View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by CONICET Digital Article Type: Full Paper Phytochemical Study of the Genus Salpichroa (Solanaceae). Chemotaxonomic Considerations and Biological Evaluation in Prostate and Breast Cancer Cells. Ana Valentina Basso,a Segundo Leiva González,b Gloria Estela Barboza,*,a Valeria Pilar Careaga,c,d Juan Carlos Calvo,d Paula A. Sacca,d and Viviana Estela Nicotra*,a a Facultad de Ciencias Químicas, Instituto Multidisciplinario de Biología Vegetal (IMBIV-CONICET), Universidad Nacional de Córdoba, CC 495, 5000 Córdoba, Argentina, [email protected] (V. Nicotra), [email protected] (G. Barboza) b Museo de Historia Natural, Universidad Privada Antenor Orrego, CC 1075, Trujillo, Perú c Departamento de Química Orgánica, Universidad de Buenos Aires, Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (UMYMFOR-CONICET), Facultad de Ciencias, Exactas y Naturales, Ciudad Autónoma de Buenos Aires, Argentina Article d Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina Twelve Salpichroa taxa have been phytochemically analyzed. From the aerial parts of S. scandens, the four known salpichrolides A, C, I, S, and an unreported withanolide named salpichrolide V (1), were isolated. In S. dependens, S. gayi, S. glandulosa subsp. glandulosa, S. glandulosa subps. weddellii, S. leucantha, S. micrantha, S. microloba, S. proboscidea, S. ramosissima, S. tristis var. tristis, and S. weberbauerii, no withanolides were found. The chemical content of ca. 85 % of the Salpichroa taxa is in agreement with molecular studies, which suggest that Salpichroa and Jaborosa, a genus considered morphologically close to Salpichroa, are distant in the systematic of the Solanoideae subfamily. Moreover, the in vitro cytotoxic activity of a set of natural salpichrolides and derivatives was examined against two prostate carcinoma cell lines (PC3 and LNCaP) and two human breast cancer cell lines (MCF-7 and T47D). Several compounds showed moderate activity (IC50 = 64.91 − 29.97 μM). Keywords: Salpichroa • Withanolides • Structure elucidation • Chemotaxonomic markers • Cytotoxicity Introduction The genus Salpichroa Miers (Solanaceae) comprises around 17 species, restricted mainly to the highland Puna and Prepuna of Peru, Bolivia and northern Argentina. One single species (S. tristis Miers) occurs throughout the highland grasslands in the Andes, reaching as far as Venezuela, while S. origanifolia (Lam.) Baillon has a worldwide distribution.[1,2] These species can take the forms of a scandent shrub (Figure 1 A, D), or a hanging shrub (Figure 1 C, E), or a procumbent shrub (Figure 1 B, F). D’Arcy[3] and Hunziker[1] placed Salpichroa together with Nectouxia Kunth and Jaborosa Juss. in the tribe Jaboroseae Miers, based mostly on morphological characters. Recent molecular phylogenies in Solanaceae proved that Salpichroa belongs This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/cbdv.201700118 Accepted This article is protected by copyright. All rights reserved. to a different and distant clade from Jaborosa. [4,5] Until now the phytochemical evidence was quite fragmentary, but in agreement with the molecular proposal to place Salpichroa and Jaborosa in independent clades.[6] Withanolides are steroidal lactones built on an ergostane skeleton of 28 carbons functionalized at carbons 1, 22, and 26, and commonly known as the withanolide skeleton, with their chemistry and occurrence having been the subject of several reviews.[6-8] Over 750 withanolides have been described, principally, but not exclusively, from genera belonging to the Solanoideae subfamily. Some clades of the subfam. Solanoideae also contain withanolides with exclusive interesting structural arrangements, which can be considered at different hierarchical levels chemotaxonomic markers.[6] Withanolides with a six- membered aromatic ring-D and a family of these related ergostane derivatives constitute an important group called salpichrolides. They have been isolated from S. origanifolia and S. tristis var. lehmannii (Dammer) Keel.[9-14] Many of these compounds exhibit interesting biological activities such as antifeedant,[15-17] antiproliferative,[18] and cancer chemopreventive effects.