydrocholesterol (3a), 3-O-b-D-glucopyranosyl-24(R)-ethyl-22E -dihydrocholesterol (3b), dioscin (4a) and 3-O-{a-L-rhamnopyranosyl-(1®4)-[a-L-rhamnopyranosyl-(1 ®2)]-b-D-glucopyranosyl}-25(S)-spirost-5-en-3 b-ol (4b). All these compounds were isolated for the first time from B. decumbens and compound 4b is described for the first time as far as we know.

Keywords: Brachiaria decumbens, steroidal saponins, dioscin, diosgenin, yamogenin, triterpene.

 

 

Introduction

Brachiaria decumbens Stapf belongs to a group of plants capable of inducing hepatogenous photosensitizations1-4 similar to those described for Panicum spp.,5-8 Tribulus terrestris,8,9 Agave lecheguilla10 and Narthecium ossifragum8. 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.

 

Experimental

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.

Isolation

Part of the dichloromethane extract (5 g) was chromatographed on a silica gel column and eluted with dichloromethane:ethanol (99:1; 90:10; 80:20 and 70:30, v/v). Fractions were pooled according to thin-layer chromatography (TLC) analysis. Fractions 2-10 (250 mg) containing compound 1 as main component, were further purified by silica gel column chromatography, eluting with hexane and resulting in pure compound 1 (12.5 mg). Fractions 30-40 (500 mg), 50-60 (350 mg) and 68-81 (500 mg) containing compounds 2, 3 and 4 as main components were submitted to successive silica gel column chromatography, eluting with hexane:ethyl acetate (3:1, v/v), dichlorometane:ethanol (9:1, v/v) and dichlorometane: ethanol (7:3, v/v), respectively. By these procedures compounds 2 (33 mg), 3 (15 mg) and 4 (40 mg) were obtained.

General procedures

Dichloromethane extract and fractions were chromatographed on a silica gel column (Merck, particle size: 230-400 mesh) at atmospheric pressure. TLC was carried out on silica gel (Merck, GF254) using hexane:ethyl acetate (3:1, v/v) and dichlorometane:ethanol (7:3, v/v). Compounds were visualized by heating (120 °C) using the anisaldehyde-sulfuric acid-spray. FAB-MS spectrum was performed on a MS50 spectrometer. 1H NMR and 13C NMR spectra were recorded on Bruker AMX 500 spectrometer.

3b-methoxy-lanost-9(11)-ene (1). 1H NMR (500 MHz, CDCl3): d 1.83 (H-1), 1.91, 1.75 (H-2), 2.70 (H-3), 0.89 (H-5), 1.67, 1.72 (H-6), 1.37 (H-7), 2.19 (H-8), 5.25 (H-11), 1.93, 2.09 (H-12), 1.28 (H-15), 1.65, 1.71 (H-16), 1.62 (H-17), 0.67 (H-18), 0.91 (H-19), 1.41 (H-20), 0.77 (H-21), 1.38 (H-22), 1.38 (H-23), 1.16 (H-24), 1.54 (H-25), 0.89 (H-26), 0.90 (H-27), 1.06 (H-28), 1.00 (H-29), 0.83 (H-30), 3.40 (OCH3).13C NMR (125 MHz, CDCl3) d 36.4 (C-1), 22.9 (C-2), 89.0 (C-3), 39.4 (C-4), 53.4 (C-5), 21.6 (C-6), 34.3 (C-7), 42.2 (C-8), 149.0 (C-9), 37.5 (C-10), 115.2 (C-11), 37.5 (C-12), 44.5 (C-13), 47.4 (C-14), 30.1(C-15), 28.5 (C-16), 51.4 (C-17), 14.8 (C-18), 18.8 (C-19), 36.6 (C-20), 18.9 (C-21), 36.9 (C-22), 24.5 (C-23), 39.9 (C-24), 28.4 (C-25), 22.9 (C-26), 23.2 (C-27), 22.6 (C-28), 28.6 (C-29), 16.8 (C-30), 57.9 (OCH3).

Compound 2a. diosgenin. 1H NMR and 13C NMR data (CDCl3): same as in references 15 and 17.

Compound 2b. yamogenin. 1H NMR and 13C NMR data (CDCl3): same as in references 15 and 17.

