**2. Results and Discussion**

#### *2.1. Testing for Anti-Inflammatory Activity in Algal Fractions*

Previous studies have shown that raw extracts of the diatom *C. closterium* had anti-inflammatory properties [12]. In the present study, a raw extract of *C. closterium* was pre-fractionated to obtain five fractions (Fractions A to E). These were amino acids and saccharides rich fraction (named Fraction A), nucleosides rich fraction (named fraction B), glycol- and phospholipid rich fraction (named fraction C), free fatty acids and sterols rich fraction (named fraction D), and triglycerides rich fraction (named fraction E), as reported in the solid phase extraction (SPE) protocol to fractionate organic extracts of Cutignano et al. [26]. Bioactivity testing of these fractions identified fraction C as the most active, able to inhibit TNF-α release at 100 μg/mL and 50 μg/mL concentrations (Figure 1). In particular, at 100 μg/mL, fraction C showed 60% inhibition of TNF-α release (*p* < 0.01), and 40% inhibition at 50 μg/mL (*p* < 0.001). Fraction D showed almost 40% TNF-α inhibition at 100 μg/mL (*p* < 0.01) and 30% at 50 μg/mL (*p* < 0.001). The other fractions did not show any significant TNF-α inhibition activity (*p* > 0.05). Both fractions C and D were selected for dereplication and further characterization.

**Figure 1.** Anti-inflammatory assay. Inhibition of TNF-α secretion from LPS-stimulated THP-1 cells treated with fractions A, B, C, D, and E of *Cylindrotheca closterium* extracts (*n* = 3, \*\* for *p* < 0.01 and \*\*\* for *p* < 0.001, Student's *t*-test).

#### *2.2. Anti-Proliferative Activity Assay*

In order to test if the active anti-inflammatory fractions of *C. closterium* also had antiproliferative activities, the 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed. In particular, A549, A2058, and HepG2 cells were incubated in the presence or in the absence of three different concentrations (1, 10, and 100 μg/mL) of both fractions C and D. After 72 h of incubation at 37 ◦C, cell survival was measured with the MTT assay. As shown in Figure 2, fractions C and D did not show any significant inhibition of cell proliferation (*p* > 0.05). These results suggest that these two fractions have no antiproliferative or cytotoxicity activity but specific anti-inflammatory activity.

**Figure 2.** Antiproliferative assay. The histograms show the antiproliferative effects of fractions C and D of *C. closterium* extracts, on A549, A2058, and HepG2 cell lines. Control sample, containing only DMSO, was also tested (named as control). Results are expressed as percent survival after 72 h exposure (*n* = 3).

#### *2.3. Dereplication*

Since isolation of new compounds is very time consuming and costly [27,28], it is important to perform an early dereplication to identify already known components. In order to identify the bioactive compounds in the active fractions C and D, they were analyzed by UHPLC-HR-MS/MS and compared to the inactive fractions A and B (Figure 3). In fraction C, we found a series of compounds that all had a common fragment at *m*/*z* 184.0740 corresponding to a molecular formula of C5H15NO4P (see Figure S1, Supplementary Information). This is a common fragment observed when the head group of phosphocholines is cleaved off in tandem mass spectrometry. Phosphocholines are a class of phospholipids where the phosphocholine head group can be esterified to one or two fatty acids. Phosphocholines with two fatty acids are common membrane-forming phospholipids known as phosphatidylcholines (PC). When one fatty acid is removed from a PC, either enzymatically or by spontaneous hydrolysis, lysophosphatidylcholines (LysoPCs) are formed. After calculating the elemental compositions of the related molecules in fraction C and searching the ChemSpider database for known compounds, they were all identified as LysoPCs with different fatty acids attached. From the UHPLC-HR-MS/MS data we were able to determine the length of the fatty acids and the degree of unsaturation, but we were not able to directly determine the position of any double bonds and if the fatty acid was attached to carbon one or two on the glycerol backbone. In order to confirm our identification of LysoPCs, we injected a commercial standard of a C16:0 LysoPC (1-palmiotyl-sn-glycero-3-phosphocholine). The standard had the same retention time, mass, collisional cross section, and fragmentation pattern as one of the most intense compounds in fraction C (see Figure S2 and S3, Supplementary Information). The dominating LysoPCs in fraction C were 16:0, 16:1, 18:1, and 18:2 (approximately equal amounts) and minor LysoPCs were 14:0 and 18:3 (each approximately 20% of the most intense LysoPCs). A summary of the most intense LysoPCs and their retention times is given in Table S1 (Supplementary Information). There is current interest in LysoPCs because some of these are proposed for treatment of systemic inflammatory disorders [29–35]. However, their biological roles are not completely understood and some studies even found a putative pro-inflammatory activity [29]. Plasma LysoPC levels are diminished in human patients with sepsis [31,36], and in rodent models of sepsis and ischemia, LysoPC treatments in ex vivo and in vivo studies suggesting a potential role to relieve serious inflammatory conditions [29]. LysoPCs have also been shown to prevent neuronal death both in an in vivo model of transient global ischemia and in an in vitro model of excitotoxicity using primary cultures of cerebellar granule cells exposed to high extracellular concentrations of glutamate (20 to 40 micromol/L).

