Macroalgae Specialized Metabolites: Evidence for Their Anti-Inflammatory Health Benefits

Inflammation is an organism’s response to chemical or physical injury. It is split into acute and chronic inflammation and is the last, most significant cause of death worldwide. Nowadays, according to the World Health Organization (WHO), the greatest threat to human health is chronic disease. Worldwide, three out of five people die from chronic inflammatory diseases such as stroke, chronic respiratory diseases, heart disorders, and cancer. Nowadays, anti-inflammatory drugs (steroidal and non-steroidal, enzyme inhibitors that are essential in the inflammatory process, and receptor antagonists, among others) have been considered as promising treatments to be explored. However, there remains a significant proportion of patients who show poor or incomplete responses to these treatments or experience associated severe side effects. Seaweeds represent a valuable resource of bioactive compounds associated with anti-inflammatory effects and offer great potential for the development of new anti-inflammatory drugs. This review presents an overview of specialized metabolites isolated from seaweeds with in situ and in vivo anti-inflammatory properties. Phlorotannins, carotenoids, sterols, alkaloids, and polyunsaturated fatty acids present significant anti-inflammatory effects given that some of them are involved directly or indirectly in several inflammatory pathways. The majority of the isolated compounds inhibit the pro-inflammatory mediators/cytokines. Studies have suggested an excellent selectivity of chromene nucleus towards inducible pro-inflammatory COX-2 than its constitutive isoform COX-1. Additional research is needed to understand the mechanisms of action of seaweed’s compounds in inflammation, given the production of sustainable and healthier anti-inflammatory agents.


Introduction
The seaweeds or macroalgae belong to the basic tropic level in the marine water ecosystem and are responsible, with microalgae, for the balance of the abiotic and biotic factors of marine life, either directly or indirectly [1]. Seaweeds reside in the littoral zone and are considered the oceans' principal resource in terms of economic and ecological significance [2]. Food and Agriculture Organization (FAO) data state that global seaweed output (aquaculture and wild) has increased from 2000 to 2019 nearly threefold, from 118,000 tons to 358,200 tons [3]. The world's seaweed production mainly comes from the five major continents, with Europe accounting for 0.8% of global seaweed production. In Europe, 96% of seaweed is naturally obtained, with 2010 marking the start of its cultivation. Continental Portugal and its two archipelagos (the Azores and Madeira Islands) present an exciting and diverse seaweed community mainly due to the latitudinal gradients, coast length, and climate conditions. Although Portuguese seaweeds have been relatively underexplored in terms of their economic benefits, in recent years, they are being used in various applications such as cosmetics, commercial harvest, and pharmaceuticals [4]. Many Portuguese institutions of higher education have research groups dedicated to seaweed

Specialized Metabolites with Anti-Inflammatory Activity
Macroalgae use is increasing and spreading. What was a common food ingredient in Oriental cuisine is nowadays an additive in several smart foods and folk medicine formulations. Some formulations are sold as promoters of health benefits, including antiinflammatory effects. Although several macroalgae extracts, as stated above, showed anti-inflammatory activity, their specialized metabolites must also be tested, and their amount in the formulations must be established. Moreover, in vivo studies and clinical trials are still required to validate the claimed potential in pharmaceutical formulations. Knowing the limitations of the in vitro studies, several authors are moving forward and focusing their biological assays using in vivo models. Unfortunately, anti-inflammatory clinical trials are still needed. This literature survey aims to give a critical synopsis of the current state of the art regarding the anti-inflammatory effects of essential macroalgae specialized metabolites, emphasizing their molecular mechanisms. The following sections will present and discuss specific examples chosen by the authors and consider the most promising anti-inflammatory compounds, as well as the ones for which the studies are broadening, including mechanism of action, and, if possible, in vivo studies.
In terms of in vivo assays, as far as we could find, they can be considered scarce, and more studies are needed; moreover, several aspects of the phlorotannins' mechanisms of action need to be clarified. The leukocyte adhesion of endothelial cells and transendothelial migration (TEM) of leukocytes are essential steps in the pro-inflammatory response. In in vivo assays, both compounds (2) and (3) exhibited an effectively inhibitory effect on the leakage of dye into the peritoneum in mice and decreased leukocytes count at a dose of 10 µM of concentration [47].
Phlorotannins are one of the most studied macroalgae-derived metabolites; nevertheless, their potential use as new anti-inflammatory drugs needs additional studies, such as pharmacokinetic and clinical trials.

