*Selection of Eligibility and Exclusion Criteria*

The eligibility and exclusion criteria (Figure 2) were as follows: publication type (1); matrices studied (2); and extraction method using organic solvents (3). In line with the eligibility criteria selected, only journal articles with empirical data were considered (1); only studies reporting bioactivity assays using seaweeds were considered, and studies using seaweeds and mixed were also considered (2); and studies reporting assays with extracts obtained using organic solvents (e.g., n-hexane, diethyl ether, dichloromethane, n-butanol, chloroform, ethyl acetate, acetone, ethanol, and methanol) were considered (3). The following studies were excluded: reviews, book chapters, proceeding papers, conference papers, and notes (1); studies reporting bioactivity from organisms other than seaweeds (2); and studies using water extracts (3). A total of 270 publications were considered eligible, with these subsequently being screened using the following sub-criteria: only studies identifying an isolated complex lipid group, classes, or species, or reaching a molecular structure were considered for a more in-depth analysis to assess a structure–function relationship. After applying these sub criteria, 29 publications were selected, with these being discussed in detail in Section 3.1.

**Figure 2.** Schematic review selection process performed according to PRISMA 2020 flow diagram [42].

## **3. Results and Discussion**

After applying the eligibility criteria adopted in the present work, 270 publications were considered for further analysis. These publications were evaluated taking in account the methodological approaches employed to perform bioassays, namely in vitro versus in vivo studies. Data analysis revealed that 178 publications referred to in vitro experiments, 73 to in vivo assays, and 19 included both in vitro and in vivo assays (Figure 3). It was also possible to record those in vivo assays included experimental work usually framed within two different approaches: (i) raw seaweed biomass; or (ii) organic extracts administrated intragastrical or in the diet as additives or feed supplements (Figure 3). Papers that described in vitro assays aimed to evaluate bioactive properties of organic extracts, and in some papers, complex lipids were identified or isolated. The papers that describe both in vitro and in vivo results, evaluated bioactive activities of organic extracts using in vitro assays and also the biological effects after oral administration performed mainly in animal models.

Data (270 publications) were plotted in a word cloud (Figure 4) featuring seaweed genus. This representation highlighted genera *Sargassum*, *Fucus*, *Dictyota*, and *Padina* (Ochrophyta; brown seaweeds), genera *Ulva* and *Codium* (Chlorophyta; green seaweeds), and genera *Gracilaria* (Rhodophyta; red seaweeds) as the most reported seaweeds with known bioactivities.

To assess the biological effects reported in eligible studies, data was plotted considering the most frequently prospected bioactivities in the 270 eligible publications (Figure 5). Antioxidant activity (138 studies) was the most reported bioactivity, followed by antimicrobial (61 studies), antitumor (30 studies), anti-inflammatory (19 studies) activities, fat reduction (12 studies), and growth performance (7 studies). Other bioactivities included a wide range of different actions, which was not possible to group within a specific classification. It was also possible to record that most bioactivities reported were related to antioxidant or anti-inflammatory activities; the accurate bioactivity or bioactivities reported on each of these studies are summarized in Table S1.

**Figure 3.** Number of eligible studies that recorded bioactivity on raw seaweed biomass or seaweeds organic extracts, distributed by type of performed assays (in vitro, in vivo and both in vitro and in vivo).

**Figure 4.** Word cloud assembled using the genera of seaweed species reported in the 270 eligible publications that reported bioactivity on raw seaweed biomass or seaweeds organic extracts. Genera featured with a larger size in the word cloud indicate that species within those genera were the ones mostly reported. Words in brown, green and red refer to genus within phylum Ochrophyta, Chlorophyta, and Rhodophyta, respectively (brown, green, and red seaweeds, respectively).

Data (270 publications) was also ranked based on biomass of various seaweeds, or their extracts used in the bioassays performed, being grouped in five categories: studies using seaweed as raw seaweed biomass (category 1); studies using organic extracts (category 2); studies using organic extracts with identified complex lipids (category 3); studies of extracts enriched in isolated groups or classes of complex lipids (category 4); and studies of isolated complex lipid molecular species (category 5).

In some of the selected categories (e.g., category 1 and 2) most studies did not highlight the identification of lipids, neither attributed the bioactivity reported to lipids. However, to our knowledge, the role of complex lipids in the observed bioactivity cannot be excluded. The distribution of eligible studies by category 1-5 and bioactivity assayed is summarized in Figure 6. Most studies were classified according to category 2 (177 studies), followed by category 1 (39 studies) and 3 (25 studies). Category 4 and 5 displayed a smaller number of studies (18 and 11, respectively). Category 1 included studies addressing the improvement

of growth and/or immune system/health status, fat reduction, including reduction in hyperlipidemia/cholesterolemia/triglycerides, anti-obesity/anti-adipogenic effects; antioxidant and other activities (Table S1). Studies related with categories 2 to 5 pinpoint antioxidant, antitumor, anti-inflammatory, and antimicrobial (including antibacterial, antiviral, anti-protozoal, anti-microalgal, and anti-fouling) bioactivities. It is important to highlight that several studies reported more than one single bioactivity.