3-O-b-D-glucopyranosyl-24(S)-ethyl-22E-dihydro cholesterol (3a). 1H NMR data (500 MHz, C5D5N): d 1.71, 0.98 (H-1), 2.12, 1.74 (H-2), 3.94 (H-3), 2.72, 2.46 (H-4), 5.34 (H-6), 1.88, 1.54 (H-7), 1.36 (H-8), 0.89 (H-9), 1.45, 1.45 (H-11), 1.97, 1.09 (H-12), 0.92 (H-14), 1.02, 1.53 (H-15), 1.26, 1.84 (H-16), 1.08 (H-17), 0.88 (H-18), 0.95 (H-19), 2.03 (H-20), 0.90 (H-21), 5.20 (H-22), 5.06 (H-23), 1.56 (H-24), 1.54 (H-25), 1.07 (H-26), 0.85 (H-27), 0.95 (H-28), 0.87 (H-29), 5.05 (glc H-1), 4.05 (glc H-2), 4.28 (glc H-3), 4.28 (glc H-4), 3.97 (glc H-5), 4.41, 4.56 (glc H-6); 13C NMR (125 MHz, C5D5N): d 37.6 (C-1), 30.4 (C-2), 78.2 (C-3), 39.5 (C-4), 141.1 (C-5), 122.1 (C-6), 32.3 (C-7), 32.2 (C-8), 50.5 (C-9), 37.1 (C-10), 21.5 (C-11), 40.0 (C-12), 42.5 (C-13), 57.0 (C-14), 24.7 (C-15), 28.7 (C-16), 56.2 (C-17), 12.3 (C-18), 19.2 (C-19), 41.0 (C-20), 19.6 (C-21), 139.0 (C-22), 129.6 (C-23), 51.6 (C-24), 32.4 (C-25), 21.7 (C-26), 19.4 (C-27), 25.9 (C-28), 12.7 (C-29), 102.7 (glc C-1), 75.5 (glc C-2), 78.8 (glc C-3), 71.8 (glc C-4), 78.7 (glc C-5), 63.0 (glc C-6).

3-O-b-D-glucopyranosyl-24(R)-ethyl-22E-dihydro cholesterol (3b). 1H NMR (500 MHz, C5D5N): d 1.71, 0.98 (H-1), 2.12, 1.74 (H-2), 3.94 (H-3), 2.72, 2.46 (H-4), 5.34 (H-6), 1.88, 1.54 (H-7), 1.36 (H-8), 0.89 (H-9), 1.45, 1.45 (H-11), 1.97, 1.09 (H-12), 0.92 (H-14), 1.02, 1.53 (H-15), 1.26, 1.84 (H-16), 1.08 (H-17), 0.88 (H-18), 0.95 (H-19), 2.03 (H-20), 0.87 (H-21), 5.20 (H-22), 5.06 (H-23), 1.56 (H-24), 1.54 (H-25), 0.85 (H-26), 0.92 (H-27), 1.28 (H-28), 0.66 (H-29), 5.05 (glc H-1), 4.05 (glc H-2), 4.28 (glc H-3), 4.28 (glc H-4), 3.97 (glc H-5), 4.41, 4.56 (glc H-6).13C NMR (125 MHz, C5D5N): d 37.6 (C-1), 30.4 (C-2), 78.2 (C-3), 39.5 (C-4), 141.1 (C-5), 122.1 (C-6), 32.3 (C-7), 32.2 (C-8), 50.5 (C-9), 37.1 (C-10), 21.5 (C-11), 40.1 (C-12), 42.6 (C-13), 57.1 (C-14), 24.7 (C-15), 28.7 (C-16), 56.4 (C-17), 12.3 (C-18), 19.2 (C-19), 41.0 (C-20), 20.2 (C-21), 139.0 (C-22), 129.6 (C-23), 51.6 (C-24), 32.4 (C-25), 19.4 (C-26), 21.5 (C-27), 23.6 (C-28), 12.2 (C-29), 102.7 (glc C-1), 75.5 (glc C-2), 78.8 (glc C-3), 71.8 (glc C-4), 78.7 (glc C-5), 63.0 (glc C-6).

Compound 4. FAB-MS (positive mode) m/z 907.6 [M+K]+, 891.6 [M+Na]+, 869.5 [M+H]+, 723.3 [(M+H)-146]+, 415.3 [(M+H)-146-146-162]+, 413.3 [aglycone-H]+, 395.3 [aglycone-H3O]+ .

Compound 4a. 1H NMR and 13C NMR data (C5D5N): same as in references 17 and 22.

