Isolation and structure elucidation of twelve new prenylated flavonoids from Onobrychis spp. (Leguminosae) employing LC-HRMS, HSCCC and NMR techniques

Onobrychis spp. (Leguminosae), mostly known as tanniferous forage legumes, bear great economic impact on agricultural and livestock fields. In our previous reports, the phytochemical and pharmacological evaluation of Onobrychis ebenoides, a Greek endemic plant, yielded novel prenylated compounds with remarkable cytotoxic and estrogenic activities [1]. The continuation of our studies suggested the prenyl moiety position, as the decisive component of the cytotoxicity and/or estrogenicity in a cell-dependent manner [2, 3]. Therefore, the present study was aimed firstly at a comparative phytochemical investigation between Onobrychis ebenoides and Onobrychis alba subsp. laconica, another endemic species of Greece without scientific reports. Among these lines, LC-HRMS-based dereplication approaches were employed for targeted isolation purposes. Secondly, both extracts subjected to isolation and purification of putative new compounds using High Speed Countercurrent Chromatography (HSCCC) technique. The profiling of methanol extracts from both plants was performed on a UHPLC-ESI (-)-LTQ-Orbitrap system. Chromatographic and spectrometric features were incorporated for the identification procedure [4]. Both extracts revealed a rich content in prenylated derivatives of isoflavonoids, coumestans and benzofurans. Consequent targeted fractionation with step-gradient HSCCC afforded 5 new isoflavonoids namely albalacone I-II, ebenosin I-III, 4 new coumestans albalacol, onobenol I-III, 1 new pterocarpan onopterol and 2 new benzofurans ebenfuran IV and bis-ebenfuran II, as indicated in the figure below, along with 10 known compounds belonging to the respective chemical classes. Conclusively, both Onobrychis ebenoides and Onobrychis alba subsp. laconica, found to be significantly rich in at least three different classes of phytoestrogens, known for their estrogenic as well as their cytotoxic properties.

Keywords: Onobrychis ebenoides, Onobrychis alba subsp. laconica, prenylated flavonoids, estrogenicity, cytotoxicity.

References:

[1] Papoutsi Z, Kassi E, Halabalaki M, Mitakou S, Moutsatsou P. Evaluation of estrogenic/antiestrogenic activity of Onobrychis ebenoides extract – Interaction with estrogen receptor subtypes ERα and ERβ. Toxicology in Vitro 2007; 3: 364 – 370

[2] Halabalaki M, Alexi X, Aligiannis N, Lambrinidis G, Pritsinis H, Florentin I, Mitakou S, Mikros E, Skaltsounis AL, Alexis MN. Estrogenic activity of isoflavonoids from Onobrychis ebenoides. Planta Med 2006; 6: 488 – 493

[3] Halabalaki M, Alexi X, Aligiannis N, Alexis MN, Skaltsounis A-L. Ebenfurans IV-VIII from Onobrychis ebenoides: Evidence that C-prenylation is the key determinant of the cytotoxicity of 3-formyl-2-arylbenzofurans. J Nat Prod 2008; 11: 1934 – 1937

[4] Tchoumtchoua J, Njamen D, Mbanya JC, Skaltsounis AL, Halabalaki M. Structure-oriented UHPLC-LTQ Orbitrap-based approach as a dereplication strategy for the identification of isoflavonoids from Amphimas pterocarpoides crude extract. J Mass Spectrom 2013; 48: 561 – 575

 

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