[19] As part of an ongoing program aimed at to clarify the systematic position of Salpichroa, a phytochemical study of twelve taxa is herein reported. The species studied were: S. dependens (Hook.) Miers, S. gayi Benoist, S. glandulosa (Hook.) Miers (with two subspecies: subsp. glandulosa and subsp. weddellii (Benoist) Keel), S. leucantha Pereyra, Quip. & S. Leiva, S. micrantha Benoist, S. microloba S. Keel, S. proboscidea Benoist, S. ramosissima Miers, S. scandens Dammer (two samples), S. tristis Miers var. tristis (three samples), and S. weberbauerii Dammer. Article Accepted This article is protected by copyright. All rights reserved. Article Figure 1. Salpichroa species in their natural habitat. A. S. glandulosa subsp. glandulosa. B. S. gayi. C. S. weberbaverii. D. S. ramosissima. E. S. didierana. F. S. microloba. Accepted This article is protected by copyright. All rights reserved. Results and Discussion Phytochemical study. The dichloromethane extract of the aerial parts of S. scandens from Argentina (sample 1) was subjected to chromatographic purification to yield the new compound 1, and the known salpichrolides A,[13] C,[14] I,[11] and S[9] (Figure 2). HRESITOFMS of salpichrolide V (1) showed a quasimolecular ion [M+Na]+ at m/z 509.2066, corresponding to an elemental formula of C28H35ClO5Na. From the following observations, the NMR spectroscopic data of compound 1 revealed the typical signals corresponding to a salpichrolide-type withanolide characterized by the aromatic ring-D and a δ-lactol side chain: (i) the 1H NMR spectrum displayed the characteristic signals corresponding to aromatic protons at δ 7.14, δ 6.94, and δ 5.91, assigned to H -C (15), H -C (16), and H -C (18), respectively; (ii) the presence of an epoxy lactol was evidenced by the signal corresponding to the oxymethine proton at δ 4.92 (d, J = 9.9 Hz), assigned to the hydrogen H -C (26), and also by the resonances of the two methyl groups at C (24) and C (25) at δ 1.31 (H3-27) and 1.29 (H3-28), respectively; (iii) the signals at δ 65.2 [C (24)], δ 64.0 [C (25)], δ 91.7 [C (26)], δ 16.9 [C (27)], δ and 19.1 [C (28)], observed in the 13C NMR spectrum, were in agreement with a side chain epoxy lactol arrangement. Regarding the A and B rings, the 1H NMR spectrum of 1 exhibited two olefinic proton signals at δ 5.91 (dd, J = 10.2 and 2.8 Hz) and 6.57 (ddd, J = 10.2, 5.1, and 2.2 Hz), typical of a 1-oxo-2-en system in ring A, while the signal at δ 4.07 (t, J = 2.9 Hz), bearing in mind that the molecule has a chlorine atom by HRESITOFMS, suggested the presence of a chlorine substituent at C (6) position. The substitution pattern in ring B was further confirmed by the signals at δ 77.5 and 64.0 in the 13C NMR spectrum, assigned to C (5) and C (6), respectively, and by the cross-correlation peaks between the signal of H -C (6) with the signals of C (5), C (10) (δ 52.9), and C (8) (δ 32.9) in the HMBC spectrum. The β orientation of Article the chlorine group was established through the multiplicity and coupling constant value of H -C (6) (t, J = 2.9 Hz), and by the cross-correlation peak observed between H -C (6) and H -C (9) (δ 2.16) in the NOESY experiment (see Supporting Information). Thus, the structure of compound 1 was established as (20S,22R,24S,25S,26R)-6β-chloro-22,26:24,25-diepoxy- 5α,26-dihydroxy-17(13 → 18) abeo-5α-ergosta-2,13,15,17-tetraen-1-one. Compound 1 is one of the examples of chlorinated withanolides found in other species of Solanaceae. To date, at least thirty-five withanolides with chlorine substituent on ring B have been reported from the Jaborosa, Nicandra Adans., Physalis L., Tubocapsicum (Wettst.) Makino, and Withania Pauquy genera.[6,8,20] Following a similar procedure, the dichloromethane extract of S. scandens from Bolivia (sample 2) was analyzed and shown to yield the salpichrolides A, C, S, and V (1) by comparison with published spectroscopic and physical data. 21 12 18 20 O OH 13 17 O 26 OH O OH O O 19 11 22 O 9 14 23 25 2 1 16 O 10 8 15 24 27 5 O O 3 4 6 7 28 HO HO O OH Cl salpichrolide V (1) salpichrolide A salpichrolide C O OH HO O OH O OR O OH OH O HO OH O O R= -glc salpichrolide S salpichrolide I Figure 2. Withanolides isolated from Salpichroa species. The dichloromethane and ethyl acetate extracts of the remainder eleven Salpichroa taxa studied were analyzed by TLC and 1H NMR but no withanolides were detected from any of these. Withanolides have been reported in the majority of the genera within subfam. Solanoideae and in most species of some of these genera (eg. Datura L., Jaborosa, Physalis, Accepted This article is protected by copyright. All rights reserved. Withania).[6] However, in other genera such as Solanum L. (ca. 1500 species), withanolides have been reported only in two species to date.[21-24] In addition to consulting the published papers related to phytochemical studies of the Solanum species, members of our working group conducted a phytochemical study in two Solanum species (S. comptum C.V. Morton and S. atropurpureum Schrank), but no withanolides were found in any of them.25 Therefore, the absence of withanolidos in some Salpichroa species would not be so strange.