title: High-speed countercurrent chromatographic recovery and off-line electrospray ionization mass spectrometry profiling of bisdesmodic saponins from Saponaria officinalis possessing synergistic toxicity enhancing properties on targeted antitumor toxins.
abstract: Saponaria officinalis L. (Caryophyllaceae), also known as fuller's herb or soapwort is a medicinal plant, which grows from Europe to Central Asia. Medicinal properties attributed to this plant include its antitussive and galactogogue properties. Recently, bisdesmodic saponins with very specific structural features from S. officinalis have been shown to strongly enhance the efficacy of specific targeted toxins (anti-tumor antibodies connected to protein toxins) in-vitro and in-vivo in a synergistic manner. In the presently reported novel approach we used preparative all-liquid high-speed countercurrent chromatography (HSCCC) to recover a total of 22 fractions using biphasic solvent system tert-butylmethylether/n-butanol/acetonitrile/water 1:3:1:5 (v/v/v/v) from a complex precipitated crude saponin mixture. Out of these 22 fractions, 3 fractions had the enhancer effect on anti-tumor toxins out of which one fraction (F7) was further tested elaborately in different cell lines. The molecular weight distribution and compound profiles of separated saponins were monitored by off-line injections of the sequentially collected fractions to an electrospray ion-trap mass-spectrometry system (ESI-IT-MS). The functional saponin fractions were mainly bisdesmosidc and contained saponin m/z 1861 amongst other. Using the bio-assay guided monitoring, the highly active fractions containing 2 to 3 bisdesmodic saponins (5μg/mL) were screened for their effectiveness in enhancing the anti-tumor activity of targeted toxin Sap3-EGF, which was determined using the impedance based real-time cell cytotoxicity evaluation. This novel combination of HSCCC fractionation, MS-target-guided profiling procedure and bio-assay guided fractionation yielded 100mg of functional saponins from a 60g crude drug powder in a rapid and convenient manner.
authors.affiliation: Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Campus Virchow-Klinikum, 13353 Berlin, Germany. Electronic address: mayank.thakur@charite.de.
authors.forename: Mayank
authors.initials: M
authors.lastname: Thakur
authors.affiliation: Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany. Electronic address: g.jerz@tu-bs.de.
authors.forename: Gerold
authors.initials: G
authors.lastname: Jerz
authors.affiliation: Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany; Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Tuwalska
authors.affiliation: Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Campus Virchow-Klinikum, 13353 Berlin, Germany.
authors.forename: Roger
authors.initials: R
authors.lastname: Gilabert-Oriol
authors.affiliation: Department of Analytical Chemistry, Institute C-1, Faculty of Chemical Engineering and Technology, Cracow University of Technology, ul. Warszawska 24, Cracow 31-155, Poland.
authors.forename: Sławomir
authors.initials: S
authors.lastname: Wybraniec
authors.affiliation: Institute of Food Chemistry, Technische Universität Braunschweig, Schleinitzstrasse 20, 38106 Braunschweig, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Winterhalter
authors.affiliation: Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Campus Virchow-Klinikum, 13353 Berlin, Germany.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.affiliation: Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Campus Virchow-Klinikum, 13353 Berlin, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Weng
mesh_terms: D000970:Antineoplastic Agents; D003377:Countercurrent Distribution / Q000379:methods*; D004357:Drug Synergism; D000922:Immunotoxins; D010936:Plant Extracts / Q000737:chemistry*; D029750:Saponaria / Q000737:chemistry*; D012503:Saponins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000970:Antineoplastic Agents; D000922:Immunotoxins; D010936:Plant Extracts; D012503:Saponins
keywords: Bisdesmodic saponins; Caryophyllaceae; Off-line ESI-MS continuous injections; Preparative high-speed countercurrent chromatography; Saponaria officinalis; Synergistic toxicity enhancers