Bromophenols
Bromophenols are phenols bearing bromine and hydroxy groups in one or more benzene rings and are amongst the specialized metabolites produced by macroalgae [48]. Actually, bromophenols are ubiquitous in the three types of macroalgae [49,50], although they were first found in red Rhodomela larix (Turner), C. Agardh, 1822, (the current accepted name is Neorhodomela larix (Turner), Masuda, 1982) [51]. Several pharmaceutical potentials have been reported for these natural compounds [50]; however, their anti-inflammatory properties were scarcely explored.
Considering the number of bromophenols found in macroalgae, their anti-inflammatory potential is scarcely studied; more toxicological and in vivo studies are needed, and, in some cases, clinical trials would be appreciated.  [53,54]) that in LPS-stimulated RAW 264.7 murine macrophages can suppress the production of IL-6, a pro-inflammatory cytokine, in a dose-dependent manner. BDB also had an inhibitory effect on the phosphorylation of nuclear factor NF-κB, a signal transducer and activator of transcription 1 (STAT1; Tyr 701), which are two major signalling molecules involved in cellular inflammation. The in vivo assay of BDB (12) on atopic dermatitis (AD) in BALB/c mice induced by 2,4-dinitrochlorobenzene (DNCB) showed that treatment (100 mg/kg) resulted in suppression of the development of AD symptoms compared with the control treatment. 3-BDB (12) also reduced immunoglobulin E levels in serum, smaller lymph nodes with reduced thickness and length, decreased ear oedema, and reduced levels of inflammatory cell infiltration in the ears [55]. With a similar structure to BDB (12), 3-bromo-5-(ethoxymethyl)benzene-1,2-diol (BEMB) (13) (Figure 2), also isolated from the red algae P. morrowii. BEMB (13) demonstrated anti-inflammatory effects by inhibiting the production of NO, the expression of iNOS, and COX-2 in the LPS-activated RAW 264.7 cells and zebrafish embryos without cytotoxicity. It suppressed the protein and mRNA expression levels of nuclear factor NF-KB in the LPS-activated RAW 264.7 cells and zebrafish model [56]. The last example is bromophenol bis(3-bromo-4,5dihydroxybenzyl) ether (BBDE) (14) (Figure 2) isolated from the same algae, which can inhibit inflammation by reducing inflammatory mediators, such as NO, prostaglandin E2, iNOS, COX-2, and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), in LPS-induced RAW 264.7 macrophage cells [57].
Considering the number of bromophenols found in macroalgae, their anti-inflammatory potential is scarcely studied; more toxicological and in vivo studies are needed, and, in some cases, clinical trials would be appreciated.

Chromenes
Chromenes or benzopyrans represent the basic nucleus of various seaweed compounds with an anti-inflammatory potential, which includes the inhibition of COX and lipoxygenase, enzymes linked to inflammatory manifestations.

