Steroidal and Triterpenoidal Glucosides from Passiflora alata

Cinco glicosídeos foram isolados a partir das folhas de P. alata. Após extensivas análises espectroscópicas, as estruturas 1-5 foram identificadas como sendo o 3-Oβ-D-glicopiranosilestigmasterol ( 1), o ácido 3-Oβ-D-glicopiranosil-oleanólico ( 2), o ácido 3-Oβ-D-glicopiranosil(1→3)-β-D-glicopiranosil-oleanólico ( 3), o ácido 3-Oβ-Dglicopiranosil-(1→2)-β-D-glicopiranosiloleanólico (4) e 9,19-ciclolanost-24 Z-en-3β,21,26-tri-hidróxi-3,26-di-O-gentiobiose ( 5). Adicionalmente, foram analisados, através de CCD, extratos hidroetanólicos de espécies de Passiflora existentes no sul do Brasil ( P. actinia, P. caerulea, P. edulis var. flavicarpa, P. elegans , P. foetida, P. misera e P. tenuifila). A acumulação de saponinas foi verificada somente em Passiflora alata.


Introduction
Passion fruits that are nowadays grown throughout the tropics are native to Latin America and Brazil is the leading exporter of passion fruit juice 1 .Additionally to this alimentary function as flavor and as juice in food industries, passionflower extract has an ancient tradition in the folk medicine of American and even European countries due its reputed sedative and tranquilizing properties 2 .
Although Passiflora alata is an official drug of the Brazilian Pharmacopoeia 3 and its leaf extract is included as an active component in many Brazilian registered pharma-ceutical preparations 4 , there are only a few investigations on its chemical components 5 and pharmacological properties 6,7 .
In view of its pharmaceutical utilization, the knowledge of the chemical components of P. alata is important for the development of methods for the quality control of drug and phytopharmaceutical preparations.Moreover, since it has recently been shown that P. alata could induce respiratory allergy 8 , quality control methods could be extended to analyze pretended hypoallergenic drugs or beverages containing passionflower extracts.Also, the phytochemical study of P. alata is justified in view of searching for the still unknown therapeutically active or allergenic substances.
This paper describes the isolation and structure elucidation of one steroid glycoside (1) and four triterpene saponins (2-5) from the leaves of P. alata.The use of these compounds as natural markers is also discussed.

Chromatographic analysis
Analytical TLC aluminum sheets coated with Si gel GF 254 (Merck) were used.Air-dried powdered leaves of Passiflora species were extracted, separately, by maceration in EtOH (plant:solvent, 1:10, w/v) (2 x 10 days).After evaporation of the ethanolic extracts, the gummy residues were dissolved, separately, in MeOH for TLC comparison.Saponins were analyzed using CHCl 3 :EtOH:AcOH (60:40:6, v/v) as the mobile phase and the spots were visualized by heating (100 °C) the anisaldehyde-H 2 SO 4sprayed plates.

General
FAB-MS analysis were performed in positive mode on a Kratos MS 80 instrument.NMR spectra were recorded on a Bruker AM 400 spectrometer.Compounds 2, 3 and 4 were hydrolyzed as described by Kartnig and Wegschaider 10 in order to analyze the aglycone and sugar components.