In fraction D, trace amounts of the same LysoPCs were present. However, the most intense peak in fraction D had a *m*/*z* value of 593.2752 with a corresponding elemental composition of C35H37N4O5 ([M + H]+). When searching in the ChemSpider database, the elemental composition, as well as the fragmentation pattern, indicated that the compound was pheophorbide a, a breakdown product of chlorophyll (see Figure S4, Supplementary Information). Another peak in fraction D was identified as a related breakdown product of chlorophyll, hydroxypheophorbide a (C35H36N4O6, *m*/*z* 609.2708 [M + H]+), see Figure S5 (Supplementary Information). Both pheophorbide a and its derivatives are known to have anti-inflammatory and anticancer properties [37–43], but, to our knowledge, this is the first case in a marine microalgae where the bioactivity was attributed to pheophorbide a. Pheophorbide a has already been extracted from a range of different marine organisms. Examples are the seaweed *Grateloupia ellittica* [40], the brown alga *Saccharina japonica* [39], marine diatoms [44,45], and the freshwater glaucophyte *Cyanophora paradoxa* [37]. Hydroxypheophorbide a has been previously isolated from the terrestrial plants *Clerodendrum calamitosum*, *Neptunia oleracea*, the freshwater unicellular green alga *Chlorella* sp., and from the marine tunicate *Trididemnum solidum*, but never from a marine diatom species. It is mainly known to have anticancer but not anti-inflammatory activity ([38,41], Patents No. 185220/82 and US4709022A). Hence we suggest that the possible anti-inflammatory activity observed in our experiments was due to the presence of LysoPCs and the known anti-inflammatory pheophorbide a. In fact, pheophorbide a is known to induce a dose-dependent inhibition against lipopolysaccharide (LPS)-induced nitric oxide (NO) production at nontoxic concentrations in RAW 264.7 murine macrophage cells and to suppress the expression of nitric oxide synthase (iNOS) [39].

**Figure 3.** Base peak intensity chromatograms of fraction A, B, C, and D from the UHPLC-HR-MS/MS analysis using positive electrospray.

#### *2.4. Anti-Inflammatory Activity of 1-Palmitoyl-sn-glycero-3-phosphocholine*

Considering that the most active fraction mainly contained various phosphocholines, we tested one of these, 1-palmitoyl-sn-glycero-3-phosphocholine (which was the most abundant compound in fraction C) in our AIF-assay. The effect of 1-palmitoyl-sn-glycero-3-phosphocholine on secretion of TNF-α showed a dose-response relationship and was active at 25 μg/mL (*p* < 0.05) and 50 μg/mL (*p* < 0.01), as shown in Figure 4.

**Figure 4.** Anti-inflammatory assay. Inhibition of TNF-α secretion from LPS-stimulated THP-1 cells treated with 3.13, 6.25, 12.5, 25, and 50 μg/mL of 1-Palmitoyl-sn-glycero-3-phosphocholine (*n* = 3; \* for *p* < 0.05 and \*\* for *p* < 0.01, Student's *t*-test).

#### **3. Conclusions**

Considering that inflammation plays a crucial role in the pathogenicity of several diseases, marine drug discovery is often directed to finding new natural products with anti-inflammatory properties [46,47]. Our results indicate that lysophosphatidylcholines (lysoPCs) and a breakdown product of chlorophyll, pheophorbide a, were probably responsible for the observed anti-inflammatory activity of the diatom *C. closterium*, giving new insights into microalgal compound bioactivities and their possible applications. This is the first time that a marine diatom is reported to produce these anti-inflammatory compounds. Pheophorbide a is known to inhibit the production of NO via inhibition of iNOS protein expression, thereby suggesting its potential use in the treatment of various inflammatory diseases (37), but there is no further information on its anti-inflammatory mechanism of action. Hence our study shows, for the first time, that it can inhibit TNF-α release in THP1 cells.

LysoPCs are products of phospholipase A2 enzyme activity, and similar to the enzyme, have a direct role in pro-inflammatory [48] and anti-inflammatory responses, in a variety of organ systems. Our results indicate that one of these LysoPCs, 1-palmitoyl-sn-glycero-3-phosphocholine (which was the most abundant compound in fraction C), had a strong anti-inflammatory activity which has not been demonstrated before. Microalgae, and in particular diatoms, therefore, can be considered potentially important producers of compounds to prevent and treat different human pathologies. In recent years they have been shown to have anti-inflammatory, antimicrobial, anticancer, antidiabetic, antiepileptic and even antituberculosis properties [10–12,49–55]. A better understanding of the potential health benefits from these marine organisms, the compounds they produce, and the environmental conditions affecting their production should allow for the sustainable development of these valuable marine resources in the future.

#### **4. Materials and Methods**

#### *4.1. Cell Culturing and Harvesting*

The diatom *Cylindrotheca closterium* (FE2), which has previously been shown to have anti-inflammatory activity [12], was cultured in Guillard's f/2 medium [56] in ten-liter polycarbonate carboys (4 replicates). Cultures were constantly bubbled with air filtered through 0.2 μm membrane filters and kept in a climate chamber at 19 ◦C and a 12:12 h light:dark cycle (100 μmol photons m−<sup>2</sup> s<sup>−</sup>1). Initial cell concentration was about 5000 cells/mL per bottle; culture growth was monitored daily by fixing 1 ml of culture with one drop of Lugol (final concentration of about 2%) and counting cells in a Bürker counting chamber under an Axioskop 2 microscope (20×) (Carl Zeiss GmbH, Jena, Germany). At the end of the stationary phase, cultures were centrifuged for 15 min at 4 ◦C at 3900 g using a cooled centrifuge with a swing-out rotor (DR 15P, Braun Biotechnology International, Allentown, PA, USA). The supernatant was discarded, and pellets freeze-dried and kept at −80 ◦C until chemical extraction.