Terpenoids
Terpenoid is a general term for hydrocarbons and their oxygen-containing derivatives obtained through isoprene unit polymerization. They are usually classified into monoterpenes, sesquiterpenes, diterpenes, and polyterpenes according to their structural units [62] and are recognized for their biological activities, from which anticancer can be highlighted [63].
Terpenoid is a general term for hydrocarbons and their oxygen-containing derivatives obtained through isoprene unit polymerization. They are usually classified into monoterpenes, sesquiterpenes, diterpenes, and polyterpenes according to their structural units [62] and are recognized for their biological activities, from which anticancer can be highlighted [63].
Su et al. [82] demonstrated that fucoxanthin has a tremendous anti-inflammatory effect in a mouse sepsis model. LPS was used to induce sepsis in mice; when treated with 1 mg/kg (b.w.) of fucoxanthin, the survival rate can duplicate (20% to 40%). Fucoxanthin is related to the reduced levels of the pro-inflammatory cytokines' TNF-α and IL-6 and the inhibition of the NF-ƘB inflammatory pathway [82].
Knowing the anti-inflammatory properties of fucoxanthin, Wu et al. [83] produced a nanofiber membrane named PLA/PEGDA-EDT@rGO-fucoxanthin (PPGF) that can capture ROS. Poly(ethyleneglycol)diacrylate(PEGDA)-1,2-ethanedithiol (EDT) copolymer (PEGDA-EDT) is responsible for the ROS capture, reduced graphene oxide (rGO) is the drug carrier, and fucoxanthin (26) attenuates osteoarthritis (OA) [83]. In response to hydrogen peroxide, the nanofiber membrane exhibited sustained and long-term fucoxanthin release behaviour in vitro (at least 66 days). Moreover, it showed low cytotoxicity and Recent studies showed that fucoxanthin has a significant pharmacological effect on diseases related to oxidative stress injury. Its mechanism of action is primarily related to nuclear factor-erythroid 2-related (Nrf2) signal transduction pathway and gut microbiota regulation [80]. Zheng et al. [81] showed that fucoxanthin increased the phosphorylation level of the Akt/Nrf2 pathway as well as its effect on increased the mRNA and proteins levels of glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS) in human keratinocytes (HaCaT) [81].
Su et al. [82] demonstrated that fucoxanthin has a tremendous anti-inflammatory effect in a mouse sepsis model. LPS was used to induce sepsis in mice; when treated with 1 mg/kg (b.w.) of fucoxanthin, the survival rate can duplicate (20% to 40%). Fucoxanthin is related to the reduced levels of the pro-inflammatory cytokines' TNF-α and IL-6 and the inhibition of the NF-KB inflammatory pathway [82].
Sun et al. [91] demonstrated the protective mechanisms of fucosterol (27) on cobalt chloride (CoCl2)-induced hypoxia damage to keratinocytes (HaCaT). It attenuates CoCl2induced excess expression of IL-6, IL-1β, and TNF-α and suppresses the phosphorylation of PI3K and Akt and the accumulation of HIF1-α simulated by CoCl2 [91]. On the other hand, Mo et al. [92] showed that (27) attenuated serum liver enzyme levels, hepatic necrosis, and apoptosis induced by TNF-α, IL-6, and IL-1β. It also showed the effect of this compound in the reduction in P38 MAPK, and NF-κB signalling was accompanied by PPARγ activation [92].
Lucenna et al. [98] also reported the caulerpin (28) anti-inflammatory effect on the murine model of peritonitis and ulcerative colitis. The authors established that caulerpin (28) at 4 mg/kg triggered improvement of the Disease Activity Index (DAI) and attenuated the colon shortening and damage. This dose reduced the TNF-α, IFN-γ, IL-6, IL-17, and NFκB p65 levels and increased the levels of IL-10 in the colon tissue [98].

