Effects of Kaempferol and Myricetin on Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production in Rats 1 Evita Rostoka 1 , Larisa Baumane 1 , Sergejs Isajevs 2 , Aija Line 3 , Maija Dzintare 1 , Darja Svirina 2 , Jelena Sharipova 1 , Karina Silina 3 , Ivars Kalvinsh 1 and Nikolajs Sjakste 1,2 1 Latvian Institute of Organic Synthesis, Riga, Latvia, 2 Faculty of Medicine, University of Latvia, Riga, Latvia, 3 Latvian Biomedical Research and Study Centre, Riga, Latvia (Received 10 July 2009; Accepted 18 October 2009) Abstract: When administered as drugs or consumed as food components, polyphenolic compounds synthesized in plants interfere with intracellular signal transduction pathways, including pathways of nitric oxide synthase expression. However, effects of these compounds in vivo do not always correlate with nitric oxide synthase-inhibiting activities revealed in experi- ments with cultured cells. The initial goal of this work was to compare effects of flavonoids kaempferol and myricetin on inducible nitric oxide synthase mRNA and protein expression monitored by real-time RT-PCR and immunohistochemistry and to evaluate the impact of these effects on nitric oxide production in rat organs measured by means of electron paramag- netic resonance spectroscopy. Kaempferol and myricetin attenuated the lipopolysaccharide-induced outburst of inducible nitric oxide synthase gene expression; kaempferol also significantly decreased the lipopolysaccharide-induced outburst of inducible nitric oxide synthase protein expression in the liver. Myricetin decreased nitric oxide production in intact rat liver. Kaempferol did not decrease nitric oxide production neither in intact rats nor in the lipopolysaccharide-treated animals. Kaempferol even enhanced the lipopolysaccharide-induced increase of nitric oxide production in blood. Myricetin did not interfere with lipo- polysaccharide effects. As both kaempferol and myricetin are known as inhibitors of inducible nitric oxide synthase expres- sion, our results suggest that modifications of nitric oxide level in tissues by these compounds cannot be predicted from data about its effects on nitric oxide synthase expression or activity. Flavonoid intake influences mortality from nitric oxide- dependent processes: ischemic heart disease, stroke, diabetes mellitus and cancer [1]. This implies significance of flavonoid and other natural compound uptake for functions of cardio- vascular, immune and nervous systems. Impact of a given compound on nitric oxide production is usually deduced from in vitro nitric oxide synthase expression and nitrite pro- duction. However, modification of the nitric oxide synthesis by drugs in animals and humans appears to be complicated and dependent on numerous factors. Modifications of nitric oxide synthesis is often organ-specific; data of in vitro and in vivo experiments happen to be contradictory [2,3]. In our opinion, only direct measurements of nitric oxide production in vivo can reveal nitric oxide-dependent effects of a given drug. The goal of this work was to monitor modification of nitric oxide production in rat organs by the flavonoids, kaempferol and myricetin. Chemical structures of the com- pounds are given in fig. 1. According to published data, ka- empferol produces inhibition of both inducible nitric oxide synthase mRNA and protein in several cell cultures [4–7]. Myricetin inhibits nitric oxide release and inducible nitric oxide synthase expression in cultured macrophages [8–10]. Taken together, these data indicate that the two compounds should decrease lipopolysaccharide-induced nitric oxide pro- duction in vivo. No data about modification of nitric oxide production by these compounds are accessible. Thus, our study was aimed to fill a significant gap in knowledge about biological activities of natural compounds. Experimental Procedures Natural compounds. Kaempferol and myricetin were purchased from Dayang Chemical Co., LTD (Taiwan 2 ). Chemicals. Lipopolysascharide, diethylthiocarbamate, ferrous sul- fate, sodium citrate and all other chemicals were from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). Experimental design and drug administration. Animals were purchased from the laboratory animal suppliers ‘Gailezers’ (Riga, Latvia). All manipulations with animals were performed in accor- dance with Republic of Latvia regulations, being in agreement with European Union rules; permission from the Ethics Commission of the Latvian Council for Science was obtained to perform this study. Male Wistar rats weighing 200–300 g were used in the experi- ments. The rats were assigned to the following experimental groups (table 1). Group 1 (n = 24) served as a control for nitric oxide detec- tion experiments. 30 min. after spin trap injection, the rats were decapitated under slight ether narcosis. In Groups 2–4, correspond- ing substances were administered per os in concentrations indicated in table 1. After 3.5 hr of substance administration, spin trap was injected, after 30 min., the rats were decapitated under slight ether narcosis. In Group 5 (n = 28), the rats were intraperitoneally injected lipopolysascharide (10 mg ⁄ kg), spin traps were administered 3.5 hr B C P T 5 2 6 B Dispatch: 17.12.09 Journal: BCPT CE: Balaji prasad Journal Name Manuscript No. Author Received: No. of pages: 6 PE: Bhagyalakshmi Author for correspondence: Nikolajs Sjakste, Latvian Institute of Organic Synthesis, Aizkraukles Street 21, Riga, LV1006, Latvia (fax +371-7553142, e-mail nikolajs.sjakste@lu.lv). Ó 2009 The Authors Doi: 10.1111/j.1742-7843.2009.00526.x Journal compilation Ó 2009 Nordic Pharmacological Society. 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