**1. Introduction**

*Tisochrysis lutea* (*T. lutea*) is a marine microalga belonging to Haptophyta, originally isolated from tropical seawater (Tahiti, French Polynesia), and currently used in aquaculture [1,2]. The presence of n-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), vitamins, proteins, and xanthophylls such as fucoxanthin [3,4], makes this microalga an interesting source of compounds with anti-inflammatory and hypolipidemic activities [5–7]. Among the marine microalgae, *Tisochrysis* contains a high amount of the pigment fucoxanthin (FX) (1.8% w/w) [8]. In several in vitro and in vivo models, FX exerts anti-inflammatory effects by inhibiting proinflammatory cytokines and enzymes [9–12]. FX also attenuates alcohol-induced oxidative lesions and inflammatory responses [13]. However, *Tisochrysis* is also a source of phenolic compounds [14] which possess a high spectrum of biological activities including antioxidant, anti-aging, and anti-inflammatory effects [15–20]. Despite the anti-inflammatory and antioxidant effects of *T. lutea* that have been mostly attributed to FX, positive pharmacodynamic synergisms among various components, acting on different targets, cannot be excluded. Indeed, superior bioactivity of either the single component or the mixture was reported in studies on natural products [21].

Cinci, L.; Niccolai, A.; Biondi, N.; Rodolfi, L.; Dos Santos Nascimiento, L.B.; Tredici, M.R.; Luceri, C. A Comparative In Vitro Evaluation of the Anti-Inflammatory Effects of a *Tisochrysis lutea* Extract and Fucoxanthin. *Mar. Drugs* **2021**, *19*, 334. https://doi.org/10.3390/md19060334

**Citation:** Bigagli, E.; D'Ambrosio, M.;

Academic Editor: Hitoshi Sashiwa

Received: 24 May 2021 Accepted: 8 June 2021 Published: 11 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The aim of this study was to perform a direct comparison between the antiinflammatory activity of a methanolic extract of *T. lutea* F&M-M36 and FX at equivalent concentrations in order to explore potential interactions among the components and pharmacological mechanisms involved. Lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophages were used as an in vitro model of inflammation.

#### **2. Results**

#### *2.1. Characterization of the T. lutea F&M-M36 Methanolic Extract*

The amount of FX in the *T. lutea* F&M-M36 methanolic extract was 4.7 mg/g dry weight. The extract was also analyzed for the total soluble phenolic content, using gallic acid as a reference. The *T. lutea* F&M-M36 methanolic extract contained 6.22 ± 0.05 mg GAE/g dry weight.

HPLC characterization showed that the *T. lutea* F&M-M36 methanolic extract contains phenolic compounds (Figure 1), with maximum absorption at 255–280 nm which eluted early in the chromatogram (retention time between 4 and 20 min, Figure 1). The spectral absorption and chromatographic behavior of these compounds are typical of simple C6 or C6-C1 phenolic skeletons, such as derivatives of hydroxybenzoic and gallic acids, as well as some aromatic amino acids. The *T. lutea* F&M-M36 methanolic extract (Figure 1) showed a little variety of phenolics, with almost all compounds showing a similar UV spectrum, compatible with the structure of simple phenolics. The putative identification was conducted based on UV-vis absorption, retention time, in comparison with standards and literature data.

**Figure 1.** Chromatograms obtained by high-performance liquid chromatography coupled to a diode array detector—HPLC-DAD (280 nm) of methanolic extract of *T. lutea* F&M-M36. Peak 1–13: phenolic acid derivatives; pick 14: catechin derivative. The putative identification was conducted based on UV-vis absorption and retention time, in comparison with standards and literature data.

#### *2.2. Effects of T. lutea F&M-M36 Methanolic Extract and FX on RAW 264.7 Macrophages Viability*

In order to evaluate the effects of *T. lutea* F&M-M36 methanolic extracts on cell viability, preliminary experiments were carried out using the MTS test. Unstimulated RAW 264.7 cells macrophages were exposed to different extract concentrations for 24 h. *T. lutea* F&M-M36 extract caused a significant reduction of cell viability (about 40%) when treatments were performed at 1 mg/mL, but was not toxic at concentrations in the range of 1–100 μg/mL (data not shown). On the basis of these results, 100 μg/mL was selected as the highest non-toxic concentration of *T. lutea* F&M-M36 extract for further analyses. FX was tested at concentrations equivalent to those measured in the microalgal extract at the same dilution (4.7–470 ng/mL).