3-O-{a-L-rhamnopyranosyl-(1® 4)-[a-L-rhamnopyranosyl-(1®2)]- b-D-glucopyranosyl}-25(S)-spirost-5-en-3b -ol (4b). 1H NMR (500 MHz, C5D5N): d 1.72, 0.97 (H-1), 2.06, 1.84 (H-2), 3.86 (H-3), 2.75, 2.75 (H-4), 5.30 (H-6), 1.84, 1.45 (H-7), 1.55 (H-8), 0.89 (H-9), 1.42, 1.42 (H-11), 1.66, 1.07 (H-12), 1.03 (H-14), 2.01, 2.01 (H-15), 4.48 (H-16), 1.77 (H-17), 1.06 (H-18), 1.05 (H-19), 1.87 (H-20), 1.13 (H-21), 1.44, 1.87 (H-23), 2.13, 1.34 (H-24), 1.80 (H-25), 4.03, 3.34 (H-26), 1.12 (H-27), 4.95 (glc H-1), 4.22 (glc H-2), 4.18 (glc H-3), 4.39 (glc H-4), 3.62 (glc H-5), 4.08, 4.21 (glc H-6), 6.39 (rha H-1), 4.84 (rha H-2), 4.67 (rha H-3), 4.37 (rha H-4), 4.95 (rha H-5), 1.75 (rha H-6), 5.85 (rhaI H-1), 4.62 (rhaI H-2), 4.53 (rhaI H-3), 4.33 (rhaI H-4), 4.93 (rhaI H-5), 1.62 (rhaI H-6).

13C NMR (125 MHz, C5D5N): d 37.6 (C-1), 30.3 (C-2), 78.1 (C-3), 39.1 (C-4), 140.9 (C-5), 121.9 (C-6), 32.4 (C-7), 31.8 (C-8), 50.4 (C-9), 37.2 (C-10), 21.2 (C-11), 39.9 (C-12), 40.5 (C-13), 56.7 (C-14), 32.3 (C-15), 81.2 (C-16), 63.0 (C-17), 16.4 (C-18), 19.5 (C-19), 42.5 (C-20), 15.0 (C-21), 109.4 (C-22), 26.5 (C-23), 26.3 (C-24), 27.6 (C-25), 65.2 (C-26), 15.2 (C-27), 100.4 (glc C-1), 77.9 (glc C-2), 78.2 (glc C-3), 78.6 (glc C-4), 77.0 (glc C-5), 61.3 (glc C-6), 102.1 (rha C-1), 72.6 (rha C-2), 72.7 (rha C-3), 74.2 (rha C-4), 69.6 (rha C-5), 18.8 (rha C-6), 103.0 (rhaI C-1), 72.9 (rhaI C-2), 72.8 (rhaI C-3), 74.0 (rhaI C-4), 70.5 (rhaI C-5), 18.6 (rhaI C-6).

 

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 13C 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 13C NMR spectrum by the signals at dc 149.0 (Cq) and dc 115.2 (CH) and, by one-proton absorbance at dH 5.25 (H-11) in the 1H NMR spectrum. These data suggest a D9(11)-lanostene skeleton. The 1H NMR spectrum of 1 showed the presence of a singlet at dH 3.40 (3H) correlated with the carbon at dc 57.9 (OCH3), demonstrating the presence of a methoxyl group. Long range correlation peaks were detected between the methoxyl signal and C-3 (dc 89.0). The others signals of 1 were established by HMBC, HMQC and 1H,1H COSY experiments. Through detailed comparison of these data with those from literature,12-14 1 was identified as 3b-methoxy-lanost-9(11)-ene.

Compound 2 presented the same Rf value of an authentic sample of diosgenin on silica gel plates (TLC). The IR spectrum showed an hydroxy group (3445 cm-1). The 13C NMR spectrum showed some duplicated signals indicating the presence of an epimeric mixture. All the signals of 2 and their connectivity were established by HMBC, HMQC and 1H,1H COSY experiments. It was possible to demonstrate a spiroketal ring system through the presence of carbon quaternary signals at dc 109.7 (C-22, 2a) and 110.1 (C-22, 2b), -OCH2 group at dc 67.2 (C-26, 2a) and 65.5 (C-26, 2b), and OCH group at dc 81.2 (C-16, 2a) and 81.3 (C-16, 2b). The epimeric mixture was also characterized through the 1H NMR spectrum by the different multiplicity of the H-26 methylene protons caused by the methyl group at C-25. In this way, compound 2a showed signals at dH 3.40 (t, J 11.0, H-26a) and at dH 3.49 (ddd, J 10.9, 4.1, 2.1 Hz, H-26b), and compound 2b presented signals at dH 3.98 (dd, J 10.7, 2.5 Hz, H-26a) and dH 3.32 (d, J 10.7 Hz, H-26b), demonstrating the equatorial and axial protons. Regarding the 13C NMR spectrum, the configuration 25R or 25S can be differentiated by the chemical shifts for C-23, C-24, C-25, C-26 and C-27. Careful comparison of 1H NMR and 13C NMR spectral data of 2 with those of the literature15-18 allowed to establish 2 as the 25-epimeric mixture of diosgenin [(25R)-spirost-5-en-3b-ol] 2a and yamogenin [(25S)-spirost-5-en-3b-ol] 2b.

