Studies on Triterpene Saponins from Saponaria vaccaria> Seed and their Apoptosis-Inducing Effect on Human Cancer Cell Lines
A phytochemical analysis of the methanol extract from the seed of Saponaria vaccaria L. cultivated in Saskatchewan was performed which resulted in the detection of several bisdesmosidic saponins. A high-performance liquid chromatographic method using photodiode array and single quadrupole electrospray mass detection for analysis and profiling was developed. Due to their structural similarities, purification of bisdesmosidic saponins was challenging. However, monodesmosidic saponin Vaccaroside B and cyclopeptides Segetalin A, Segetalin B, and a new cyclopeptide, segetalin I [whose structure was proposed to be cyclo(Gly1-Pro2-Tyr3-Tyr4-Pro5-Phe6)], were purified employing various chromatographic techniques such as HPLC, VLC, PTLC).
Crude methanol extracts of S. vaccaria seed were evaluated for cytotoxic activity using the methyl-thiazol-tetrazolium non-radioactive cell proliferation assay (MTT assay). Various concentrations of the extract (2-50 ug/mL) were tested against a series of four human cancer cell lines (WiDr, colon; MDA-MB-231, breast; NCI-417, lung and PC-3, prostate). The human foreskin (BJ)-derived normal human fibroblast cell line CRL-2522 was included as a non-cancerous control. Results showed that cytotoxic activities from the seed extract were greater than commercially available Quillaja saponaria saponin.
The human cancer cell lines were also exposed to fractions containing high titers of saponins as well as semi-purified saponins (ca. 80%). All bisdesmosidic saponins and fractions thereof showed cytotoxicity against the cell lines studied. The effect was particularly strong in breast and prostate cancer cell lines with IC50 values in the range 14 ug/mL. Monodesmosidic saponins, phenolics and cyclopeptides did not show activity even at the highest concentration (50 ug/mL) tested in this study. Chemical modifications of the saponin structures resulted in an overall decrease in activity, but an increase in selectivity in comparison to CRL-2522. Time and concentration-dependent studies using the nuclear stains propidium iodide and Hoechst 33342, demonstrated that the stimulation of apoptosis was the mechanism of cytotoxic action. When breast and prostate cell lines were exposed to small amounts (4-7 mM) of bisdesmosidic saponins Segetalin H (MW 1448) and Segetalin I (MW 1464), it was observed that apoptosis was induced at an early incubation time (4-10 h). Activation of caspases and changes in membrane potential were determined by flow cytometry.
As a result of this study, we propose that triterpene bisdesmosidic saponins from the seed of Saponaria vaccaria L. represent a new type of drug having potential antitumor/anticancer activity due to their ability to induce apoptosis in vitro in human cancer cell lines at low concentrations. These compounds are extracted from a plant that can be easily cultivated using conventional agricultural equipment in Western Canada.
Advisor:Arnason, John T.; Dimmock, Jonathan R.; Gopalakrishnan, Venkat; Warrington, Rob C.; Tuchek, John M; Balsevich, John; Hickie, Robert A.; Wu, Lingyun
School:University of Saskatchewan
School Location:Canada - Saskatchewan
Source Type:Master's Thesis
Keywords:triterpene saponins saponaria vaccaria apoptosis bioassay cancer
Date of Publication:08/13/2008