Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock Hwi-Ho Lee, †,‡ Ji-Sun Shin, †,§,⊥ Woo-Seok Lee, †,‡ Byeol Ryu, ‡ Dae Sik Jang,* ,‡ and Kyung-Tae Lee* ,†,‡ † Department of Pharmaceutical Biochemistry, College of Pharmacy, ‡ Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, § Reactive Oxygen Species Medical Research Center College of Pharmacy, and ⊥ Department of Physiology, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea * S Supporting Information ABSTRACT: Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) in RAW 264.7 macrophages were evaluated, and 1 (IC 50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC 50 > 60 and 46.0 μM). The suppression of NO and PGE 2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1. I nflammation is a damaging component of many human diseases, including arteriosclerosis, inflammatory bowel disease (IBD), arthritis, infectious diseases, and cancer. 1 The pathogenesis of inflammation is a complex process that is regulated by cytokine networks and many pro-inflammatory products such as nitric oxide (NO), prostaglandin E 2 (PGE 2 ), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6. NO is produced enzymatically by inducible nitric oxide synthase (iNOS) at inflammatory sites and acts as a cytotoxic agent during immune and inflammatory responses. 2 Large amounts of PGE 2 are generated during the inflammatory process due to the increased expression of cyclooxygenase-2 (COX-2) and play an important role in the regulation of inflammatory responses such as fever, pain hypersensitivity, and edema. 3,4 Cytokines like TNF-α, IL-1β, and IL-6 have been reported to be crucial mediators of inflammation during acute response to injury. 5 TNF-α is induced by a wide range of pathogenic stimuli and performs a key role during the orchestration of inflammatory responses. 6,7 IL-6 is a pivotal pro-inflammatory cytokine and a crucial checkpoint regulator of neutrophil trafficking, which is achieved by orchestrating chemokine production and leukocyte apoptosis. 8 Macrophages are the major cellular source of these factors, which, in turn, participate in the mediation of acute phase responses to injury. 5,9 Therefore, reducing activation signals in activated macrophages has been suggested as a therapeutic strategy for various inflammatory diseases. 10 Stimulation of Toll- like receptor 4 (TLR4) on macrophages by lipopolysaccharide (LPS) promotes the recruitment of myeloid differentiation factor 88/IL-1 receptor-associated kinase (IRAK)/TNF receptor- associated factor 6 (TRAF6) to the receptor’s intracellular complex and results in the activation of transforming growth factor-β-activated kinase 1 (TAK1) in macrophages. 11 Activated TAK1 subsequently induces the activation of transcription factors such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), signal transducers and activators of transcriptions (STATs), and interferon response factors (IRFs). 12,13 These Received: July 10, 2015 Article pubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.5b00609 J. Nat. Prod. XXXX, XXX, XXX-XXX