Fatty Acids
Fatty acids (FAs) are classified according to their carbon-chain length and sometimes the number of double bonds present. Long-chain fatty acids should have more than twelve carbons in the chain, whereas very long-chain should contain more than twenty-two. In the case of the polyunsaturated fatty acids, a further classification of omega-3 (ω-3) and omega-6 (ω-6), based on the position of the first double bond on the methyl terminal end, can be found in the literature. Polyunsaturated fatty acids (PUFAs) are known to play a vital role in body homeostasis. In general, higher levels of ω-6 polyunsaturated fatty acids are associated with constriction of blood vessels, inflammation, and platelet aggregation, whereas ω-3 may help to resolve inflammation and alter the function of vascular biomarkers [99]. It is known that ω-3 PUFAS has an important role in the reduction of depressive symptoms and exerts an anti-inflammatory action by the production of distinct metabolites, such as resolvins D (RvD) and E series, and maresins (MaR) and protectins (PD). The Z-4,7,10,13,16,19-docosahexaenoic acid (DHA)-derived trihydroxydocosahexanoic acid mediators termed RvD are produced by a series of reactions involving COX-2 and 5-LOX or by a pathway involving lipoxygenase enzymes and other reactions. The metabolism of DHA initially occurs by 15-lipoxygenase and then a series of other reactions generates a dihydroxy derivative termed protectin D1. The trihydroxyeicosapentaenoic acid mediators, termed RvE, form from Z-5,8,11,14,17eicosapentaenoic acid (EPA) by a similar series of reactions involving COX-2 and 5-LOX [100]. These mediators appear to act as a potent anti-inflammatory in psychiatric, neurodegenerative, and neurological diseases. On a cellular level and in a depression model, RvDs increased serotonin levels; on the other hand, they decreased gliosis in neurodegenerative disorders. Protectins prevented neurite and dendrite retraction and apoptosis in models of neurodegeneration, whereas maresins reduced cell death [101].
Our final choice relay on two enones (E)-9-oxooctadec-10-enoic acid (34) and (E)-10oxooctadec-8-enoic acid (35) fatty acids isolated from Gracilaria verrucose (Hudson) Papenfuss, nom. rejic., 1950, (the current accepted name is Gracilariopsis longissimi (S. G. Gmelin) Steentoft, L. M. Irvine and Farnham, 1995), and for which the inhibition of the production of the inflammatory markers' nitric oxide, TNF-α, and IL-6, in a dose-dependent manner and LPS-stimulated RAW264.7 cells, was reported. They suppressed NF-κB Recently, in a randomized, controlled, double-blind, and crossover study, 21 subjects (9 men and 12 postmenopausal women) with chronic inflammation and some characteristics of metabolic syndrome received a 10-week supplementation with EPA (30) or DHA (29) (3 g/day), relative to a 4-week lead-in phase of high oleic acid sunflower oil (3 g/day, defined as baseline). This study showed that both EPA (30) and DHA (29) significantly lowered the tricarboxylic acid (TCA) cycle intermediates, the interconversion of pentose and glucuronate, alanine, aspartate, and glutamate pathways (FDR < 0.05), and that DHA (29) had a greater effect on the TCA cycle than EPA (30). This study demonstrated a significant impact of both compounds on the cell metabolism of individuals with chronic inflammation [106].

