Metabolites with Antioxidant Activity from Marine Macroalgae

Reactive oxygen species (ROS) attack biological molecules, such as lipids, proteins, enzymes, DNA, and RNA, causing cellular and tissue damage. Hence, the disturbance of cellular antioxidant homeostasis can lead to oxidative stress and the onset of a plethora of diseases. Macroalgae, growing in stressful conditions under intense exposure to UV radiation, have developed protective mechanisms and have been recognized as an important source of secondary metabolites and macromolecules with antioxidant activity. In parallel, the fact that many algae can be cultivated in coastal areas ensures the provision of sufficient quantities of fine chemicals and biopolymers for commercial utilization, rendering them a viable source of antioxidants. This review focuses on the progress made concerning the discovery of antioxidant compounds derived from marine macroalgae, covering the literature up to December 2020. The present report presents the antioxidant potential and biogenetic origin of 301 macroalgal metabolites, categorized according to their chemical classes, highlighting the mechanisms of antioxidative action when known.


Introduction
In all aerobic organisms, oxygen is a crucial element in their metabolic pathways. A high redox potential milieu stimulates the production of free radicals, defined as chemical species with unpaired valence electrons [1]. The most common reactive species in biological systems are oxygen radicals or oxygen-derived species, such as superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (·OH) [2,3], collectively named reactive oxygen species (ROS). Still, other forms of radicals, such as nitric oxide (NO·) and transition metal ions, can also be produced. ROS are generated as products of normal cellular functioning and oxygen metabolism and have essential functions in various important biochemical processes, such as the defense against infections, vasodilation, neurotransmission, gene regulation, and oxidative signaling [3,4].
The defense system of living organisms against free radicals comprises both enzymatic and non-enzymatic antioxidants [18]. Enzymes either prevent the formation of or neutralize free radicals (e.g., superoxide dismutases (SOD), catalases (CAT), lactoperoxidases, and glutathione peroxidases (GPx)), or indirectly neutralize free radicals by supporting the Figure 1. Causes and effects of oxidative stress (adapted from [6]).
The defense system of living organisms against free radicals comprises both enzymatic and non-enzymatic antioxidants [18]. Enzymes either prevent the formation of or neutralize free radicals (e.g., superoxide dismutases (SOD), catalases (CAT), lactoperoxidases, and glutathione peroxidases (GPx)), or indirectly neutralize free radicals by supporting the activity of other endogenous antioxidants (e.g., glutathione reductase (GR) and glucose-6-phosphate dehydrogenase) [19]. On the other hand, non-enzymatic antioxidants are compounds, other than enzymes, that act on free radicals and can be either produced by the stressed living organism or delivered through the diet, e.g., via the consumption of ascorbic acid (vitamin C), tocopherol (vitamin E), β-carotene, flavonoids, and polyphenols [20]. The most effective and extensively used strategy to diminish oxidative stress is the supplementation of exogenous antioxidants [21]. In recent years, safety and health concerns have been raised for synthetic antioxidants. Therefore, natural antioxidants have attracted attention and are being widely used [1]. Since 2007, antioxidants have been defined as "any substance that delays, prevents or removes oxidative damage to a target molecule'' [2].
Oceans, covering about 70% of Earth's surface and hosting an immense array of macro-and microorganisms, constitute a renewable resource of potential therapeutic agents. The diverse and antagonistic marine environment triggers the production of a wide variety of bioactive compounds. Marine organisms have adapted remarkably to extreme environmental conditions, such as high salinity, low or high temperature, high pres- Oceans, covering about 70% of Earth's surface and hosting an immense array of macro-and microorganisms, constitute a renewable resource of potential therapeutic agents. The diverse and antagonistic marine environment triggers the production of a wide variety of bioactive compounds. Marine organisms have adapted remarkably to extreme environmental conditions, such as high salinity, low or high temperature, high pressure, low availability of nutrients, and low or high exposure to sunlight [22], and can, therefore, provide an outstanding reservoir of bioactive compounds, many of which are unprecedented in terrestrial organisms [23][24][25][26][27].
