The aqueous extract of C. ficifolia was obtained and standardized with regard to its DCI content. RINm5F pancreatic β-cells were incubated with different concentrations (50, 100, 200 and 400 μM) of DCI alone or C. ficifolia (9, 18, 36 and 72 µg of extract/mL), and the [Ca2+]i of the cells was quantified. The cells were preloaded with the Ca2+ fluorescent dye fluo4-acetoxymethyl ester (AM) and visualized by confocal microscopy. Insulin secretion was measured by an ELISA method. Subsequently, the effect of C. ficifolia on the K+ATP channel was evaluated. In this case, the blocker activator diazoxide was used to inhibit the C. ficifolia-induced calcium influx. In addition, the inositol 1,4,5-trisphosphate (IP3)-receptor-selective inhibitor 2-amino-thoxydiphenylborate (2-APB) was used to inhibit the influx of calcium from the ER that was induced by C. ficifolia.
It was found that DCI alone did not increase [Ca2+]i or insulin secretion. In contrast, treatment with C. ficifolia increased [Ca2+]i 10-fold compared with the control group. Insulin secretion increased by 46.9%. In the presence of diazoxide, C. ficifolia decreased [Ca2+]i by 50%, while insulin secretion increased by 36.4%. In contrast, in the presence of 2-APB, C. ficifolia increased [Ca2+]i 18-fold, while insulin secretion remained constant, indicating an additive effect. Therefore, C. ficifolia was not found to block the K+ATP channel. However, it did exert an effect by increasing [Ca2+]i from the ER, which may partly explain the insulin secretion observed following treatment with C. ficifolia.
The hypoglycemic properties of C. ficifolia can be explained in part by its effect as a secretagogue for insulin through an increase in [Ca2+]i from the calcium reservoir in the ER. Therefore, the mechanism of action of C. ficifolia is different to those of the currently used hypoglycemic drugs, such as sulfonylureas. These results support that C. ficifolia may be a potential natural resource for new agents to control T2D.