Xanthane sesquiterpenoids from the roots and flowers of Xanthium cavanillesii

Abstract The sesquiterpene lactones xanthanodiene, 4-epi-xanthanol, 4-epi-isoxanthanol, and 4-epi-xanthinin, as well as the xanthanolide derivative 4-oxo-bedfordia acid were isolated from the chloroform extracts of roots and flowers of Xanthium cavanillesii Schouw. The identities of these compounds were corroborated through comparison of their spectroscopic data, including IR, MS, and 1H and 13C NMR assignments, with literature reports. In addition, the structural characterization of 4-oxo-bedfordia acid was revisited and a comprehensive spectroscopic study of the compound is presented. This is to our knowledge the first phytochemical investigation of the roots of X. cavanillesii, and of flowers in the whole Xanthium genus.


Introduction
The genus Xanthium L. of the Asteraceae Dum. family (tribe Heliantheae) comprises annual coarse herbs and shrubs that bear small heads of greenish flowers followed by characteristic burrs with hooked bristles. Many of the species in the genus, including Xanthium spinosum and Xanthium strumarium, are popularly considered as medicinal plants and used for the treatment of a variety of ailments (Kamboj & Saluja 2010). extracts and pure compounds isolated from species of the genus have shown antiulcerogenic, antileishmanial, antifungal, antiinflammatory, and antinociceptive activity (Favier et al. 2005;Kim et al. 2005;Lavault et al. 2005;Han et al. 2007), as well as vasorelaxant properties due to inhibitory activity on NO, PGe 2 , and NF-κB activation (Bader et al. 2013). In Uruguay, where it grows wild, the infusion of Xanthium cavanillesii Schouw, a species native of South America commonly known as 'Abrojo' or 'Abrojo grande, ' is popularly used as antiseptic (Lombardo 1983).
The genus has been the subject of numerous phytochemical investigations (Bohlmann & Zdero 1981;Omar et al. 1984;Ahmed et al. 1990;de Riscala et al. 1994), being sesquiterpene lactones with guaiane or secoguaiane frameworks the main secondary metabolites (Fraga 2011;Vasas & Hohmann 2011). However, most of these studies were carried out on the species recognized as medicinal plants, and almost completely carried out on leaf extracts. There are very few studies on the root extracts of the genus (Maitra et al. 2006;Sridharamurthy & Yogananda 2010;Kan et al. 2011;Chen et al. 2014). The only ones which refer to sesquiterpene lactones are those by Tsankova and Bader, who isolated xhantodiene and zinolide from Xanthium italicum roots, respectively (Tsankova et al. 1993;Bader et al. 2013). Furthermore, and to the best of our knowledge, no studies on flowers of the genus have been performed. Continuing with our work on Xanthium cavanillesii (Cerdeiras et al. 2007;Olivaro & Vazquez 2009;Olivaro et al. 2014), we herein present the first report of xanthane sesquiterpenoids in the roots of this species, as well as in flowers of the whole Xanthium genus.

Results and discussion
The fractionation of the chloroform extract of X. cavanillesii roots through repeated chromatographic separation steps yielded a single product. Comparison of its spectroscopic data with literature reports allowed us to conclusively identify it as xanthanodiene (1, Figure 1), a sesquiterpene lactone with an eremophilane backbone first described by Bohlmann and coworkers (1982). When applied to the flowers of the shrub, the same process afforded the previously reported xanthanolides 4-epi-isoxanthanol (2), 4-epi-xanthanol (3), and 4-epixanthinine (4) (Bohlmann & Jakupovic 1981;Marco et al. 1993;Tsankova et al. 1993), all shown in Figure 1.
Following a number of column chromatography and preparative TLC purification steps, the chloroform extract of X. cavanillesii flowers afforded an additional compound that developed differently than xanthanolides 2-4 when treated with p-hydroxylbenzaldehyde. The HR-eSIMS of this slightly yellow waxy solid showed a quasimolecular ion The 1 H NMR spectrum of this compound presented two geminal olefinic protons at 6.28 and 5.64 ppm, which were both coupled to a proton at 2.50 ppm and showed HMBC correlations to quaternary alkene and carboxylate carbons at 146.1 and 171.7 ppm, respectively. These signals correspond to 1 H and 13 C nuclei from a fragment bearing a carboxylic acid with an exocyclic α,β-unsaturation that was previously inferred from IR data. An additional olefinic 1 H signal at 5.42 ppm showed TOCSY correlations to protons on 6 carbons that are part of a seven-membered ring, including the base of the aforementioned α,β-unsaturated carboxylate sidechain and a methyl group at 1.11 ppm. The ketone carbonyl resonance at 208.9 is part a 2-butanoyl moiety attached to the remaining olefinic carbon of the ring, and presents 1 H signals for two methylenes and one methyl group at 2.25, 2.53, and 2.16 ppm, respectively, and their corresponding 13 C signals at 33.9, 42.7, and 30.0 ppm. Together with detailed analysis of 3 J HH data (Table S1), this information allowed us identify the compound as 4-oxo-bedfordia acid (5, Figure 1), a xanthanolide derivative found in the Xanthium and Bedfordia genera. It was reported originally by Bohlmann and Le Van (1978), who, likely due to the instrumental limitations of the time, presented only limited 1 H data for the molecule. Despite some work on 4-oxo-bedfordia acid was done since then (Bohlmann et al. 1982), particularly regarding its biological activity as antifungal, antileishmanial, and antiulcerogenic (Favier et al. 2005;Lavault et al. 2005), none of these studies revisited its structural characterization. The work presented here remedies these shortcomings, which included 8 unassigned proton signals and no 13 C data. Our studies also help corroborate the relative configuration of the two chiral centers on the 7-membered ring. Indeed, strong NOe correlations observed between protons H7 and H10 in the 1D-NOeSY spectra of compound 5 are consistent with the proposed cis arrangement of the substituents on positions 7 and 10.

Conclusions
In summary, we described the isolation of five xanthane sesquiterpenoids form the roots and flowers of X. cavanillesii Schouw, including xanthanodiene, 4-epi-xanthanol, 4-epi-isoxanthanol, and 4-epi-xanthinin, and 4-oxo-bedfordia acid. We also revisited the structural characterization of the latter xanthanolide derivative, presenting a comprehensive spectroscopic study of the compound and confirmation of its relative stereochemistry. As stated earlier, this work constitutes the first phytochemical study of the roots of X. cavanillesii and of flowers in the whole Xanthium genus.

Supplementary material
Supplementary material relating to this article is available online together with Table S1.