Results and Discussion
The n-BuOH extract of P. alata was submitted to Si gel CC to yield compounds 1-5 and their identification was achieved through the combined use of mass and NMR spectroscopy.
The 1 H NMR spectrum of 1a displayed signals at δ 0.69 and 0.98 (s, 3 H each) for two tertiary methyl groups, two doublets (3 H each) at δ 0.79 (J 7.0 Hz) and 0.84 (J 6.5 Hz) assignable to an isopropyl group and the presence of another secondary methyl at δ 1.01 (d, J 6.5 Hz, 3 H).A triplet centered at δ 0.80 (J 7.0 Hz, 3 H) was attributed to a primary methyl.These characteristic signals suggested a steroid skeleton.Three olefinic proton signals at δ 5.35 (br, d, J 5.1 Hz), δ 5.15 (m) and at δ 5.05 (m) were attributed to H-6, H-22 and H-23, respectively, and together with the signal at δ 3.49 (m, 1 H, Hα-3) they are characteristic for ∆ 5,22 -3βhydroxysterols 12,13,14 .Comparison of 13 C NMR spectrum (Table 1) and the 2D-HSQC spectrum with spectral data of related sterols found in the literature indicated that compound 1 is the sterol glycoside 3-O-β-D-glucopyranosylstigmasterol isolated as a peracetylated derivative.Its occurrence in nature has only been reported several times although the aglycone is widely found.
Acid hydrolysis of 2, 3 and 4 furnished oleanolic acid and glucose (Glc).Both were identified through co-TLC with authentic samples.
The FAB-MS (positive ion mode) spectrum of 2 indicated the molecular mass 618 due to the quasimolecular ion peak at m/z 641 (M+Na) + .In addition, there were two characteristic peaks at m/z 203 and 248 denoting the retro-Diels-Alder fragmentation commonly found in the spectra of oleanane or ursane derivatives 15 .Careful comparison of the 13 C NMR data of peracetylated compound 2a (Table 1) with those of peracetylated dumosasaponin 6 9 , allowed the unambiguous assignment of the signals of the oleanolic acid aglycone.The presence of one β-D-Glc was evidenced through the anomeric signal at δ c 102.9 (δ H 4.55, d; J 8.0 Hz, 1 H) and the sugar substitution at C-3 of the aglycone was established (δ C-3 90.5).These conclusions were confirmed by 1 H-1 H COSY, 1 H- 13 C COSY and ROESY experiments.Therefore, compound 2 was identified as 3-O-β-Dglucopyranosyl-oleanolic acid, already isolated from Chenopodium quinoa 16 .
For compound 3 the molecular formula C 42 H 68 O 13 was deduced based on the FAB-MS spectrum, which displayed a pseudo-molecular ion peak at m/z 803 (M+Na) + and it also showed fragments at m/z 203 and m/z 248.Detailed comparison of 13 C NMR data of 3a and 2a showed that 3a structurally differs from 2a only by the presence of signals for another hexose.Thus, as in the case of 2a, compound 3a presented a free carboxyl group at position C-28 while a Glc,Glc-constituted disaccharide was substituted at C-3.
The great similarity of the 13 C NMR spectrum of 4 with that of 3a (Table 1) showed that 4 was also a derivative of oleanolic acid with two Glc residues substituted at carbon 3. The FAB-MS spectrum of 4 exhibited a peak at m/z 803 (M+Na) + and was consistent with the presence of these two Glc residues.The interglycosidic linkage of this disaccharide was deduced to be Glc(1→2)Glc from the deshielding in the 13 C NMR spectrum of 4 of one of the CH units of this moiety (δ 83.4).Thus, 4 was determined to be 3-O-β-D-gluco-pyranosyl-(1→2)-β-D-glucopyranosyl-oleanolic acid, already isolated from Passiflora quadrangularis 17 and Luffa acutangula 18 .
This study allowed us to identify five glycosides from the leaves of P. alata.Although none of them are specific to P. alata, their association in a single plant is unique, so we wonder if we could use them as a phytochemical tracer to either authentify the exclusive use of P. alata in pharmaceutical preparations, or to certify the lack of P. alata in therapeutic preparations elaborated from other passionflower species.Furthermore, the TLC profiles of the hydroethanol extracts from the leaves of Passiflora species found in the State of Rio Grande do Sul (south of Brazil), P. actinia, P. alata, P. caerulea, P. edulis var.flavicarpa, P. elegans, P. foetida, P. misera and P. tenuifila showed accumulation of saponins only in P. alata.As far as we know, saponins have only been reported for P. quadrangularis L. 17,19 and P. edulis Sims 20 .
Since secondary metabolite content can vary as a function of multiple factors (such as environmental conditions and harvest period), reproduction of this analysis over a long period of time is of course needed before the effectiveness of our method is totally demonstrated.We are currently pursuing this goal.
1997.A herbarium specimen (ICN 8344) is on deposit in the Herbarium of the Botany Department of the Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.Aerial parts of native P. actinia Hooker, P. caerulea L., P. edulis Sims var.flavicarpa, P. elegans Masters, P. foetida L., P. misera H.B.K. and P. tenuifila Killip were collected in different cities of the State of Rio Grande do Sul.