Punicalagin Induce the Production of Nitric Oxide and Inhibit Angiotensin Converting Enzyme in Endothelial Cell Line EA.hy926

Introduction: punicalagin, a hydrolysable tannin polyphenol from pomegranate, reported to have a protective effect against many diseases due to its high antioxidant and free radical scavenging activities. Aim, this study investigated the potential antihypertensive activity of punicalagin in human derived EA.hy926 endothelial cell model, via two mechanisms. In first mechanism, punicalagin enhance the nitric oxide production through scavenging reactive oxygen specious (ROS) and activating the endothelial nitric oxide synthase enzyme (eNOS). In second mechanism, punicalagin showed effect activity by inhibiting the angiotensin converting enzyme (ACE). Methods: The effect of different concentrations (1-100 μM) of punicalagin on EA.hy926 cells was measured using MTT assay. Induction of ROS was done using Ang II and scavenging activity of punicalagin was assessed by flow cytometer and fluorimeter. NO production was measured in order to determine the effective dose of punicalagin followed by measuring the ability of punicalagin to induce eNOS activity and the enzyme expression by Western blotting cellular Ca concentration and ACE inhibition were also determined. Results: Punicalagin (1-60 μM) reduced ROS production in EA.hy926, which induced by added angiotensin II, as shown by flow cytometry and by fluorometry. In addition, at the same concentration the nitric oxide production was increased in a dose-dependent manner due to increased eNOS activation. The activation of eNOS enzyme was promoted by an increase of cellular calcium concentration at the tested concentrations. Examined punicalagin concentrations significantly inhibited ACE activity, possibly due to zinc binding. Conclusion: punicalagin clearly exhibits the potential for reducing hypertensive activity by a dual mechanism of nitric oxide synthase induction by increasing nitric oxide levels, and ACE inhibition.