Marine algae constitute a rich source of structurally diverse natural products, often exhibiting significant biological activities [28,29]. Algae are growing in ecosystems with intense exposure to sunlight and high concentrations of oxygen, conditions that favor the production of free radicals. However, the absence of oxidative damage in structural fatty acid membranes suggests that these organisms synthesize compounds with antioxidant activity [30]. In recent years, several studies highlight the antioxidant potential of seaweeds, attributed to natural products belonging to different structural classes [31][32][33][34][35][36]. Table 1. A list of the most commonly used in vitro assays for the determination of antioxidant activity (adapted from [50]).

Phenolic Compounds
Phenols comprise a class of chemical compounds containing an aromatic ring bearing a hydroxyl group. Phenolic compounds are classified either as simple phenols or polyphenols based on the number of phenol units in their molecule. Bromophenols (BPs) are marine secondary metabolites containing one or several phenols with one or more bromine atoms in their molecule. Many BPs have been isolated and identified from a variety of marine species, including red, brown, and green algae, as well as ascidians and sponges [73]. Phlorotannins constitute another important and diverse group of naturally occurring polyphenolic secondary metabolites, restricted though to marine algae. Table 2 presents the phenolic compounds, including BPs, phlorotannins, and flavonoids (Figures 2-8), isolated so far from marine macroalgae that exhibit significant antioxidant activities.       Recent studies reveal BPs to be one of the most promising candidates in the prevention of diseases associated with free radical attack [73]. Hitherto, more than 60 BPs, mainly isolated from marine red algae, have been reported to exert antioxidant activity in vitro. Their antioxidant activity has been primarily determined by the DPPH radical scavenging method. In general, the BPs shown in Table 2 exhibited better activity than that of butylated hydroxytoluene (BHT, IC 50 = 82.1 µM), a synthetic antioxidant often used as positive control, with BPs isolated from the red algae Polysiphonia urceolata, Rhodomela confervoides and Symphyocladia latiuscula, as well as the green alga Avrainvillea sp. possessing the highest activities in the DPPH assay (IC 50 < 10.0 µM).
The degree of bromination does not appear to affect the antioxidant activity in a consistent manner.  (44) caused ERK inactivation and an inflammatory reaction [90]. Therefore, BBDE (44) inhibits LPS-induced inflammation by inhibiting the ROS-mediated ERK signal-ing pathway in RAW 264.7 macrophage cells and thus can be useful for the treatment of inflammatory diseases [90].
The structure-activity relationship of phlorotannins, although not fully elucidated, suggests that the hydroxyl group availability influences phlorotannins' antioxidant capacity to a far greater extent than polymerization and the size of the molecule.
Flavonoids are another important class of polyphenolic secondary metabolites often exhibiting potent antioxidant activity, found predominantly in plants and fungi, but also to a lesser degree in algae. The flavonoids acanthophorin A (88) and acanthophorin B (89), isolated from the red alga Acanthophora spicifera, were shown to exert significant antioxidant activity by preventing lipid peroxidation and inhibiting the generation of MDA in liver homogenates of rat in vitro. Compounds 88 and 89, with IC 50 values 1.0 × 10 −2 and 1.5 × 10 −2 µM, respectively, displayed almost 10,000 times higher activity than vitamin E (IC 50 = 160 µM) [124].
Moreover, the abeo-oleanenes 110 and 111 were isolated from the red alga Gracilaria salicornia and their antioxidant activity was evaluated employing the DPPH and ABTS + radical scavenging assays [138]. Compound 110 exhibited higher radical scavenging activities (DPPH IC 50  Fucosterol (104), frequently isolated from brown algae, was confirmed to exert antioxidant activity on hepatic cells via an increase in the hepatic levels of GSH and a decrease in ROS production, therefore preventing hepatic damage and the resultant increase in alanine transaminase and aspartate transaminase activities [136]. Hence, fucosterol is considered an effective hepatoprotective agent that could be useful for preventive therapies against oxidative stress-related hepatotoxicity. Among terpenoids, carotenoids, a family of lipophilic pigments synthesized by plants, algae, fungi, and microorganisms, but not animals, exhibit high levels of antioxidant activity. In red, brown, and green algae, carotenoids play a key role in their protection against photo-oxidative processes [6]. Their antioxidant action is based on their singlet oxygen quenching properties and their free radicals scavenging ability, which mainly depends on the number of conjugated double bonds, the nature of substituents and the end groups of the carotenoids [6].
