Inhibition of Nitric Oxide Synthesis in Mouse Macrophage Cells by Feverfew Supercritical Extract Anat Aviram, 1 Nikolaos M. Tsoukias, 2 Steven J. Melnick, 1,3 Anna P. Resek 1 and Cheppail Ramachandran 1,3 * 1 Miami Children’s Hospital, Department of Pathology, Miami, FL 33155, USA 2 Florida International University, Department of Biomedical Engineering, Miami, FL 33174, USA 3 Dharma Biomedical LLC, 12777 Old Cutler Road, Miami, FL 33156, USA Feverfew is the most commonly used medicinal herb against migraine headache. The antimigraine mechanism of feverfew supercritical extract was investigated in vitro using the mouse macrophage cell line (RAW 264.7). Mouse macrophage cells were treated with lipopolysaccharide in the presence and absence of feverfew extracts. Inhibition of lipopolysaccharide-induced nitric oxide and TNF-a synthesis were quantified by ELISA. The mRNA and protein expression of iNOS and eNOS genes were analysed by RT-PCR and western blot analysis, respectively. The feverfew extract inhibited both nitric oxide (NO) and TNF-a production in a dose-dependent manner with complete inhibition of NO occurring at 5 mg/mL of feverfew extract. Both eNOS and iNOS mRNA levels were unchanged with the feverfew treatment. However, eNOS and iNOS proteins were significantly down- regulated by the feverfew extract. Feverfew inhibition of NO is due to the down-regulation of both eNOS and iNOS enzymes at the translational and/or post-translational level. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: feverfew; migraine; lipopolysaccharide; nitric oxide synthase; tumor necrosis factor a; macrophage. INTRODUCTION Feverfew (Tanacetum parthenium) is the most commonly used medicinal herb for the treatment of migraine head- ache. It is also a rich source of parthenolide, considered to be the major bioactive chemical (Tassorelli et al., 2005). Because of inadequate treatment of the disease and the desire to avoid side effects of current medications, many sufferers have turned to natural medicines such as feverfew herb for migraine management. The method of extraction of feverfew has been shown to affect its stability (Pfaffenrath et al., 2002). A commercial supercritical CO 2 extract of feverfew (FF) known as MIG-99 was shown to produce a stable product with an enriched fraction of parthenolide (Pfaffenrath et al., 2002; Evans and Taylor, 2006). Clinical studies examining the effect of FF for migraine treatment have been inconclusive in its findings. Some of the major problems faced during these trials included the use of different methodology, small popula- tions and an overall poor trial design (Tassorelli et al., 2005; Ernst and Pittler, 2000; Maizels et al., 2004). Never- theless, most of the clinical trials of feverfew extracts have shown clinical benefits when compared with placebo (Pfaffenrath et al., 2002; Vogler et al., 1998). The precise mechanism by which feverfew exerts its antimigraine effects is not fully understood. Feverfew has been shown to inhibit the production of prostaglandins, the release of serotonin and the inhibition of nuclear factor-kB (NF-kB) activity in vitro (Pfaffenrath et al., 2002; Tassorelli et al., 2005; Maizels et al., 2004). However, these properties are not specifically associated with the initiation of migraine from the outset. Nitric oxide (NO) in the brain, involved in the regulation and maintenance of dural vasodilation and neurotransmission, is considered to be an important mediator of migraine (Cady, 2007; Olesen, 2008). Nitric oxide is a small unstable free radical produced naturally by the body during the degradation of L-arginine to L-citrulline by the catalytic action of nitric oxide synthase (NOS). Several studies have described NO as a key player in the initiation of migraine pain (Olesen, 2008; Kurul et al., 2008; Thomsen and Olesen, 2001; Van der Kuy and Lohman, 2003). This hypothesis is also supported by the efficacy of current medications (not including analgesic) for the treatment of migraine and other headaches that exert their effects on the nitric oxide–cyclic guanosine monophosphate pathway (NO– cGMP) (Olesen, 2008; Fidan et al., 2006). Moreover, acti- vation of the NO–cGMP pathway, by exogenous sources of NO, such as nitroglycerin, is known to trigger a head- ache indistinguishable from a regular migraine. This has led investigators to focus their attention on molecules that can interact with the NO-pathway in designing drugs that can treat migraine (Olesen, 2008; Fidan et al., 2006). To understand the antimigraine mechanism of feverfew further, the inhibitory effects of a supercritical CO 2 extract of FF on NOS and NO were investigated in mouse macro- phage cells. MATERIALS AND METHODS Feverfew extract. A supercritical CO 2 extract of fever- few was prepared by Flavex Naturextrakte, Rehlingen, Germany, which contained all CO 2 -soluble lipophilic * Correspondence to: Cheppail Ramachandran, Miami Children’s Hospital, Department of Pathology, Miami, FL 33155, USA. E-mail: cheppail.ramachandran@mch.com PHYTOTHERAPY RESEARCH Phytother. Res. 26: 541–545 (2012) Published online 16 September 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.3594 Copyright © 2011 John Wiley & Sons, Ltd. Received 10 February 2010 Revised 11 May 2011 Accepted 20 May 2011