Taraxastane-type triterpene saponins isolated from Pittosporum angustifolium Lodd.

1 Introduction

Pittosporum angustifolium Lodd. (Pittosporaceae) is a small-sized tree that is distributed regularly but never frequently in Australia’s inland areas. Some of the plants known trivial names used colloquially are “cumby cumby”, “weeping pittosporum” or “butterbush” [1]. In addition to a reported economic significance, a variety of medical and therapeutic agents, prepared from different parts of the plant are described in the Aboriginal ethnomedicine [1]. Furthermore, a complementary usage of preparations as a supplement for the treatment of malignant diseases has been described in recent years [2].

Previously, we have phytochemically investigated the leaves and the seeds of P. angustifolium, resulting in the isolation and characterization of a number of secondary metabolites, such as known polyphenols [3], as well as novel and known structures of the class of triterpene saponins [2, 4, 5]. We found that acyl residues, especially attached to C-21 and C-22 of the aglycone moiety, are essential for a cytotoxic activity [2, 4]. In addition, from a few Pittosporum species, structurally very similar composed triterpene saponins, possessing A₁- and R₁-barrigenol as well as oleanolic acid backbones, have been isolated [6–10], while other types of aglycone structures of genuine saponins, e.g., the unusual 17,22-seco-skeleton found in two saponins from P. angustifolium [2], have not been described from another Pittosporum species. In consequence of a phylogenetic study in 2000, P. angustifolium was reinstated as a separate species that was previously considered as P. phillyr(a)eoides or as variety P. phillyre(a)oides var. microcarpa [1]. Under the taxonomic classification P. phillyraeoides taraxastane-type sapogenins featuring a δ-lactone substructure (phillyrigenin and derivatives) have been obtained after acidic hydrolysis [11, 12]. During our ongoing studies of the phytochemical composition of P. angustifolium two new taraxastane-type triterpene saponins were identified. The structure elucidation and the results of an investigation for cytotoxicity, as well as a supposed chemical formation mechanism leading to phillyrigenin derivatives that were described in former studies [11, 12], are presented in this work.

2 Results and discussion

A part of the crude extract (80 % EtOH) of the leaves was purified and fractionated by successive chromatographic techniques, using Sephadex LH20, silica gel and RP18 solid phase extraction. From the obtained subfraction A_{5_100 %} [5] compounds 1 and 2 (Fig. 1) were isolated by semipreparative HPLC and named pittangretosides L (1) and C₁ (2).

Fig. 1:

Taraxastane-type triterpene saponins from the leaves of Pittosporum angustifolium.

The high-resolution ESI mass spectrum of pittangretoside L (1) revealed a quasimolecular ion peak [M–H]⁻ at m/z = 955.4884, indicating a molecular formula of C₄₈H₇₆O₁₉. The ¹H NMR spectrum exhibited signals for four singlet methyl groups (δ_H = 0.89, 0.92, 0.99, 1.12 ppm), one olefinic methyl group (δ_H = 1.64 ppm), one doublet methyl group (δ_H = 1.02 ppm, d, J = 6.7 Hz), one oxygenated methyl group, resp. hydroxymethylene group, at δ_H = 3.78 (d, J = 12.0 Hz) and 4.22 (d, J = 12.0 Hz) ppm, as well as an olefinic proton resonance at δ_H = 5.28 (d, J = 7.4 Hz) ppm, suggesting a pentacyclic triterpene skeleton. Furthermore, in the downfield region, three signals of anomeric proton resonances at 4.47 (d, J = 7.6 Hz), 4.81 (d, J = 7.6 Hz), and 5.18 (br s) ppm were assigned, implying the presence of three sugar units. A detailed look at the two-dimensional NMR spectroscopic data (H-H COSY, HMQC, and HMBC) confirmed one β-glucuronopyranose, one β-galactopyranose, and one α-rhamnopyranose unit, while their absolute configuration was deduced from the corresponding thiazolidine carboxylates by GC-MS experiments to be d, d, and l, respectively. As in the HMQC spectrum cross-peaks were observed between δ_H = 4.47 ppm (H-1, GlcA) and δ_C = 91.1 ppm (C-3), δ_H = 4.81 ppm (H-1, Gal) and δ_C = 78.3 ppm (C-2, GlcA), and δ_H = 5.18 ppm (H-1, Rha) and δ_C = 76.7 ppm (C-2, Gal) as well, the glycosidic linking and its attachment at C-3 of the aglycone moiety were elucidated unequivocally. The same sugar chain has already been described for saponins from P. angustifolium [2] and Stylosanthes erecta [13]. In comparison with literature [14–16], the NMR spectroscopic data of the aglycone part showed similarities to a taraxastane backbone. In contrast to those skeletons reported, a characteristic shift for a carboxylic acid function was assigned at δ_C = 178.8 ppm and its position turned out to be at C-28, while a hydroxymethylene group (δ_C = 60.3; δ_H = 3.78 ppm, d, J = 12.0 Hz; 4.22 ppm, d, J = 12.0 Hz) was assigned at C-27. Finally, the taraxastane configuration was evidenced by carefully evaluated ROESY correlations observed between CH₃-26 (δ_H = 0.99 ppm), H-13 (δ_H = 2.50 ppm) and H-19 (δ_H = 2.09 ppm), indicating these protons to be oriented in the same direction, while CH₂-27 (δ_H = 3.78 ppm), H-18 (δ_H = 1.35 ppm) and CH₃-29 (δ_H = 1.02 ppm) show correlations, corroborating they face into the other direction. The ROESY walk H-13–H-12β–H-12α–H-9–H-1α–H-2α–H-3–H-5–H-7α–CH₂-27 also confirmed the configuration of H-9, H-3, and H-5 to be as found for CH₂-27. Thus, the new natural product pittangretoside L (1) was elucidated as 3β-[α-l-rhamnopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)]-β-d-glucuronopyranosyloxy-27-hydroxy-20-taraxastene-28-oic acid.