Specifically, astaxanthin (116) activates the phosphatidylinositol 3-kinase (PI3K)/Akt and ERK signaling pathways, and thus facilitates the dissociation and nuclear translocation of Nrf2, which leads to upregulation of the expression of Nrf2-regulated enzymes (e.g., HO-1, NQO-1, and GST-α1) [147]. Astaxanthin (116) inhibits the production of intracellular ROS by negatively regulating the Sp1/NR1 signaling pathway [149,150] and modulating the expression of oxidative stress-responsive enzymes, such as HO-1, which is a marker of oxidative stress and a regulatory mechanism involved in cell adaptation against oxidative damage [143]. In addition, astaxanthin activates the Nrf2/HO-1 antioxidant pathway by generating small amounts of ROS [145,146]. In agreement with these studies, Xue et al.
(2017) observed that astaxanthin upregulated Nrf2 expression, as well as Nrf2-targeted proteins HO-1 and antioxidative enzymes SOD2, CAT, and GPx1 in irradiated cells [151]. Thus, astaxanthin (116) exerts noteworthy antioxidant activities via both direct radical scavenging, and activation of the cellular antioxidant defense system through modulation of the Nrf2 pathway. Furthermore, a recent study in a rat deep-burn model demonstrated astaxanthin's protective role in early burn-wound progression by controlling ROS-induced oxidative stress. In that case, the regulation of free radical production is due to the influence of xanthine oxidase and the reduced form of nicotinamide adenine dinucleotide phosphate oxidase, both contributing to the generation of ROS [144]. Moreover, the abeo-oleanenes 110 and 111 were isolated from the red alga Gracilaria salicornia and their antioxidant activity was evaluated employing the DPPH and ABTS + radical scavenging assays [138]. Compound 110 exhibited higher radical scavenging activities (DPPH IC50 = 1.33 mM; ABTS + IC50 = 1.09 mM), when compared to those displayed by compound 111 (DPPH IC50 = 1.56 mM; ABTS + IC50 = 1.24 mM) and α-tocopherol that was used as positive control (DPPH IC50 = 1.46 mM; ABTS + IC50 = 1.72 mM).
Among terpenoids, carotenoids, a family of lipophilic pigments synthesized by plants, algae, fungi, and microorganisms, but not animals, exhibit high levels of antioxidant activity. In red, brown, and green algae, carotenoids play a key role in their protection against photo-oxidative processes [6]. Their antioxidant action is based on their singlet oxygen quenching properties and their free radicals scavenging ability, which mainly depends on the number of conjugated double bonds, the nature of substituents and the end groups of the carotenoids [6].
The study of Taira et al. (2017) demonstrated that fucoxanthin (118), through the Nrf2 activation, exerts either cytoprotective activity or induction of apoptosis, depending on the concentrations employed [153]. At a low concentration range (1-4 µM), fucoxanthin provides a cytoprotective effect due to its antioxidant activity, as exerted by its peroxyl radical scavenging capacity, involving the antioxidant HO-1 protein expression increase through the activation of the Nrf2/ARE pathway. On the other hand, high concentration (>10µM) treatment of cells induces apoptosis with caspase -3/7 activation during the suppression of anti-apoptotic proteins, such as Bcl-xL and pAkt.
