**1. Introduction**

Inflammatory bowel diseases (IBDs), are chronic and non-resolving intestinal inflammatory diseases, which include two clinical entities: Crohn's disease (CD) and ulcerative colitis (UC). Their etiology is far from being fully understood, although it is widely recognized that genetic, environmental, microbial, and immune factors are functionally integrated in the IBD pathogenesis [1]. A pivotal role seems to play a worsening and inappropriate mucosal immune response. In particular, NF-κB activation, production of pro-inflammatory cytokines such as IL-6 and IL-1β [2–4], myeloperoxidase (MPO), expression of cycloxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) collectively enhance inflammatory response in the colon and lead to a concomitant reduction in antioxidant levels. These molecular events could culminate in a serious damage of epithelial cell and disruption of the mucosal barrier [5,6].

Conventional IBD therapies have various side effects, and many patients do not respond to these treatments [7]. Thus, to identify effective new therapeutic options for improving colitis signs with fewer or no side effects, has becoming essential.

In recent years, attention was increased on complementary medicine approaches based on natural ingredients.

Various bioactive compounds, such as phycocyanins, carotenoids, γ-linolenic acid, fibers, and plant sterols, have been reported to clinically improve IBD-related symptoms [8]. However, the efficacy and mechanism of action of these products require further studies in vitro and in vivo models. The cyanobacteria blue-green algae (BGA), are photosynthetic prokaryotes present in aquatic ecosystems, rich in these bioactive molecules [9,10]. Recent studies highlight that their consumption is associated with ameliorative effects on the components of human metabolic syndrome [11], to the decrease of inflammation markers in hypertensive patients [10], as well as in obese patients. Moreover, immunomodulatory effects of BGA supplementation has been shown in healthy volunteers [12]. Aphanizomenon flos-aquae (AFA) also known as "Klamath algae", is as cyanobacterial dominant species growing in the Upper Klamath Lake (Oregon, USA) [13].

AFA, consumed as a "superfood", is a rich source of mycosporine-like amino acids (MAAs) and phycocyanins (PCs), likely responsible for its various health benefits. AphaMax® is a unique patented extract from AFA, manufactured by Nutrigea, containing a concentrated amount of AFA-phycocyanins and AFA-phytochrome, the Klamath microalgae components showing the greatest antioxidant, anti-inflammatory, anticancer, and cardiovascular properties. These molecules, which are unique to Klamath microalgae, increased the beneficial proprieties compared to other blue-green algae. Indeed, AFA-PCs due to their peculiar structure [14] have up to 200 times higher antioxidant power compared to other PCs [15]. AFA-PCs have neuroprotective effects and ameliorate psychological stress and on menopausal well-being [16] In addition, AphaMax® is rich in some smaller molecules, endowed with similar antioxidant properties, as many as the AFA-mycosporines (MAAs, or mycosporine-like amino acids) [17], 15 carotenoids, including zeaxanthin, lutein, canthaxanthin, a wide spectrum of polyphenols as caffeic acid, and an high content of chlorophyll.

Given the well-demonstrated anti-inflammatory and antioxidant effects of the majority of the molecules present in Klamath algae, as phycocyanins that selectively inhibit key enzymes in inflammatory disease as COX-2, and iNOS, and the high concentration of these components in AphaMax®, the aim of our study was to investigate the possible protective potential of this extract in an animal model of IBD, the 2,4-dinitrofluorobenzenesulfonic acid (DNBS) -induced rat colitis. DNBS rat colitis shares many of the pathological features of human Crohn's disease and is considered useful for studying the etiopathogenesis of IBD as well as for providing an inexpensive model useful for investigating new potential treatments [18].

#### **2. Materials and Methods**
