The protective effects of ginsenosides on human erythrocytes against hemin-induced hemolysis

Abstract

Panax ginseng has been used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress. Many reports have been devoted to the effects of ginsenosides on many in vitro or in vivo experimental systems. The major aim of this work is to investigate the protective effects of 12 individual ginsenosides including Rb1, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, R1 and pseudoginsenoside F11, together with the central structures of aforementioned ginsenosides, 20(S)-protopanaxadiol (PD) and 20(S)-protopanaxatriol (PT), on hemin-induced hemolysis of human erythrocytes. This is because hemin can induce hemolysis by accelerating the potassium leakage, dissociating skeletal proteins and prohibiting some enzymes in the membrane of erythrocyte. Thus, the structure-activity-relationship (SAR) between ginsenosides and protective effects has been screened in this in vitro experimental system. It is found that Rh2 and Rg3 intensify hemolysis in the presence of hemin, and initiate hemolysis even in the absence of hemin. All the other ginsenosides protect human erythrocytes against hemin-induced hemolysis more or less. The overall sequence is Rc > Rd > Re ∼ Rb1 > Rg1 ∼ Rh1 > Rb3 ∼ Rg2 ∼ R1 ∼ F11 ∼ PT. In addition, the protective effects of PD and PT have been detected, and found that PD promotes hemolysis appreciably, whereas PT protects erythrocytes efficiently. Moreover, the protective effects of PT ginsenosides are similar to PT itself, and the protective effects of PD ginsenosides vary remarkably, demonstrating that the positions of the sugar moieties make the protective activities of ginsenosides complicated. Especially, sugar moiety at 20-position is critical for PD ginsenosides to inhibit hemolysis, whereas hydroxyl group at 3-position is important for PT ginsenosides. The present result may be useful for understanding the SAR of ginsenosides.

Introduction

Heme is a prosthetic group in hemoglobin and myoglobin that carry oxygen in the bloodstream and in muscles (Streitwieser and Heathcock, 1985) (see Scheme 1). However, heme may behave as injurant in the case of oxidative stress because the hydrophobic hemin is able to intercalate into lipid part of membranes, resulting in the collapse of the bilayer structure of cells (Fitch et al., 1983, Shaklai et al., 1985, Balla et al., 1991), namely, hemin destroys the membrane of erythrocytes by accelerating the potassium leakage, dissociating skeletal proteins in membrane and prohibiting some erythrocyte enzymes, leading to hemolysis eventually (Liu et al., 1985, Harvey and Beutler, 1982, Zaidi et al., 1995).

Therefore, hemin-induced hemolysis can be an in vitro experimental system to detect whether a compound can eliminate the destroying function derived from hemin. For example, Zou et al. have reported that vitamin E protects sheep erythrocytes against hemin-induced hemolysis by stabilizing membrane (Wang et al., 2006). And glutathione, a cellular antioxidant, inhibits hemin-induced hemolysis by promoting hemin degradation (Atamna and Ginsurg, 1995). Desferrioxamine, an iron chelator, suppresses hemin-induced hemolysis by binding to hemin (Sullivan et al., 1992). These results motivate us to investigate whether some compounds extracted from natural medicinal herbs can protect human erythrocytes against hemin-induced hemolysis.

Panax ginseng has been used in traditional Chinese medicine since the Han Dynasty some 2000 years ago (Gillis, 1997). The major active components are called ginsenosides that are derivatives of the dammarane-type triterpene attached by some sugar moieties at 3-, 6-, and 20-position. According to the position of sugar moieties, ginsenosides are divided into 20(S)-protopanaxadiol (PD) group and 20(S)-protopanaxatriol (PT) group (Liu et al., 2003). As can be seen in Table 1, Table 2 , the sugar moieties in the PD group attach to 3-position of dammarane-type triterpene including Rg3, Rd, Rc, Rh2, Rb1 and Rb3, whereas the sugar moieties in the PT group attach to the 6-position of the dammarane-type triterpene involving Rg1, Rg2, Rh1, Re and R1. Although the carbon chain at 20-position is replaced by a tetrahydrofuran ring, pseudoginsenoside F11 belongs to PT group as well.

Some research works have focused on the total extraction from ginseng root, and found that the total ginsenosides inhibited iron-mediated peroxidation of arachidonic acid significantly, and suppressed the formation of hydroxyl radical efficiently (Zhang et al., 1996). However, the total ginsenosides cannot lead us to clarify the structure-activity relationship (SAR) of individual ginsenosides. With the development of the isolation technology (Soldati and Sticher, 1980, Shibata et al., 1985, van Breeman et al., 1995), about 20 individual ginsenosides have been isolated, and the SAR can be carried out in various experimental systems. We have reported the antioxidative effects of 11 individual ginsenosides together with 20(S)-protopanaxadiol (PD) and 20(S)-protopanaxatriol (PT) on human erythrocytes against 2,2′-azobis (2-amidinopropane dihydrochloride) (AAPH) induced hemolysis (Liu et al., 2002, Liu et al., 2003). Presented here is the study on the protective effects of total ginsenoside, 12 individual ginsenosides, i.e. Rb1, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, R1 and pseudoginsenoside F11, and PD and PT on human erythrocytes against hemin-induced hemolysis, and the SAR in the case of hemin-induced hemolysis is also the major concern in this work.