title: Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica.
abstract: The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3 µg/mL (CI95: 25.9-28.9 µg/mL) corresponding to 9.6 µM (CI95: 9.1-10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR.The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4-6″) and single linked B-type units.This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.
authors.affiliation: Institute of Pharmacy, Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Schäfer
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Salcher
authors.affiliation: Department of Pediatrics I, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Seiter
authors.affiliation: Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Ranninger
authors.affiliation: Department of Zoology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Möst
authors.affiliation: Department of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: P
authors.initials: P
authors.lastname: Obexer
authors.affiliation: Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria.
authors.forename: C G
authors.initials: CG
authors.lastname: Huber
authors.affiliation: Department of Pediatrics I, Medical University Innsbruck, Innsbruck, Austria.
authors.forename: M J
authors.initials: MJ
authors.lastname: Ausserlechner
authors.affiliation: Institute of Pharmacy, Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.
authors.forename: S
authors.initials: S
authors.lastname: Schwaiger
authors.affiliation: Institute of Pharmacy, Pharmacognosy and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Stuppner
mesh_terms: D029790:Ephedra sinica / Q000737:chemistry*; D005454:Fluorescence Polarization; D057809:HEK293 Cells; D006801:Humans; D035261:Plant Components, Aerial / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D044945:Proanthocyanidins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding; D000072417:Protein Domains; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors*
chemical_list: D010936:Plant Extracts; D044945:Proanthocyanidins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human