Active pharmaceutical ingredients and mechanisms underlying phasic myometrial contractions stimulated with the saponin extract from Paris polyphylla Sm. var. yunnanensis used for abnormal uterine bleeding.

BACKGROUND: Total steroidal saponins of Paris polyphylla Sm. var. yunnanensis (TSSP) have been widely used in China for the treatment of abnormal uterine bleeding (AUB). But until now, the main active constituents and the mechanisms underlying the pharmacological actions on uterine activity have not been described. METHODS: Total steroidal saponins were extracted with EtOH and purified by chromatography. In vitro isometric contraction studies were performed using myometrial strips from estrogen-primed or pregnant rats. Intracellular calcium was monitored under a confocal microscope using Fluo-3 AM-loaded myometrial cells. RESULTS: TSSP dose-dependently induced phasic myometrial contractions in vitro. Experiments with calcium channel blockers or kinase inhibitors demonstrated that the TSSP-stimulated myometrial contraction was mediated by an increase in [Ca(2+)](i) via influx of extracellular calcium and release of intracellular calcium. Through bioassay-guided separation, it was found that total spirostanol saponins exhibited contractile activity in myometrium and Pennogenin-3-O-alpha-L-arabinofuranosyl(1-->4)[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-glucopyranoside (PARG) was identified as the active ingredient of TSSP. Furthermore, the contractile response of rat myometrium to PARG was significantly enhanced with advancing pregnancy. CONCLUSIONS: These data provide evidence that myometrial contractility stimulated by TSSP results from [Ca(2+)](i) increase and supports the possibility that some spirostanol gylcosides may represent a new type of contractile agonist for the uterus.

Pennogenin glycosides with a spirostanol structure are strong platelet agonists: structural requirement for activity and mode of platelet agonist synergism.

BACKGROUND: Steroidal saponins have long attracted scientific attention, due to their structural diversity and significant biological activities. For example, total steroidal saponins extracted from the rhizome of Paris polyphylla Sm. var. yunnanensis (TSSPs) constitute an effective treatment for abnormal uterine bleeding. OBJECTIVE: To determine the active constituents in TSSPs and elucidate the mechanisms that underlie their in vivo pharmacologic actions on hemostasis. METHODS: Steroidal saponins were purified by chromatography, and their effects upon hemostasis and platelet function were evaluated by tail bleeding time in mice and rats, aggregometry, flow cytometry and Western blotting. RESULTS: TSSPs promoted hemostasis in vivo and dose-dependently induced rat or human platelet aggregation in vitro. Using bioassay-guided separation, four known pennogenin glycosides with a spirostanol structure were identified as the active ingredients of TSSPs. A structure-activity assay showed that the aglycone and sugar moieties of pennogenin glycosides are both essential for their aggregatory activity. Their synergistic actions on platelet aggregation were observed with pennogenin glycosides and with other known platelet agonists, suggesting that these glycosides are platelet agonists. Aggregation in response to the pennogenin glycosides involved alpha(IIb)beta(3) activation, was inhibited by cAMP, was dependent upon extracellular calcium, secreted ADP and thromboxane synthesis, and was mediated by phosphatidylinositol-3-kinase. CONCLUSION: We identified pennogenin glycosides with a spirostanol structure as the active ingredients of Paris polyphylla Sm. var. yunnanensis in promoting hemostasis in vivo. Their mode of their action on platelets suggests that they represent a new type of platelet agonist.