**Figure 5.** Number of eligible publications that reported bioactivity on raw seaweed biomass or seaweeds organic extracts.

**Figure 6.** Ranking of eligible studies that reported bioactivity of raw seaweed or seaweeds organic extracts distributed by distinct categories.

> Antioxidant activity was most studied in categories 1 (13 studies out of 39), 2 (113 studies out of 177), and 3 (11 studies out of 25). In category 1, most studies that evaluated the antioxidant activity tested the inclusion of the raw seaweed biomass on diet, with no specification of the bioactive compound. In category 2, most studies tested organic extracts and were oriented towards phenolic compounds, which were recognized by their antioxidant properties. In category 3, the antioxidant activity was evaluated testing organic extracts with identified complex lipids, assigning the bioactivity to the whole extract

and the synergic effect between molecules. The in vitro assays of antioxidant evaluation using free radical scavenging activities were one of the bioactivities more intensively investigated, likely because of well-established and easy-to-use methodologies. However, these *in chemico* assays have limited biological relevance considering the effect in the modulation of redox homeostasis of in vivo organisms. Therefore, additional studies are still needed using in vivo models, and measuring biologically relevant biomarkers of redox homeostasis, such as catalase, and superoxide dismutase enzymes, or addressing the proper value of seaweeds lipid antioxidant bioactivities.

Antimicrobial and antitumor activities were mostly studied on categories 4 (11 studies out of 18) and 5 (5 studies out of 11), respectively. Several studies reported the antimicrobial properties of lipid extracts from seaweeds. However, the majority of the studies reported only the estimation of inhibition of bacterial growth, lacking information on the identification of the bioactive lipids promoting such response and/or elucidating the mechanism of antimicrobial action. Interestingly, some studies reported antibacterial and antiviral activity of lipid extract from specific seaweeds and activities seem to be dependent on their origin. As society urgently needs new antibiotics to overcome the current scenario of antibiotic resistance, along with powerful new antiviral drugs to face future pandemics [7], it is urgent to further explore these bioactivities in seaweeds. Concerning antitumor activity, information is also scarce and lacks key information on putative structure function relationship.

To unravel the most studied phyla of seaweeds, data (270 publications) were ranked considering how reported bioactivities were distributed over the phyla Ochrophyta, Chlorophyta, and Rhodophyta (Figure 7). Seaweed species belonging to the Ochrophyta were the most reported on antioxidant, antimicrobial, antitumor, and anti-inflammatory activities, followed by species within the Rhodophyta. Species within the Chlorophyta were the less studied.

**Figure 7.** Number of eligible studies that reported bioactivity on raw seaweed biomass or seaweeds organic extracts distributed by bioactivities and seaweed phyla.

Bioactivity distributed by phylum combined with the five categories selected in the present study is plotted in Figure 8. Seaweeds within the Ochrophyta were the most screened to evaluate antioxidant, antimicrobial, antitumor, and anti-inflammatory bioactivities on category 2–4. On the other hand, seaweeds from the Rhodophyta were the most investigated to screen for growth performance, fat reduction, and antioxidant activity over criteria 1. Although with a lower number of studies on category 5, seaweed species within the Chlorophyta and Ochrophyta appeared as the most screened for antitumor activity. Seaweed species within the Rhodophyta were the most studied for anti-inflammatory activity under category 5.

**Figure 8.** Number of eligible studies that reported bioactivity distributed over the five categories and seaweed phyla.

In most studies (Table S1), the bioactivity reported for seaweed lipids was often associated with the most abundant molecules identified in organic extracts, or with other molecules detected by the methodology used for structural characterization (e.g., fatty acid identification by Gas Chromatography–Mass Spectrometry (GC-MS)). PUFA have been frequently identified as bioactive lipids in many studies because FA identification was the only approach used for extract characterization on those publications [31,43–46]. Nevertheless, this is an inadequate approach since FA commonly exist in low amounts as free FA and they are mostly esterified in complex lipids. Other studies tested extracts obtained with organic solvents, which also extract complex lipids. However, these studies only focused on the identification of well-known phytochemicals, which are present at a lower abundance in seaweeds, such as phenolic compounds, excluding the putative role of lipids and/or the synergic effect of other lipid-soluble compounds [47–49].

Knowledge progression of natural bioactive products and their application depends on the isolation of pure molecules to achieve a possible structure–function relationship [50,51]. While this is a very laborious and time-consuming task, it is also essential to understand specific biological effects of these biomolecules. Moreover, this task will also provide a new perspective to plan chemical synthesis and subsequent applications on different fields, such as in the pharmaceutical industry, aiming to add-value to seaweeds as natural sources of bioactive compounds. To date, few studies have tried to overcome this drawback. New studies being performed on bioassays using specific groups or class of seaweed lipids are scarce; although, they are paramount to isolate molecules to address a proper clarification of structure–bioactivity relationship. These studies are detailed bellow.