Besides, the cytoprotective effect of fucoxanthin (118) has been investigated against H 2 O 2 -induced cell damage [154,158]. It was shown that fucoxanthin effectively inhibited intracellular ROS formation, DNA damage, and apoptosis induced by H 2 O 2 . Finally, the protective effect of fucoxanthin was investigated against UVB-induced cell injury in human fibroblasts and showed significant decrease in intracellular ROS formation and increase in cell survival rate in a dose-dependent manner [155].
Comparative studies of the radical scavenging efficiency of fucoxanthin (118) and its stereoisomers (119-121) isolated from Laminaria japonica have also been conducted [162]. All three stereoisomers had stronger hydroxyl radical scavenging activities than α-tocopherol but showed weaker scavenging activities toward DPPH and superoxide radical, while their radical scavenging activities were not remarkably different, indicating that the differences in the geometry of the double bonds had very little effect on their activity.

Meroterpenoids
Meroterpenoids are natural products of mixed biosynthesis containing a terpenoid part that exhibit a variety of biological activities. Metabolites belonging to this class that display antioxidant activity have been isolated from various macroalgae ( Table 4, Figures 13-19), the majority of which belong to the phylum Ochrophyta, and especially to the genera Cystoseira and Sargassum.       Overall, meroterpenoids from marine macroalgae have exhibited moderate to remarkable antioxidant activity. Specifically, the brominated compound cymopol (125), isolated from the green alga Cymopolia barbata, exerted noticeably high DPPH scavenging activity with an IC 50 (159), and 11-hydroxyamentadione (160), which exhibited antioxidant activity in the ABTS assay in the range of 77-115% compared to Trolox that was used as a standard.  (127, 128, 146-155), isolated from the brown alga Cystoseira crinita, that showed very high levels of radical scavenging at a concentration of 230 μM (92.5-96.7% as compared to 95.2% scavenging for α-tocopherol) [177]. In contrast, the co-occurring quinones 197 and 198 showed DPPH radical scavenging activities significantly less than that of α-tocopherol and the hydroquinones, but still comparable to that of BHT, i.e., 29.0% for 197 and 38.6% for 198 as compared to 35.6% scavenging observed for BHT at a concentration of 230 μM. the other hand, in the TBARS assay, potent inhibition of linolenic acid methyl ester peroxidation was observed for all hydroquinones, i.e., 66.5-74.9% inhibition for compounds 127, 128, and 146-155 at a concentration of 164 μM. These activities were comparable to those of α-tocopherol (72.7%) and BHT (69.3%). Additionally, these compounds showed activities between 13% (153) and 59% (149) of α-tocopherol in the TEAC test and between 40% (152) and 112% (198) of α-tocopherol in the PCL assay [177].        (127, 128, 146-155), isolated from the brown alga Cystoseira crinita, that showed very high levels of radical scavenging at a concentration of 230 µM (92.5-96.7% as compared to 95.2% scavenging for α-tocopherol) [177]. In contrast, the co-occurring quinones 197 and 198 showed DPPH radical scavenging activities significantly less than that of α-tocopherol and the hydroquinones, but still comparable to that of BHT, i.e., 29.0% for 197 and 38.6% for 198 as compared to 35.6% scavenging observed for BHT at a concentration of 230 µM. The observed differences in the values obtained in the DPPH assay for the tested compounds were attributed to the existence of small impurities in the samples (e.g., due to autoxidation) and the handling of small amounts rather than to structural variations. On the other hand, in the TBARS assay, potent inhibition of linolenic acid methyl ester peroxidation was observed for all hydroquinones, i.e., 66.5-74.9% inhibition for compounds 127, 128, and 146-155 at a concentration of 164 µM. These activities were comparable to those of αtocopherol (72.7%) and BHT (69.3%). Additionally, these compounds showed activities between 13% (153) and 59% (149) of α-tocopherol in the TEAC test and between 40% (152) and 112% (198) of α-tocopherol in the PCL assay [177].
In an effort to elucidate the mechanism of antioxidant activity of zonarol (134), Shimizu et al. (2015) studied its effect on neuronal cells and proved that zonarol protects them from oxidative stress by activating the Nrf2/ARE pathway and inducing phase-2 enzymes [180].
