Direct scavenging of nitric oxide and superoxide by green tea

Abstract

In the present study, we investigated the free radical scavenging effects of green tea extract and green tea tannin mixture and its components using a nitric oxide (NO) and superoxide (O₂⁻) generating system in vitro. Green tea extract showed direct scavenging activity against NO and O₂⁻ and green tea tannin mixture, at the same concentration, showed high scavenging activity. Comparison of the activities of seven pure compounds isolated from green tea tannin mixture showed that (−)-epigallocatechin 3-O-gallate (EGCg), (−)-gallocatechin 3-O-gallate (GCg) and (−)-epicatechin 3-O-gallate (ECg) had higher scavenging activities than (−)-epigallocatechin (EGC), (+)-gallocatechin (GC), (−)-epicatechin (EC) and (+)-catechin (C), showing the importance of the structure of flavan-3-ol linked to gallic acid for this activity. Among the gallate-free tannins, EGC and GC were more effective O₂⁻ scavengers than EC and C, indicating the O-trihydroxy structure in the B ring is an important determinant of such activity. However, this structure did not affect the NO scavenging activity. These findings confirm that green tea tannin has excellent antioxidant properties, which may be involved in the beneficial effect of this compound.

Introduction

Green tea, which is a widely consumed drink, has received much attention due to the beneficial biological effects attributable to its excellent antioxidative activity (Ho et al., 1992, Jovanovic et al., 1994, Salah et al., 1995, Guo et al., 1996, Pannala et al., 1997, Yokozawa et al., 1998). Our research has focused on the protective effect of green tea against renal damage, because renal failure is considered to be a condition that develops as a result of oxidative stress (Yokozawa et al., 1996, Yokozawa et al., 1997, Yokozawa et al., 1998, Yokozawa et al., 2000). Renal ischemia-reperfusion injury is a frequently encountered phenomenon in renal transplantation, and certain disease states and the proximal tubular cells appear to be the major target of circulatory damage in the kidney. In a previous study of the effects of green tea against ischemia-reperfusion injury, we established a cell culture model system in which renal epithelial LLC-PK₁ cells, which have the nature of proximal tubules, were cultured under hypoxic conditions and then re-oxygenated. We examined the effects of green tea tannin on this system and observed that it protected renal cells against ischemia-reperfusion injury (Yokozawa et al., 1997).

It is well accepted that ischemia-reperfusion increases the generation of O₂⁻ in renal proximal tubule cells, which then increases lipid peroxidation and leads to cell and tissue injury (Paller et al., 1984). In addition, recent studies have indicated that NO is also produced in rat proximal tubules as a result of hypoxia-reperfusion (Yu et al., 1994). Under physiological conditions, NO plays important roles as a vasodilator, neurotransmitter and in the immunological system as a defense against tumor cells, parasites and bacteria. In the kidney, NO contributes to the regulation of renal function, including renal hemodynamics, sodium excretion and renin release, tubuloglomerular feedback, pressure natriuresis and tubular function (Galle & Wanner, 1996, Lahera et al., 1997). However, excess NO produced during ischemia-reperfusion is considered to act as a toxic radical and to cause renal dysfunction as well as O₂⁻ (Paller et al., 1984, Yu et al., 1994). NO and O₂⁻ cause ischemic renal injury individually and they work together to bring about further damage. Indeed, the toxicity and damage caused by NO and O₂⁻ was found to be multiplied considerably as a result of their reaction to form peroxynitrite (ONOO⁻), which is extremely reactive and leads to serious toxic reactions with biomolecules, such as proteins, lipids and nucleic acids (Moncada et al., 1991, Radi et al., 1991a, Radi et al., 1991b, Yermilov et al., 1995). Therefore, it is thought that the generation of NO and O₂⁻ is a trigger for ischemic renal injury and reducing their levels, which will suppress the formation of ONOO⁻, would be an important avenue of protection against cell injury. This raises the possibility that the ability of green tea tannin to reduce NO and/or O₂⁻ generation is involved in the protective mechanisms of this compound observed in our previous study.

In this study, to explore this possibility, we used a NO and O₂⁻ generating system in vitro to examine the direct scavenging activities of green tea extract and its components. Seven pure tannins isolated from green tea were also tested.