Protective effects of five compounds from R. Brown leaves against hypoxia/reoxygenation, HO, or adriamycin-induced injury in H9c2 cells.

Discovering new antimyocardial ischemia drug candidates that are highly efficient, have low toxicity, and originate from natural products is a popular trend for new cardiovascular drug development at present. The ethanol extract of leaves showed a favorable antioxidant activity in our preliminary screening test. This study aims to screen out antioxidants from the herb leaves further and evaluate their efficacy in acute myocardial ischemia treatment at the cellular level. Guided with online 1, 1-diphenyl-2-picrylhydrazyl (DPPH)-high-performance liquid chromatography (HPLC) screening, antioxidants were first separated and isolated from the ethanol extract of leaves by preparative-HPLC. Subsequently, offline DPPH approach was used to validate the free radical scavenging activity of the components. Ultimately, the resulting antioxidants were evaluated against the hypoxia/reoxygenation (H/R)-, HO-, or adriamycin (ADM)-induced injury in H9c2 cells to verify their cardioprotective effects in vitro. Five antioxidant ingredients, namely, orientin, isoorientin, vitexin, isovitexin, and tricin, were quickly distinguished and isolated from leaves. The IC values of these ingredients were further examined by offline DPPH assay, as follows: 15.51±0.22, 6.64±0.38, 11.86±0.24, 8.89±0.66, and 31.86±0.24 μg/mL, respectively. Out of these ingredients, isoorientin showed the strongest antioxidation, which was equivalent to that of the positive control drug (vitamin C, IC: 6.99±0.62 μg/mL). Using H/R-, HO-, and ADM-induced H9c2 cell injury models, the five ingredients had different extents of cardioprotective effects in vitro. In particular, isoorientin showed the strongest protection. All the five ingredients also showed insignificant cytotoxic effect to normal H9c2 cells. The ethanol extract of leaves contained five antioxidants with low cardiac cytotoxicity. Isoorientin possessed the strongest antioxidation, which can predominantly account for the myocardial protection effects within the extract.