Atherosclerosis 139 (1998) 333 – 339 Effect of allicin and ajoene, two compounds of garlic, on inducible nitric oxide synthase Verena M. Dirsch a, *, Alexandra K. Kiemer a , Hildebert Wagner b , Angelika M. Vollmar a a Institute of Pharmacology, Toxicology and Pharmacy, Ko ¨niginstr. 16, D-80539 Munich, Germany b Institute of Pharmaceutical Biology, Karlstr. 29, D-80333 Munich, Germany Received 24 November 1997; received in revised form 2 March 1998; accepted 30 March 1998 Abstract Inducible nitric oxide synthase (iNOS) has recently been shown to be present in human atherosclerotic lesions and to promote the formation of deleterious peroxynitrite. Allicin and ajoene are discussed as active compounds with regard to the beneficial effects of garlic in atherosclerosis. The aim of this study was to investigate the effect of allicin and ajoene on the iNOS system in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ajoene (IC 50 2.5–5  M) and allicin (IC 50 15–20  M) dose dependently reduced nitrite accumulation, a parameter for NO synthesis, in supernatants of LPS-stimulated (1  g/ml, 20 h) macrophages. Accordingly, reduced iNOS enzyme activities were measured by conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline in homogenates of LPS-activated cells treated with ajoene or allicin. None of these compounds, however, showed a direct effect on the catalytic activity of iNOS. Consequently, iNOS protein and mRNA expression in ajoene (10  M) or allicin (50  M) treated cells were evaluated by Western blot and Northern blot analysis, respectively. Markedly reduced iNOS protein as well as mRNA levels were demonstrated. These observations indicate that allicin and ajoene inhibit the expression of iNOS in activated macrophages. The possible link of this effect to the beneficial features attributed to garlic is discussed. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Nitric oxide; Atherosclerosis; Ajoene; Allicin; Garlic 1. Introduction The medicinal use of garlic is an extremely old and wide spread tradition, illustrated by its quotation in the oldest preserved medicinal text book, the Papyrus Ebers (1550 B.C.). As a consequence, garlic is one of the most studied medicinal plants, which is demonstrated by more than 1300 research articles during the past 100 years [1]. A major part of these publications deals with the beneficial effect of garlic on the cardiovascular system, mainly related to atherosclerosis. Most findings demonstrated lipid-lowering and antithrombotic effects for garlic [1,2]. Although the data available today sug- gest that garlic contains biologically active compounds which are beneficial in cardiovascular disease like atherosclerosis, the question about the active principles and their mechanism of action is still not settled [1,2]. Great attention has been focused on the molecule nitric oxide (NO), with regard to the pathophysiology of atherosclerosis, as it is implicated in the maintenance of vascular homeostasis. Numerous reviews have been published regarding this issue [3–6] and one major finding was that endothelial dysfunction in atheroscle- rosis is related to the decreased biological activity of nitric oxide. On the other hand, there is increasing evidence that the inflammatory environment in human atherosclerotic lesions results in an expression of the inducible form of nitric oxide synthase (iNOS) and subsequently in the formation of peroxynitrite [7]. Per- oxynitrite is a potent oxidant, which is formed when the synthesis of large amounts of NO coincides with super- * Corresponding author. Tel.: +49 89 21802664; fax: +49 89 342316; e-mail: dirsch@pharmtox.vetmed.uni-muenchen.de 0021-9150/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0021-9150(98)00094-X