Black soybean seed coat polyphenols promote nitric oxide production in the aorta through the Akt/eNOS pathway

Abstract

Background: Black soybean seed coat contains an abundance of flavan-3-ols and possesses various bioregulatory functions. Nitric oxide (NO) is produced by endothelial nitric oxide synthase (eNOS) in vascular endothelial cells and regulates vascular function through vasodilation and the inhibition of platelet aggregation in blood vessels. It has been reported that flavan-3-ols increase NO production, but many previous reports used a high concentration of flavan-3-ols. In the present study, we investigated the effect of flavan-3-ol-rich black soybean seed coat extract (BE) on NO production at a lower concentration that is close to the concentration after permeation through the monolayer of Caco-2 cells.Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with BE, and then NO production in the medium and eNOS phosphorylation in the cells were examined. Intestinal epithelial Caco-2 cells on the upper side of a transwell filter were co-cultured with HUVEC on the basolateral compartment of the transwell apparatus. BE was added from the upper side, and the basolateral medium was collected to measure the concentration of NO and the content of flavan-3-ols. Furthermore, HUVEC were incubated with each flavan-3-ol in order to individuate the most effective compound in BE.Results: BE significantly increased NO production in the medium of HUVEC. When polyphenols in BE were removed from the basolateral medium by ethyl acetate extraction, increased NO production from HUVEC was not observed. Additionally, BE increased phosphorylation of eNOS and Akt in HUVEC. A portion of flavan-3-ols in BE had permeated through intestinal epithelial cells. Among the flavan-3-ols that had permeated, procyanidin C1 had the strongest effect on NO production in HUVEC at the concentration that had permeated the monolayer of Caco-2 cells. Procyanidin C1 (0.05 µM) also induced phosphorylation of eNOS and Akt in HUVEC without affecting the cAMP level. Conclusion: A portion of flavan-3-ols in BE directly promoted NO production through the Akt/eNOS pathway in vascular endothelial cells. These findings suggest that flavan-3-ols in the black soybean seed coat may contribute to improve the vascular function.Keywords: Black soybean seed coat polyphenols; NO; eNOS; Akt; vascular endothelial cells