*4.10. Effect Calcium Dependence Extracellular Calcium Ionic and Effect of Barium chloride, Tetraethylammonium (TEA), and Bay K6844*

To study the role of extracellular calcium, experiments were performed with a calcium-free KRB containing (× <sup>10</sup>−<sup>3</sup> M) 4.2 KCl, 1.19 KH2PO4, 125 NaCl, 25 NaHCO3, 1.2 MgSO4, and 5 D-glucose (pH 7.4). The aortic rings were first pre-incubated in a normal KRB for 30 min; then the normal KRB was replaced with KRB calcium-free for 5 min before PE (10−<sup>6</sup> M) was added. Five min after contraction with PE (10−<sup>6</sup> M), cumulative concentrations of CaCl2 (0.1 to 1.0 × <sup>10</sup>−<sup>3</sup> M) were added to the medium. In other experiments, the contraction was induced by 5 × <sup>10</sup>−<sup>3</sup> M BaCl2 or 5 × <sup>10</sup>−<sup>3</sup> M TEA for 10 min. BaCl2 and TEA are used because they increases vasoconstriction by blocking of potassium channels, thus depolarizing the plasma membrane. In other protocols, the contractile response was induced by an agonist of voltage-dependent calcium channels (10−<sup>8</sup> M Bay K6844). The aortic rings were pre-incubated with 8-oxo-9-dihydromakomakine (10−<sup>5</sup> M) for 20 min before the experiment.

#### *4.11. Effect Accumulative KCl and Phenylephrine Modulated by 8-Oxo-9-Dihydromakomakine*

In the first curve, the aortic rings were stimulated with accumulative KCl doses (10 mM to 60 mM) or PE (10−<sup>9</sup> to 10−<sup>5</sup> M). In the second curve, the aortic rings were pre-incubated with 8-oxo-9-dihydromakomakine (10−<sup>5</sup> M or 10−<sup>4</sup> M) for 20 min and then stimulated with accumulative KCl doses (10 mM to 60 mM) or PE (10−<sup>9</sup> to 10−<sup>5</sup> M).

#### *4.12. Statistical Analysis*

Data shown in figures and tables are expressed as average ± standard errors of the mean. Statistical analysis was performed by means of one-way and two-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test, and some cases Student's *t*-test. Results are given in the text as probability values, with *p* < 0.05 adopted as the criterion of significance. The graphics and linear regression were done by the least square method, using GraphPad Prism software, version 6.0 (GraphPad Software, Inc, La Jolla, CA, USA).

#### **5. Conclusions**

This study demonstrates that the natural alkaloid 8-oxo-9-dihydromakomakine obtained from leaves of *Aristotelia chilensis*, a medicinal tree widely employed in Chilean traditional medicine, is able to decrease the tone of arterial smooth muscle. The vasodilator effect of this alkaloid involves responses independent of endothelium, probably due to calcium channels blockage and/or activation of potassium channels, whose mechanism of action remains to be clarified. Our data suggest that Chilean medicinal plants constitute an important reservoir of bioactive compounds that deserves intensive scientific exploration.

**Supplementary Materials:** The following are available online, Figure S1. Numbered structure of 8-oxo-9-dihydromakomakine. Figure S2: 1H-NMR (600 MHz, CD3OD) spectra of 8-oxo-9-dihydromakomakine. Figure S3. 13C-NMR (150 MHz, CD3OD) spectra of 8-oxo-9-dihydromakomakine. Figure S4. gs-H,H-COSY (CD3OD) spectra of 8-oxo-9-dihydromakomakine. Figure S5. edited HSQC (CD3OD) spectra of 8-oxo-9-dihydromakomakine. Figure S6. gs-HMBC (CD3OD) spectra of 8-oxo-9-dihydromakomakine.

**Author Contributions:** F.C and A.P. conducted the pharmacological assays. A.P., C.R.N., and J.P. contributed to the preparation and writing the manuscript. C.P. isolated the tested compound and prepared the manuscript.

**Funding:** This research was supported by the grant [DI17-0049] from the Universidad de La Frontera, Chile and Network for Extreme Environments Research project (NEXER; Project [ANT1756], Universidad de Antofagasta, Chile).

**Acknowledgments:** The authors wish to express their gratitude to the Universidad de La Frontera and the Rectoria y Vicerrectoria de Investigacion, Innovacion y Postgrado Universidad de Antofagasta for their financial and technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.
