Antioxidant effects of ginsenoside Re in cardiomyocytes

Abstract

We have previously demonstrated that American ginseng berry extract exhibited significant protection against oxidant-mediated injury in cardiomyocytes. To extend this work, we sought to investigate the antioxidant effects of Re, a protopanaxatriols-type and single chemical integrant present in American ginseng berry extract, using the same chick cardiomyocyte model of oxidant injury as well as ESR spectroscopy in a cell-free chemical system. In cells exposed to 2 h of H₂O₂ (0.5 mM), pretreatment with Re (0.05, 0.1, or 0.5 mg/ml for 2 h) significantly attenuated 2′,7′-dichlorofluorescein (DCF) fluorescence by 51% (from 1345 ± 67 to 658 ± 46 a.u., P < 0.001), and remarkably reduced cell death (from 51.5 ± 3.0% to 11.8 ± 1.5%, P < 0.001, compared to the control). Similar results were also observed in cells exposed to antimycin A (100 μM), a mitochondrial electron transport chain site III inhibitor which increases endogenous oxidative stress. In the ESR study, however, Re failed to reduce the formation of the superoxide/DMPO adduct and DPPH radicals. These results suggest that ginsenoside Re functions as an antioxidant, protecting cardiomyocytes from oxidant injury induced by both exogenous and endogenous oxidants, and that its protective effects may be mostly attributed to scavenging H₂O₂ and hydroxyl radicals.

Introduction

Ginseng is a perennial herb of the Araliaceae family and a highly valued medicinal plant in the Far East that has gained popularity in the West during the past decade (Blumenthal et al., 2000, Barnes et al., 2004, Helms, 2004). Asian ginseng (Panax ginseng C. A. Meyer) cultivated in China, Japan, Korea and Russia has been used as a medicinal plant in China for thousands of years (Chevallier, 2000, Helms, 2004). American ginseng (Panax quinquefolium L.), grown in the United States and Canada, is a more popular herbal, nutritional supplement used throughout the world (Harkey et al., 2001, Barnes et al., 2004). Ginsenosides are the bioactive principals in ginseng and belong to a family of steroids named dammarane saponins which are separated into protopanaxadiols and protopanaxatriols (Attele and Xie, 2002, Helms, 2004).

A large number of reports suggest that both American and Asian ginseng have diverse components and multifaceted pharmacological functions (Attele et al., 1999, Lee et al., 2005, Liou et al., 2005, Xie et al., 2005a). Among the diverse constituents of ginseng, saponins have been found to be the major components responsible for its biochemical and pharmacological actions (Lee et al., 1999). The majority of ginseng's pharmacological activities have been closely linked to its antioxidant property (Zhang et al., 1996, Keum et al., 2000, Kitts et al., 2000, Bae and Lee, 2004, Mehendale et al., 2004). In vitro experiments indicated that the constituents of the North American ginseng extract exhibit both lipid-soluble and water-soluble antioxidant activity (Kitts et al., 2000). It has also been revealed by pharmacological investigations that ginseng extract and/or some of its ginsenosides exert preventative effects on myocardial ischemia and reperfusion damage induced by hyperbaric oxygen (Zhan et al., 1994, Maffei Facino et al., 1999), protective effects on endothelial cells against damage by lipid peroxidation (Mei et al., 1994), and hepatoprotective effects against oxidative stress induced by exhaustive exercise (Voces et al., 1999). Recent research has shown as well that cultivated and wild Korean ginseng leaf extracts have antioxidant properties (Jung et al., 2005). Coincidently, our studies provide evidence that American ginseng berry extract also is a potent antioxidant and has a significantly stronger antioxidant activity compared to that of the ginseng root (Shao et al., 2004).

As alluded to above, the pharmacological properties of ginseng are mainly attributed to ginsenosides, the bioactive constituents that are found in the extracts of different parts of ginseng (Lee et al., 1999). We analyzed six major ginsenosides (Rb₁, Rb₂, Rc, Rd, Re, and Rg₁) in American ginseng berry extract by using high performance liquid chromatography (HPLC) and found that the rank order of the total ginsenosides concentration is leaf > berry > root (Xie et al., 2004). Our results also indicated that the specific ginsenoside Re is more abundant in the leaf and berry than in the root (Xie et al., 2004). So far, there are no data available in the literature with regard to the antioxidant property of Re. Therefore, the aim of the present study is to further explore the antioxidant activity of ginsenoside Re in cultured chick cardiomyocytes and cell-free chemical systems.

In order to study the antioxidant effect of Re, first a cell-free experiment was carried out using ESR spectroscopy. For further investigation of antioxidant properties, we used chick cardiomyocytes exposed to exogenous H₂O₂ or endogenous antimycin A, a mitochondrial electron transport chain (ETC) site III inhibitor. This cardiac cell model of acute oxidant stress has been successfully used in previous studies (Vanden Hoek et al., 1996, Shao et al., 2002, Shao et al., 2003). Acute oxidant injury can be generated consistently in this model as shown by significantly increased oxidant-dependent fluorescence and decreased cardiomyocyte viability. Effective protection by administration of antioxidant components to the cardiomyocyte culture prevented the pathological consequences of acute oxidant injury (Vanden Hoek et al., 1997b, Duranteau et al., 1998, Shao et al., 1999, Shao et al., 2004). We applied this model, therefore, to further investigate the antioxidant effects of Re in the present study, and demonstrated the novel finding that Re is a potent antioxidant in cardiomyocytes.