A minor product, rebaudioside M2 (2), from the bioconversion reaction of rebaudioside A (4) to rebaudioside D (3), was isolated and the complete structure of the novel steviol glycoside was determined. Rebaudioside M2 (2) is considered an isomer of rebaudioside M (1) and contains a relatively rare 1→6 sugar linkage. It was isolated and characterized with NMR (1H, 13C, COSY, HSQC-DEPT, HMBC, 1D-TOCSY, and NOESY) and mass spectral data. Additionally, we emphasize the importance of 1D and 2D NMR techniques when identifying complex steviol glycosides. Numerous NMR spectroscopy studies of rebaudioside M (1), rebaudioside D (3), and mixture of 1 and 3 led to the discovery that SG17 which was previously reported in literature, is a mixture of rebaudioside D (3), rebaudioside M (1), and possibly other related steviol glycosides.
In the present study, we have investigated potential cardioprotective properties of Isosteviol analogue we recently synthesized and named JC105. Treatment of heart embryonic H9c2 cells with JC105 (10 μM) significantly increased survival of cells exposed to hypoxia-reoxygenation. JC105 (10 μM) activated ERK1/2, DRP1 and increased levels of cardioprotective SUR2A in hypoxia-reoxygenation, but did not have any effects on ERK1/2, DRP1 and/or SUR2A in normoxia. U0126 (10 μM) inhibited JC105-mediated phosphorylation of ERK1/2 and DRP1 without affecting AKT or AMPK, which were also not regulated by JC105. Seahorse bioenergetic analysis demonstrated that JC105 (10 μM) did not affect mitochondria at rest, but it counteracted all mitochondrial effects of hypoxia-reoxygenation. Cytoprotection afforded by JC105 was inhibited by U0126 (10 μM). Taken all together, these demonstrate that (a) JC105 protects H9c2 cells against hypoxia-reoxygenation and that (b) this effect is mediated via ERK1/2. The unique property of JC105 is that selectively activates ERK1/2 in cells exposed to stress, but not in cells under non-stress conditions.