Moreover,  elucidated the role of sargachromanol G (208), isolated from the brown alga S. siliquastrum, in receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation [193]. Compound 208 was found to inhibit RANKL-induced osteoclast differentiation from RAW264.7 cells without signs of cytotoxicity. Additionally, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase 9 (MMP9), and calcitonin receptor (CTR), was also strongly inhibited. It was concluded that sargachromanol G inhibits RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α protein degradation, and therefore the phosphorylation of mitogen activated protein kinases (p38, JNK, and ERK).   , respectively) indicated the pivotal role of the second free hydroxyl group in the phenol ring for enhanced radical scavenging activity. Along this trend, the absence of a free phenolic hydroxyl group resulted in lack of scavenging activity [187].
Another investigation conducted by Jang et al. (2005) reported the isolation of meroterpenoids belonging to the subclasses of chromenes and chromenols (201, 202, 204-212, 218,  221-224) from the brown alga S. siliquastrum that exhibited over 87% radical scavenging activity at a concentration of 0.23 to 0.29 mM (0.1 mg/mL) [191]. Moreover, the antioxidant activity of compounds 205, 206, and 209, along with that of 203, 216, and 217, was evaluated in various assays, including scavenging effects on the generation of intracellular ROS, increments of intracellular GSH levels, and inhibitory effects on lipid peroxidation in human fibrosarcoma HT 1080 cells [192]. All tested compounds significantly decreased the generation of intracellular ROS, while increasing the levels of intracellular GSH at a concentration of 5 µg/mL, and inhibited H 2 O 2 -induced lipid peroxidation at a concentration of 50 µg/mL.
In an effort to elucidate the mechanism of antioxidant activity of zonarol (134), Shimizu et al. (2015) studied its effect on neuronal cells and proved that zonarol protects them from oxidative stress by activating the Nrf2/ARE pathway and inducing phase-2 enzymes [180].
In an effort to elucidate the mechanism of action of dictyospiromide (231), neuronlike PC12 cells were treated with H 2 O 2 , and its cytoprotective effect against the induced oxidative damage was evaluated [199]. Treatment with dictyospiromide increased cell survival in a dose-dependent manner and reduced H 2 O 2 -induced lactate dehydrogenase (LDH) production at a concentration as low as 0.5 µM. Additionally, compound 231 was investigated regarding its implication in the Nrf2/ARE signaling pathway, which regulates the expression of genes involved in cellular antioxidant defense. It was found that dictyospiromide (231) exhibited a cytoprotective antioxidant effect in PC12 cells that involved activation of the Nrf2/ARE signaling pathway and enhanced expression of HO-1. The characteristic pigments of the light harvesting proteins phycoerythrobilin (239), pheophorbide a (240), chlorophyll β (241) and pyropheophytin α (242) have been found to exert antioxidant activity [141,[201][202][203][204][205][206]. It seems that the porphyrin ring system is important for the expression of antioxidative activity in the dark. Indeed, phycoerythrobilin (239) showed potent antioxidant activity in in vitro experiments and significantly inhibited the release of β-hexosaminidase in rat basophilic leukemia cells [207], suggesting that phycoerythrobilin exhibits anti-inflammatory activity. Pheophorbide a (240) demonstrated antioxidant activity (88.6 ± 1.3% DPPH scavenging) higher than that of α-tocopherol, and comparable to that of butylated hydroxyanisol (BHA, 85.3 ± 0.2% DPPH scavenging) at a concentration of 0.1 mg/mL [202], while pyropheophytin α (242) demonstrated antioxidant activity higher than that of α-tocopherol [206].