Macroalgae Commercially Available Products
Early seaweed was mainly collected in its natural form; however, wild seaweed resources are limited with the constantly growing market for the food industry, medical and cosmetic uses, and energy sources. Alternatives pass to seaweed culture on land, sea, desert, and even in integrated aquaculture systems. The Financial Times has reported that the global population will rise to 10 billion by 2050. Furthermore, algae could supply the protein needed for people while conserving natural resources. It can be part of the solution by providing an excellent alternative to traditional crops as they do not require arable land and can grow on minimal nutrients [108]. The global seaweed market size was, in 2017, around USD 4097.93 million, and it is projected to reach USD 9075.65 million by 2024, registering a compound annual growth rate (CAGR) of 12.0% from 2018 to 2024 [109,110]. So, it is clear that seaweeds have emerged as one of the most promising resources due to their remarkable adaptability, short development period, and resource sustainability. The advantages of using seaweed in the food, cosmetic, and medical fields are huge in terms of economics and sustainability.
The European Algae Biomass Association (EABA) was founded in 2009 and, from the beginning of its formation, started to promote synergies between academia, industry, and decision-makers, aiming to establish the algae industry. Since the EABA's creation, some EU countries have created their associations; the Fédération des Spiruliniers in France and PROALGA (Associação Portuguesa de Produtores de Algas) in Portugal are excellent examples. Currently, around 420 companies from 23 countries produce 36% of seaweed in Europe [111]. As a result, algae or algae products are nowadays used, usually in the EU, as food or food ingredients. For instance, cookies, pasta, bread, and beverages, that are produced using algae are increasing in the European market, holding a 1.34% share of the new European foods and drinks launched in 2017 [112,113].
Algae are considered appreciated components in the medical field, particularly algal hydrogels and hydrocolloids, which are actually polysaccharide-based hydrogels. These algal hydrogels are widely used in wound healing, drug delivery, in vitro cell culture, and tissue engineering. From the structural point of view, these gels are similar to the tissues' extracellular matrix and can be manipulated to perform several vital roles. Some drug-specific gels have been clinically used for wound healing and have proven efficient and safe. In fact, wound healing and drug delivery applications are excellent examples of continuous and sequential drug release [114]. So, the development of new products with this specificity is vital in the future. One example is ACTIVHEAL ® ALGINATE, which is already used in medical treatment [115].
Cosmetics is a field in which seaweed has consolidated its use. A wide variety of products, from slimming creams to perfumes, shampoos, sunscreens, and bath salts, can be found on the market. In terms of seaweed-based products on the market, Revertime™, Sealgae ® , Codiavelane ® , Algowhite ® , Pheofiltrat ® , and Actiseane ® are some examples of products and trademarks, most of them used in cosmetics, algotheraphy, and thalassotherapy [116].
Although the products mentioned above obtained from macroalgae have exciting applications, with some possible anti-inflammatory ones [114], it is evident that the isolated compounds' applications are far from being commercially available. This is primarily due to the lack of proper toxicological assays and clinical trials.

Conclusions
The analysis of the macroalgae specialized metabolites' anti-inflammatory potential, herein presented and discussed, shows that the attributes commonly known for these marine species may be due to their specialized metabolites. In particular, the established anti-inflammatory activities for macroalgae [23,85] must be owing to some of the metabolites mentioned above. Moreover, these metabolites may explain some use of macroalgae in the production of biomaterials [117]. The chosen specialized metabolites present exciting activities, in some cases lowering concentrations and having different biological targets (Table 1), which make them suitable leader compounds for developing new anti-inflam matory drugs. Moreover, some compounds were also tested in in vivo assays and maintained their activity (Table 1). Naturally, toxicological assays and clinical trials are essential to establish the compound's potential. In this regard, it is worth mentioning the more studied compounds, such as fucoxanthin (26), fucosterol (27), and caulerpin (28), for which clinical trials are needed. Nevertheless, we hope this survey will incentivize future investigations concerning the specialized metabolites herein discussed and the search for other bioactive compounds isolated from macroalgae.
In our final comments, we highlight that the isolation of these bioactive is still problematic and prevent their industrial use. Mostly, they are costly procedures and allow for only small amounts of pure compounds, so using all macroalgae is still more economical.
The old traditional technique of solid-liquid extraction is still the most employed because it is easier to use and less expensive. However, it also involves more energy consumption and the use of less environmentally friendly solvents. The use of non-conventional extraction methods is highly recommended. In this regard, several authors are investigating alternative methods, such as microwave-assisted extraction, ultrasonic-assisted extraction, pressurized solvent extraction, supercritical fluid extraction, and enzyme-assisted extraction [118][119][120][121][122][123][124], to obtain the bioactive compounds in more environmentally friendly conditions. Some of these methods still need extra optimizations to incentive their use.
Note: The macroalgae full names are accordingly the Algaebase (https://www. algaebase.org/) and were confirmed on 18 December 2022.