As the chromatographic, ATR-IR- and NMR-spectroscopic, as well as mass spectrometric data of pittangretoside C₁ (2) were quite identical to those of compound 1, it was supposed to be a structurally closely related compound. With respect to this, the same molecular formula of C₄₈H₇₆O₁₉ was derived from its high-resolution ESI mass spectrum, displaying a quasimolecular ion peak [M–H]⁻ at m/z = 955.4898. Since the one and two-dimensional NMR spectra revealed three resonances of anomeric protons at 4.45 (d, J = 7.3 Hz), 4.81 (d, J = 7.6 Hz), 5.17 (br s) ppm and HMBC cross peaks were observed between δ_H = 4.45 ppm (H-1, GlcA) and δ_C = 92.4 ppm (C-3), δ_H = 4.81 ppm (H-1, Gal) and δ_C = 78.9 ppm (C-2, GlcA), δ_H = 5.17 ppm (H-1, Rha) and δ_C = 76.8 ppm (C-2, Gal) as well as the results of TLC and GC-MS (as corresponding thiazolidine carboxylates) analysis were identical to those of compound 1, it was confirmed that compound 2 possesses the same sugar chain as found in 1. Instead, aglycone resonances were partially different. In the ¹H NMR spectrum four singlet methyl signals were assigned at δ_H = 0.86, 0.91, 1.00, 1.09 ppm, and one methyl doublet at δ_H = 1.06 (d, J = 6.7 Hz) as well, while signals of an olefinic methyl singlet in the region of δ_H = 1.64 ppm (CH₃-30, compound 1), and an olefinic methine proton resonance at δ_H = 5.28 ppm (H-21, compound 1) were missing. Furthermore, the carbon atom resonance of C-20 (δ_C = 152.1 ppm) was deshielded compared to 1 (δ_C = 143.2 ppm), as well as two proton signals at δ_H = 2.21 and 2.50 ppm, corresponding to C-21 (δ_C = 27.2 ppm) in the HMQC spectrum, implying allylic proton resonances of a methylene group. Moreover, as two proton singlets appeared at δ_H = 4.58 and 4.60 ppm (CH₂-30), it was deduced that these resonances represent a terminal double bond, resp. an exo-methylene group [14]. Finally, since the same ROESY correlations were observed as found in compound 1, the aglycone configuration of a taraxastane skeleton was elucidated, as well. The new structure of pittangretoside C₁ (2) was thus determined as 3β-[α-l-rhamnopyranosyl-(1→2)-β-d-galactopyranosyl-(1→2)]-β-d-glucuronopyranosyloxy-27-hydroxy-20(30)-taraxastene-28-oic acid.

In recent publications we described pronounced cytotoxic activitiy of acylated triterpene saponins of the oleanane-type isolated from Pittosporum angustifolium [2, 4], as it was also shown for acylated saponins from other Pittosporum species [6, 9, 10]. Although compounds 1 and 2 do not possess an acylation and feature a structurally differing taraxastane backbone, they were evaluated for their cytotoxic potential against three tumourigenic and one non-tumourigenic cell line. Up to a concentration of 130 μm, neither compound 1, nor compound 2 showed cytotoxicity against the investigated cell lines.

Concerning a structural characterization of the aglycones of compounds 1 and 2, this is the first time that saponins with a taraxastane-type skeleton were isolated from a Pittosporum sp. However, some studies on P. phillyraeoides described taraxastane-type aglycones with the additional structural element of a δ-lactone (Fig. 2) obtained after acidic hydrolysis that were named phillyrigenins [11, 12]. Even though lactonic ring structures are known features of a few triterpenes [17], until now – to the best of our knowledge – the original existence of these phillyrigenin structures has never been confirmed again. Looking at the genuine compounds 1 and 2 obtained in this study (Fig. 1), it seems worth discussing, that the δ-lactone possessing phillyrigenin backbones could theoretically be products of 1 and/or 2 or similar, not yet identified structures, formed by the acidic conditions in aqueous solution. By an electrophilic addition a proton attachment at C-21 (1) or C-30 (2) could take place, followed by a hydroxylation at C-20 (1 and 2). In a subsequent step, reaction conditions could possibly lead to the lactone formation between that hydroxyl group (C-20) and the carboxyl group at C-28 (Fig. 2). Due to the small amount of substance and usage for biological screenings, this issue has not been proven experimentally by us, but it might be a likely formation mechanism of the phillyrigenin derivatives – at least as long as it has not been shown that the lactones are also present in the original plant material.

Fig. 2:

Suggested mechanism of phillyrigenin formation of compounds 1 and 2 by electrophilic addition under acidic aqueous conditions of hydrolysis (refs. [11, 12]).

3 Experimental

4 Supporting information

Figures of the ROESY spectra of compounds 1 and 2 are given as Supporting Information (DOI: 10.1515/znb-2015-0005).