The 13C NMR of compound 3 displayed 46 signals including, as determined from the DEPT spectrum, 13 methyl, 11 methylene, 18 methine and 4 quaternary carbon atoms, indicating an epimeric mixture through the presence of some duplicated signals. All signals of 3 and their connectivity were established by HMBC, HMQC and 1H,1H COSY experiments. The NMR data showed the presence of one sugar identified as glucose (dH 5.05, d, J 7.6, dc 102.7) which signals have correlation to H-3. Two insaturations at dc 141.1 (Cq, C-5) and 122.1 (CH, C-6), and dc 139.0 (CH, C-22) and 129.6 (CH, C-23) together with the corresponding olefinic protons at dH 5.34 (H-6), 5.20 (H-22) and 5.06 (H-23) are characteristic for D5,22-3b-hydroxysterols. Comparing to the literature data19,20 it was possible to verify that 3 is 24a- and 24b-stigmastane mixture that can be differentiated by the chemical shifts for C-26 and C-27, which vary about d 2 ppm. Therefore, it was concluded that 3 is the epimeric mixture of 3-O-b-D-glucopyranosyl-24(S)-ethyl-22E -dihydrocholesterol (3a) and 3-O-b-D-glucopyranosyl-24(R)-ethyl-22E -dihydrocholesterol (3b, stigmasterol glucoside).

FAB-MS (positive mode) of compound 4 exhibited ions at m/z 869.5 [M+H]+, 891.6 [M+Na]+ and 907.6 [M+K]+ indicating the molecular mass C45H72O16. Fragments ions at m/z 723.3 and m/z 415.3 indicated the elimination of one methylpentose, and two methylpentoses and one hexose, respectively.21 The 13C 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 13C 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 1H,1H-COSY experiments. Comparing the chemical shifts of sugars with the literature data7,17,22 it was possible to identify them as two rhamnoses and one glucose. Using the HMBC, HMQC and 1H,1H-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,1-4 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.

In the case of B. decumbens, the presence of saponins was detected and the aglicone was characterized by GC-MS after acid hydrolysis from butanolic extract,2 but the chemical structures of these saponins were not elucidated. In the present investigation it was possible to isolate and identify the following compounds: 3b-methoxy-lanost-9(11)-ene (1), diosgenin (2a), yamogenin (2b), 3-O-b-D-glucopyranosyl-24(S)-ethyl-22E -dihydrocholesterol (3a), 3-O-b-D-glucopyranosyl-24(R)-ethyl-22E -dihydrocholesterol (3b), 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). This last compound is the dioscin 25-epimer and, to the best of our knowledge, has not been previously described. All these compounds were isolated for the first time from Brachiaria decumbens. Diosgenin and yamogenin were already isolated from the acid hydrolysis of B. decumbens butanolic extract,2 but were not isolated in the free form from this plant. These results together with our previous work11,23 demonstrated that B. decumbens, as Panicum spp., T. terrestris, A. lecheguilla and N. ossifragum, is capable of producing photosensitization in animals due the presence of steroidal saponins and sapogenins.

 

Acknowledgements

We are grateful to Prof. Eberhard Breitmaier from Kekulé-Institut für Organische Chemie der Universität Bonn, Germany for NMR and FAB-MS measurements, to Cláudio Cruz from Department of Veterinary Pathology of the Universidade Federal do Rio Grande do Sul, Brazil for collecting, and Marcos Sobral from the Universidade Federal do Rio Grande do Sul for identifying the plant material. This work was supported by fellowships to V.S. Pires from CAPES (Brazil), and to G. Gosmann and E. P. Schenkel from CNPq (Brazil).

 

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Received: July 25, 2001
Published on the web: December 19, 2001

 

* e-mail: schenkel@ccs.ufsc.br