*4.6. Study of Vasorelaxant Mechanisms of B. monnieri Active Compounds on Intracellular Ca2*<sup>+</sup> *Release*

To stimulate initial Ca2<sup>+</sup> loading of the SR Ca2<sup>+</sup> stores, endothelial denuded mesenteric arteries were exposed to 80 mM K<sup>+</sup> solution for 5 min, and then washed out with Ca2+-free Krebs' solution containing 1 mM EGTA for 10 min. The arterial rings were then challenged with 10 μM PE (acting through phospholipase C/IP3 signaling) which release Ca2<sup>+</sup> from the SR thereby eliciting a transient contraction [29]. The same protocol was then repeated to ensure that similar transient contractions to PE could be obtained. Then, the arterial rings were challenged again with 80 mM K<sup>+</sup> solution for 5 min, and washed out with Ca2+-free Krebs' solution containing 1 mM EGTA and 10 μM active compounds for 10 min. The arterial rings were again challenged with 10 μM PE. The PE-induced contractions were compared in the presence or absence of active compounds.

#### *4.7. Statistical Analyses*

Statistical analyses used GraphPad Prism version 5.00 for Windows, (GraphPad Software Inc., La Jolla, CA, USA). Data from each concentration-effect curve was analysed using non-repeated two-way ANOVA. Curve fitting in the figures was generated by the same software using non-linear regression. EC50 and Emax were compared using unpaired Student's *t* test. Values are expressed as mean ± SEM. A *p*-value < 0.05 was considered significant. 'n' is the number of vascular rings used, each ring originating from a different animal.