Carbohydrates and Polysaccharides
Carbohydrates ranging in size from simple monosaccharides to high molecular weight polysaccharides isolated from marine macroalgae often exert antioxidant activities [208,209] (Table 6, Figure 22).  Chlorophylls are natural pigments with a well-known antioxidant activity. Although their radical scavenging activities are reported to be low [203], their inhibitory action in lipid peroxidation was found to be 95% at concentrations as low as 100 µM [204]. However, knowledge is limited regarding the yield of chlorophyll metabolites, their absorption and transportation processes, their metabolic pathways, and their precise oxidation mechanisms. At the in vitro level, only few researchers have studied the stability of chlorophylls during digestion and subsequent absorption through intestinal cells. The major outcome is that chlorophylls α and β are transformed into their corresponding pheophorbides and pheophytins and are absorbed at similar rates to those of carotenoids. Further, it has been shown that pheophorbide a is transported at the intestinal level by a protein-mediated mechanism, with scavenger receptor class B type 1 (SR-BI) being a plausible transporter. These results have been confirmed at the in vivo level, using mice as the experimental model, showing a preferential accumulation of pheophorbide in the liver along with multiple other chlorophyll compounds [205].

Carbohydrates and Polysaccharides
Carbohydrates ranging in size from simple monosaccharides to high molecular weight polysaccharides isolated from marine macroalgae often exert antioxidant activities [208,209] (Table 6, Figure 22).    The simplest sugar alcohol isolated from a plethora of macroalgae is mannitol (243), representing up to 9%, 47%, and 59% of the dry algal weight in Chlorophyta, Rhodophyta and Ochrophyta, respectively [210]. Antioxidant activity evaluation by enzymes (αglucosidase, acetyl (AChE) and butyrylcholinesterase (BuChE)) and free radicals (DPPH, NO, OH, and O 2 − ) revealed that higher contents of mannitol are closely related with cholinesterases and DPPH radical scavenging, and to a lesser extent are responsible for α-glucosidase inhibition, OH, O 2 − , and NO scavenging. Two simple glucosides, floridoside (244) and D-isofloridoside (245), have been isolated from the red alga Laurencia undulata and their free radical scavenging activity, inhibition of intracellular ROS levels, the level of membrane protein oxidation, myeloperoxidase (MPO) activity inhibition, gene expression levels of GSH and SOD, and protein expression of MMP2 and MMP9 have been determined [211]. It was found that both floridoside (244) and D-isofloridoside (245) possess significant antioxidant capacity and are potential inhibitors of MMP2 and MMP9.
Marine macroalgae are the most important source of non-animal sulfated polysaccharides (SPs), with the main categories being fucoidans isolated from brown algae, carrageenans and porphyrans isolated from red algae and ulvans isolated from green algae. SPs possess excellent in vitro antioxidant activity, including both radical scavenging capacity and metal chelating ability [212,227,228]. The antioxidant activity of SPs directly related to their structural features, such as degree of sulfation, molecular weight (MW), type of major sugar, and glycosidic branching [212,225,229]. For example, low MW SPs have shown potent antioxidant activity, stronger than that of high MW SPs [230]. The rationale for this is that low MW SPs may be incorporated into the cells more efficiently and donate proton effectively compared to high MW SPs.
Alginate oligosaccharide (AO, 246) and fucoidan oligosaccharide (FO, 247) were enzymatically produced from commercially available polysaccharides and their antioxidant activity was studied [212]. AO (246) had the highest hydroxyl radical scavenging activity as compared to FO (247), while in the Fe 2+ chelation assay, FO exhibited good chelation in contrast to AO that hardly displayed any activity.
Fucoidans of diverse MW and sulfation degree (247-264) have been isolated from various brown algae and/or chemically modified and their antioxidant activity has been tested employing OH and O 2 − scavenging, erythrocyte hemolysis inhibition, metal chelation, and anti-lipid peroxidation assays [212][213][214][215]. In the study of Zhao et al. (2008) two fractions of different MW, namely 742 kDa (254) and 175.9 kDa (255), were obtained from fucoidans extracted from L. japonica and evaluated for their OH and O 2 − scavenging activity, with the higher MW fraction exhibiting higher levels of activity [215]. Following radical process degradation, an ascophyllan-like fraction rich in glucuronic acid and a fraction rich in galactose and mannose were confirmed as responsible for the oxygen free radical scavenging activity [215]. On the contrary, Koh et al. (2019) reported on the higher antioxidant capacity of low MW (10 kDa) fucoidan (256) from Undaria pinnatifida (close to that of BHA) as compared to a high MW (300 kDa) fucoidan (257) [216].
