Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo
Graphical abstract
Arzanol causes reduced levels of leukotrienes and PGE2 in vivo, connected to anti-inflammatory effects.
Introduction
Inflammation is a complex biological response to harmful stimuli required to remove an offensive agent and to initiate the healing process. However, chronic inflammation can lead to diseases (e.g., rheumatoid arthritis, atherosclerosis, and autoimmune disorders) that still represent unmet clinical needs. The prostanoids and leukotrienes (LTs) formed from arachidonic acid (AA) via the cyclooxygenase (COX)-1/2 (EC 1.14.99.1) and 5-lipoxygenase (5-LO) (EC 7.13.11.34) pathway, respectively, mediate inflammatory responses, chronic tissue remodelling, cancer, asthma, and autoimmune disorders, but also possess homeostatic functions in the gastrointestinal tract, uterus, brain, kidney, vasculature, and host defence [1]. In fact, the clinical use of non-steroidal anti-inflammatory drugs (NSAIDs), a class of drugs that block formation of all prostanoids, is hampered by severe side effects including gastrointestinal injury, renal irritations, and cardiovascular risks [2]. Therefore, anti-inflammatory agents interfering with eicosanoid biosynthesis require a well-balanced pharmacological profile to minimize these on-target side effects [2]. Current anti-inflammatory research aims at identifying compounds that can suppress the massive formation of pro-inflammatory prostaglandin (PG)E2 without affecting homeostatic PGE2 and PGI2 synthesis, and also inhibit LT formation [3], [4]. While there is no shortage of anti-inflammatory natural products that suppress eicosanoid generation by inhibiting 5-LO and/or COX enzymes [5], [6], the activity of most of them has only been evaluated in terms of expression and/or activity of COX enzymes and 5-LO in vitro, and high concentrations, unrealistic to translate in a clinical setting, are often required for activity [5]. Moreover, in many cases the anti-inflammatory activity was not evaluated in vivo, or, if so, it was not clearly related to an interference with the biosynthesis of eicosanoids.
Arzanol, a prenylated heterodimeric phloroglucinyl α-pyrone (Fig. 1A), was identified as the major anti-inflammatory constituent of Helichrysum italicum (H. italicum), a Mediterranean plant used in folk medicine to treat inflammatory diseases and infections [7], [8]. In fact, extracts of H. italicum have been reported to exhibit anti-oxidant [7], anti-bacterial [9], anti-fungal [10] and anti-viral [11] effects. Extensive clinical research on the activity of extracts from H. italicum to manage inflammatory conditions was carried out by the Italian physician Leonardo Santini in the 40s and 50s. However, his findings were published in little known Journals, and, after his death, were largely overlooked [8]. Arzanol showed anti-oxidant activity in various systems of lipid peroxidation in vitro, and protected VERO cells against tert-butyl hydroperoxide (TBH)-induced oxidative stress [12]. Moreover, arzanol inhibited nuclear factor kappa beta (NFκB) activation and HIV-1 replication in T cells (IC50 ∼ 5 μg/ml and 5 μM, respectively) as well as the release of pro-inflammatory mediators such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)α, and PGE2 in lipopolysaccharide (LPS)-stimulated monocytes [8]. Nevertheless, additional molecular target(s) of arzanol may exist and its anti-inflammatory efficacy in vivo remains to be investigated. To this aim, we have investigated the activity of arzanol on the biosynthesis of prostanoids and LTs, and we have evaluated its anti-inflammatory efficacy in vitro and in vivo. We show that arzanol potently inhibits the inducible microsomal PGE2 synthase (mPGES)-1 (EC 5.3.99.3), COX-1, and 5-LO in various test systems with IC50 values between 0.4 and 9 μM, and significantly reduces the inflammatory response and the PGE2 levels in the carrageenan-induced pleurisy in rats. These observations validate arzanol as a novel anti-inflammatory chemotype worth further development.
Section snippets
Reagents
Arzanol was isolated from H. italicum as described [8], dissolved in dimethyl sulfoxide (DMSO) and kept in the dark at −20 °C, and freezing/thawing cycles were kept to a minimum. Arzanol was found to be stable in neutral medium in polar solvents. Thus, after 96 h at room temperature in DMSO, methanol or acetone, no degradation could be observed (NMR evidence). For animal studies, arzanol was dissolved in DMSO and diluted with saline achieving a final DMSO concentration of 4%. The thromboxane
Effects of arzanol on 5-LO product formation
Many plant-derived 5-LO inhibitors are lipophilic reducing agents that act by uncoupling the redox cycle of the active-site iron of 5-LO or by radical scavenging activity [5]. In agreement with the result from previous studies [12], we could confirm the anti-oxidant properties of arzanol. Thus, arzanol caused a concentration-dependent reduction of DPPH with similar efficiency as well-recognized antioxidants such as l-cysteine or ascorbic acid (IC50 approx. 20 μM, Fig. 1B). Based on the
Discussion
Arzanol was recently characterized as the major bioactive constituent of H. italicum with excellent anti-oxidant properties that inhibited the HIV-1 replication in T cells and the release of pro-inflammatory cytokines from stimulated monocytes, which was attributed to interference with the NFκB pathway [8], [12]. We now report that arzanol potently inhibits the biosynthesis of PGE2, TXB2, and LTs which are pivotal mediators of inflammatory reactions, and we identified 5-LO, COX-1, and mPGES-1
Conflict of interest statement
None declared.
Acknowledgments
The authors thank Bianca Jazzar and Daniela Mueller for expert technical assistance and we thank Aureliasan GmbH (Tuebingen, Germany) for financial support.
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