**1. Introduction**

Inflammation is a complex set of interactions among soluble factors and cells (e.g., chemokines, cytokines, adhesion molecules, recruitment, and activation of leukocytes) that can arise in any tissue helping to protect the host from systemic infection and to restore tissue homeostasis after injury, infection, and irritation [1–3]. Therefore, it represents a crucial defense mechanism that is important for maintenance of health [1,2]. However, if targeted destruction and assisted repair are not properly controlled by its mediators, the so-called "non-resolving inflammation", they can lead to persistent tissue damage and the insurgence of various pathologies [2]. Inflammation has important pathogenic roles in several pathologies, such as asthma, atherosclerosis, atopic dermatitis, Crohn's disease, multiple sclerosis, cystic fibrosis, psoriasis, neurodegenerative diseases, as well as cancer [1,4]. It is a protective response that involves immune cells, blood vessels, and different molecular mediators (e.g., TNF-α, IL1, nitric oxide, and prostaglandins) and anti-inflammatory assays that are used in the literature, generally include the study of one or more of these characteristics and mediators.

Oceans account for 71% of the earth's surface and represent a huge, relatively untapped, reservoir of new compounds for drug discovery [5]. One such source is the Phytoplankton, photosynthetic eukaryotes at the base of marine and freshwater food webs that are essential in the transfer of organic material to top consumers such as fish [6]. These micro-organisms have shorter generation times as compared with macro-organisms and can easily be cultivated in closed photobioreactors or in open ponds providing access to larger amounts of biomass necessary for an eco-sustainable and eco-friendly approach to drug discovery [7]. Diatoms, with over 100,000 species, constitute one of the major components of marine phytoplankton, comprise up to 40% of annual productivity at sea [8] and represent 25% of global carbon-fixation [9]. Different studies have shown that diatoms are excellent sources and producers of pigments, lipids, and bioactive compounds [7,10]. Anti-inflammatory properties have previously been found for various diatoms, such as *Porosira glacialis*, *Attheya longicornis* [11], *Cylindrotheca closterium*, *Odontella mobiliensis*, *Pseudonitzschia pseudodelicatissima* [12], and *Phaeodactylum tricornutum* [13]. The activity was assessed on lipopolysaccaride (LPS)-stimulated human THP-1 macrophages, except for *P. tricornutum* which was tested on murine RAW 264.7 macrophages. However, there is very little information available on the actual compounds responsible for the observed anti-inflammatory activity.

Anti-inflammatory properties have also been found for flagellates. Extracts of *Tetraselmis suecica* [14], *Chlorella ovalis*, *Nannochloropsis oculata,* and *Amphidinium carterae* [13], and a sterol-rich fraction of *Nannochloropsis oculata* [15] were active in LPS-stimulated RAW 264.7 macrophages. Oxylipin-containing lyophilized biomass from *Chlamydomonas debaryana* have been shown to have anti-inflammatory properties in an induced colitis rat model [16–18] and *Dunaliella bardawil* was found to protect against acetic acid-induced small bowel inflammation in rats [16,17]. Regarding studies on LPS-stimulated human THP-1 monocytic leukemia cells, lipid extracts of *Pavlova lutheri* [19] and monogalactosyldiacylglycerols (MGDGs) and digalactosyldiacylglycerols (DGDGs) mixtures, and the isolated DGDGs 11 and 12 from *Isochrysis galbana* [20] were reported as active. Regarding the compounds responsible for anti-inflammatory properties from flagellates, lycopene was purified from *Chlorella marina* and the activity was confirmed in a rat model of arthritis [21], and phytosterols from *Dunaliella tertiolecta* were tested in a sheep model of inflammation [22]. In addition, carotenoids, the most abundant lipid-soluble phytochemicals, have shown anti-inflammatory properties [23].

In this study, we investigated the capacity of extracts of *C. closterium* to inhibit the release of one of the main effectors of inflammation, TNF-α [3], in LPS-stimulated human THP-1 monocytic leukemia cells. Bioactivity-guided fractionation was performed, and chemical contents of the active fraction are described for the first time. This study is perfectly aligned with recent trends in analyzing possible microalgal properties for cancer prevention and improving general human health and well-being [24,25].