Two simple glucosides, floridoside (244) and D-isofloridoside (245), have been isolated from the red alga Laurencia undulata and their free radical scavenging activity, inhibition of intracellular ROS levels, the level of membrane protein oxidation, myeloperoxidase (MPO) activity inhibition, gene expression levels of GSH and SOD, and protein expression of MMP2 and MMP9 have been determined [211]. It was found that both floridoside (244) and D-isofloridoside (245) possess significant antioxidant capacity and are potential inhibitors of MMP2 and MMP9.   Additionally, Rodriguez-Jasso et al. (2014) isolated fucose-containing sulfated polysaccharides from Fucus vesiculosus using either microwave-assisted extraction (258) or autohydrolysis (259) and their antioxidant activity was determined [217]. Both samples presented similar sulfate contents (~21%), as well as comparable antioxidant potential as evaluated by DPPH and ABTS + scavenging, and lipid oxidation inhibition methods. Differences in the antioxidant potential could be observed only when using a differential pulse voltammetry technique, pointing to structural variations of the fucans obtained by the two different methods.
Several studies have reported the in vitro and in vivo antioxidant efficacy of fucoidan [231].  have demonstrated that low MW fucoidan (262) might block NO, as well as ROS production, suppressing therefore oxidative stress and MAPKs in RAW264.7 cells [220]. Additionally, fucoidan (263) was found to reduce the oxidative stress through Nrf2/ERK signaling mediated regulation of HO-1 and SOD1 expression in human keratinocytes [221]. More recently,  have demonstrated that fucoidans derived from U. pinnatifida (264) exhibit significant in vitro and in vivo anti-arthritic responses in rabbit articular chondrocytes and rats, respectively. Moreover, administration of fucoidan to arthritic rats ameliorated the clinical symptoms and led to the overall improvement of their health [222].
Rocha de Souza et al. (2007) reported on the isolation of iota (ι)-(265), kappa (κ)-(266), and lambda (λ)-(267) carrageenans from various red algae and their antioxidant activity as evaluated by the scavenging of OH and O 2 − radicals, and lipid peroxidation assays [213]. The results of the study indicated that, among the different carrageenans, λ-carrageenan (267) exhibited the highest antioxidant and free radical scavenging activity. Thus, a positive correlation between sulfate content and antioxidant activity was evidenced.
Acetylation, phosphorylation and benzoylation of porphyran (268) extracted from the red alga Porphyra haitanensis afforded derivatives with improved antioxidant activity, as evaluated in superoxide radical, hydroxyl radical and reducing power assays [223]. In a previous study,  obtained through anion-exchange column chromatography three sulfated polysaccharide fractions with variable sulfate content (17.4%, 20.5% and 33.5%) from the same red algal species and investigated their in vitro antioxidant activities [229]. All three showed strong scavenging effect on superoxide radical and much weaker effect on hydroxyl free radical, while lipid peroxide in the rat liver microsome was significantly inhibited. In two subsequent studies the fractions with sulfate contents 17.4% and 20.5% were evaluated in vivo in aging mice [48,49]. In both cases, intraperitoneal administration significantly decreased lipid peroxidation in a dose-dependent manner, while at the same time increasing total antioxidant capacity and the activity of SOD and GPx in all organs of the aging mice.
