**1. Introduction**

Echinochrome A (Ech A) is a dark red pigment separated from sea urchin shell and spine and has a chemical structure of 6-ethyl-2,3,5,7,8-pentahydroxy-1,4-naphthoquinone [1,2]. As a main active component of a commercial therapeutic agent called 'histochrome', Ech A has been used for the treatment of cardiovascular disorders and ophthalmopathic complications in Russia [3–5]. Among the several biological benefits of Ech A, anti-oxidant and anti-inflammatory capacity is proposed as a major underlying therapeutic mechanism. Indeed, Ech A has been shown to attenuate the oxidative stress caused by reactive oxygen species (ROS) and cardiac toxic drugs, providing mitochondrial protection of cardiomyocyte [6]. Park et al. have reported similar observations showing that Ech A reduced both cellular and mitochondrial ROS levels of patient-derived cardiac progenitors during the oxidative stress situation [7]. The anti-oxidative and anti-viral activity of Ech A has also been proved in vitro using a tick-borne encephalitis virus and herpes simplex virus type 1-infected cell models [8]. The therapeutic potential of Ech A was also evaluated in an experimental gastric ulcer model where Ech A provided anti-ulcerogenic effects by increasing endogenous enzymatic and non-enzymatic antioxidant levels in vivo [9]. In another study, Ech A treatment could reduce ROS production and pro-inflammatory tumor necrosis factor-α (TNF-α) secretion in a rat model of acute uveitis induced by lipopolysaccharide injection [10]. These previous findings imply that Ech A could exert a wide range of therapeutic impacts on other oxidative stress-related and inflammatory pathologic conditions; however, the cell-type specific regulation of Ech A on the immune system, which consists of various innate and adaptive immune cells, has not been elucidated yet.

Inflammatory bowel disease (IBD) is an intractable, chronic inflammatory disease of the digestive tract and Crohn's disease (CD) and ulcerative colitis (UC) are the major types of IBD [11,12]. The etiology and pathogenic mechanisms of IBD remain largely unknown and both environmental factors and genetic factors combined with immunological dysfunction seem to drive IBD development. To attenuate the excessive immune response, advanced immunotherapy using immune-modulators such as inflammatory cytokine blockers has been used recently; however, the presence of non-responder and uncontrolled side effects are the common challenging issues when using immunotherapy [13,14]. Therefore, there has been an unmet need to develop novel therapeutics for the effective management of IBD.

In this study, we investigated whether Ech A could exhibit a protective role in IBD progression using a chemical colitogen dextran sodium sulfate (DSS)-induced colitis mice model. To explore the therapeutic mechanism of Ech A, we also performed in vitro proliferation and polarization experiments with two major innate and adaptive immune cells, macrophage and CD4<sup>+</sup> helper T cells (Th cells), respectively. Our in vivo findings suggest that Ech A could attenuate the clinical signs, as well as histological improvement, for the first time in a colitis model which represents IBD. More importantly, in vitro results demonstrate that the anti-inflammatory function of Ech A is manifested by, in part, inducing immunomodulatory effector cells, such as M2 macrophages and Treg cells.
