Modulation of the activity of pro-inflammatory enzymes, COX-2 and iNOS, by chrysin derivatives

doi:10.1016/S1043-6618(03)00248-2

Pharmacological Research

Volume 49, Issue 1, January 2004, Pages 37-43

https://doi.org/10.1016/S1043-6618(03)00248-2 Get rights and content

Abstract

Chrysin, a natural flavone compound found in plants, has anti-inflammatory activity that has been previously explained in part by the suppression of promoter activities of pro-inflammatory enzymes, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Here we present evidence that several chrysin derivatives modulate the activities, as well as the expression, of COX-2 and iNOS enzymes. Nitrate production triggered by lipopolysaccharide (LPS) was suppressed by treatment of cultured Raw264.7 cells (mice macrophage/monocyte) with chrysin, 5-hydroxy-7-methoxyflavone (Ch-2), and 5,7-diacetylflavone (Ch-4). Interestingly, COX-2 enzyme was strongly inhibited by Ch-4 (IC₅₀=2.7 μM) but not by other derivatives. Furthermore, the inhibition of COX enzyme by Ch-4 was selective for COX-2 over COX-1. Three-dimensional modeling showed that Ch-4 fits well into the binding pocket of COX-2. The modeling suggested that a hydrogen bond exists between the oxygen of the ketone group at the 7-position of Ch-4 and the hydroxyl group of Tyr355. Docking Ch-4 into the V523I mutant of COX-2 indicated that Ile523 of COX-1 might contribute to the selectivity of COX-2 over COX-1. Ch-4 showed no effect on iNOS activity. Chrysin and Ch-2 weakly inhibited iNOS enzyme activity in the hemoglobin assay, but the underlying mechanisms of inhibition of iNOS by chrysin are not understood.

Introduction

Chrysin (5,7-dihydroxyflavone) is a natural flavonoid contained in many plant extracts [1]. Many polyphenolic compounds, including chrysin, are known to have multiple biological activities, such as anti-inflammation [2], [3], anti-cancer [4], [5], anti-oxidation [6], [7], and estrogenic effects [6]. For its anti-inflammatory effect, it has been shown that chrysin acts as an agonist of PPAR-γ which results in down-regulation of the key pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2, prostaglandin synthase-2) [8].

Nitric oxide (NO) is a highly reactive free radical that is involved in diverse physiological and pathological processes [9], [10], [11]. NO is synthesized during conversion of l-arginine to citrulline in vivo by nitric oxide synthase. NO acts as an intra- or inter-cellular second messenger, usually via guanylate cyclase, at low concentrations (up to 2 μM). When macrophages are activated by interferon (INF)-γ and bacterial lipopolysaccharide (LPS), they induce the expression of iNOS (type II, NOS2) and produce large amounts of NO. The other two NOS enzymes, endothelial NOS (eNOS, type III, NOS3) and neuronal NOS (nNOS, type I, NOS1), are constitutive enzymes. Overproduced NO acts as a major cytotoxic mediator and inhibits the growth of invading microorganisms and tumor cells. Also, excessive NO (>10 μM) interacts with oxygen radicals and forms highly reactive peroxynitrite, which in turn induces inflammatory cellular cytokines and COX-2 and also leads to cytokine-induced cell death by apoptosis and necrosis [10].

Cyclooxygenase (COX, prostaglandin H₂ synthase (PGHS)) is the key enzyme in the biosynthesis of prostaglandin from arachidonic acid. COX is a bifunctional enzyme catalyzing both the conversion of arachidonic acid into prostaglandin G₂ (PGG₂) as a cyclooxygenase and the reaction from PGG₂ to prostaglandin H₂ (PGH₂) as a peroxidase. COX exists as two isotypes. COX-1 is responsible for the production of basal levels of prostaglandin needed for gastrointestinal tract homeostasis. COX-2 is an inducible enzyme involved in inflammatory events. Well-known nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, ibuprofen, indomethacin, and naproxen act as inhibitors of COX. However, the inhibition of COX-1 enzyme provokes undesirable side effects, such as ulcerogenic and nephrotoxic activities, which justifies further efforts to develop selective inhibitors of COX-2.

A series of chrysin alkyl and aryl derivatives was synthesized [12]. Chemo-protective or anti-inflammatory effects of flavonoid compounds including chrysin have been extensively studied. This study was designed to determine if the chrysin derivatives also exerted their anti-inflammatory effect by direct inhibition of the key pro-inflammatory enzymes.

Section snippets

Materials

Chrysin derivatives (Table 1) were synthesized as described [12]. LPS (Escherichia coli Serotype 026:B6), Griess reagent, oxyhemoglobin (HbA_o), and cofactors (NADPH, FAD, FMN, H₄B) were purchased from Sigma Chemical Co. (St. Louis, MO). iNOS (murine recombinant), and COX-2 enzymes were obtained from Cayman (Ann Arbor, MI).

Measurement of NO production

Raw264.7 cells (mice macrophage/monocyte) were kindly provided by Dr. Park (Kunkook University, Seoul, Korea). They were grown in DMEM medium supplemented with 10% FBS, 25 μM

Chrysin, Ch-2, and Ch-4 inhibited LPS-induced NO overproduction

Biologically produced NO formed the stable oxidative metabolites, nitrite and nitrate, which were measured using the Griess reaction. NO induction by LPS treatment in cultured Raw264.7 cells was prevented up to 80% by chrysin (at 50 μM). Even though the inhibitory activities were weaker, Ch-2 (30% at 50 μM) and Ch-4 (50% at 50 μM) showed concentration-dependent inhibition of NO production induced by LPS (Fig. 1). Ch-3 only weakly suppressed at high concentrations. Ch-5 did not inhibit NO

Discussion

The expression of iNOS is activated by bacterial endotoxins or various cytokines. Also, excessive NO production is known to be involved in a wide range of inflammatory diseases. In order to block the pathological inflammatory events due to COX-2 and iNOS, various drugs with different mechanisms are under development. First, we can block the signaling pathways for the induction of iNOS and COX-2. iNOS is expressed in response to pro-inflammatory agents interleukin (IL)-1β, tumor necrosis factor

Acknowledgements

We thank the Center for Biological Modulators of the Korea Research Institute for Chemical Technology (Daejeon, Korea) for allowing us to use the modeling program SYBYL.

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Modulation of the activity of pro-inflammatory enzymes, COX-2 and iNOS, by chrysin derivatives

Abstract

Introduction

Section snippets

Materials

Measurement of NO production

Chrysin, Ch-2, and Ch-4 inhibited LPS-induced NO overproduction

Discussion

Acknowledgements

Phytochemistry

Bioorg. Med. Chem.

Brain Res.

FEBS Lett.

Int. Immunopharmacol.

Biochem. Pharm.

Mutat. Res.

Methods Enzymol.

Pharm. Ther.

J. Biol. Chem.

J. Biol. Chem.

Prostaglandins Other Lipid Mediat.