Ulvans of diverse sulfation degree and MW (269-271) have been isolated from the green alga Ulva pertusa and/or chemically modified and their antioxidant activity was tested employing OH and O 2 − radical scavenging, reducing power and metal chelating assays [224][225][226]. Specifically,  extracted ulvan (269) with 19.5% sulfate content and chemically prepared derivatives of higher sulfate content ranging from 23.5% to 32.8%. Upon evaluation of their O 2 − radical and OH radical scavenging activity, it was observed that the derivatives displayed higher levels of activity, ranging from 91.7% to 95.5% at a concentration as low as 23.0 µg/mL for O 2 − radical scavenging and with IC 50 values ranging from 0.46 to 1.43 mg/mL for OH radical scavenging [224].
In another study,  initially extracted ulvan (270) from U. pertusa, and subsequently, three derivatives of different MW were prepared by H 2 O 2 degradation and their antioxidant activities, including OH and O 2 − radical scavenging activity, reducing power and metal chelating ability, were investigated [225]. The MW of the natural and degraded ulvans were calculated at 151.7, 28.2, 58.0, and 64.5, kDa, respectively. All polysaccharides exhibited significant OH and O 2 − radical scavenging capacity at all concentrations tested with similar IC 50 values at about >1 mg/mL and 22.1 µg/mL, respectively. Among the natural ulvan and the obtained derivatives, the lowest MW one showed the strongest reducing power and metal chelating ability. The results indicated that MW had a significant effect on the antioxidant activity of ulvan, with low MW ulvan exerting the strongest antioxidant activity. In a further study, Qi et al. (2006) prepared derivatives of ulvan (262) after acetylation and benzoylation, which exhibited higher levels of antioxidant activity, as determined using in vitro assays, including scavenging activity against superoxide and hydroxyl radicals, reducing power, and chelating ability [226].

Conclusions
The marine environment harbors diverse biological species that can provide a vast repertoire of molecules with therapeutic properties. Forced to tolerate extreme environmental conditions, marine organisms produce structurally unique molecules as an adaptive strategy to survive in their biotopes. In particular, macroalgae contain a plethora of antioxidative compounds, such as bromophenols, phlorotannins, pigments, terpenoids, and polysaccharides, in order to protect themselves from free radicals, the production of which is favored in sublittoral zones with intense exposure to sunlight and high concentrations of oxygen.
Structural elements, such as the number of phenol rings, the number of free hydroxyl groups and conjugated systems, are in general accepted as enhancing the antioxidant activity observed. Among the metabolites presented in the current review, the most active belong to the classes of phenols and polyphenols, as well as meroterpenoids, with bromophenols and phlorotannins exerting the highest activities. In particular, the bromophenol rhodomelin A (18) isolated from the red alga R. confervoides, the phlorotannins fucodiphloroethol G (77), phlorofucofuroeckol-A (79), 974-B (83), and 2,7"-phloroglucinol-6,6 -bieckol (84) purified from brown seaweeds especially of the genus Ecklonia, as well as the meroterpenoids 174, 175, and 178-183 isolated from brown algae of the genus Sargassum exerted noticeably high DPPH scavenging activity.
Nevertheless, the most studied antioxidant compounds are the natural pigments astaxanthin (116) and fucoxanthin (118), belonging to the class of carotenoids, ubiquitous in marine macroalgae. Their antioxidant action is based on their singlet oxygen quenching properties and their free radicals scavenging ability, which mainly depends on the number of conjugated double bonds and end groups. The antioxidant activity of fucoxanthin (118) has also been evaluated in vivo. Dietary intake of fucoxanthin significantly reduced lipid hydroperoxide levels of liver and abdominal white adipose tissue of obese/diabetes KK-A y mice [243]. Fucoxanthin supplementation also significantly reduced the blood glucose level and hepatic lipid contents of the mice. Promising results were also observed in experiments on rats fed a high fat diet supplemented with fucoxanthin that improved the antioxidant capacity, depleted by a high fat diet, by activating the Nrf2 pathway and its downstream target gene NQO1 [244]. Therefore, supplementation of the diet with fucoxanthin, especially of those who consume high fat in their diet, may benefit them by reducing the risk of oxidative stress.
Although emerging evidence points to a diversity of actions and effects, which are intricate and independent from any antioxidant chemical nature, there is an urgent need