Sargassum Seaweed as a Source of Anti-Inflammatory Substances and the Potential Insight of the Tropical Species: A Review

Sargassum is recognized both empirically and scientifically as a potential anti-inflammatory agent. Inflammation is an important response in the body that helps to overcome various challenges to body homeostasis such as microbial infections, tissue stress, and certain injuries. Excessive and uncontrolled inflammatory conditions can affect the pathogenesis of various diseases. This review aims to explore the potential of Sargassum’s anti-inflammatory activity, not only in crude extracts but also in sulfated polysaccharides and purified compounds. The tropical region has a promising availability of Sargassum biomass because its climate allows for the optimal growth of seaweed throughout the year. This is important for its commercial utilization as functional ingredients for both food and non-food applications. To the best of our knowledge, studies related to Sargassum’s anti-inflammatory activity are still dominated by subtropical species. Studies on tropical Sargassum are mainly focused on the polysaccharides group, though there are some other potentially bioactive compounds such as polyphenols, terpenoids, fucoxanthin, fatty acids and their derivatives, typical polar lipids, and other groups. Information on the modulation mechanism of Sargassum’s bioactive compounds on the inflammatory response is also discussed here, but specific mechanisms related to the interaction between bioactive compounds and targets in cells still need to be further studied.


Introduction
Inflammation is known to be a protective strategy that evolves in high-level organisms in response to threats interfering with body homeostasis. Types of threats causing inflammation include microbial infections, tissue stress, and certain injuries [1,2]. The well-known symptoms of classic inflammation are redness, pain, swelling, and fever [3]. Recent literature shows that inflammation operates via an advanced system and has a broad impact on various physiological aspects and human pathology. Although inflammatory response plays a pivotal role in protecting cellular physiological conditions, this process is only needed for a short time to avoid the stages of escalation that result in undesirable conditions, such as collateral tissue damage [1]. Uncontrolled and excessive inflammatory conditions multilevel fractionation. Various Sargassum species used in several studies of anti-inflammatory activity originated from two subgenera of Sargassum, namely Bactrophycus and Sargassum.
The Sargassum genus is generally divided into four subgenera: Phyllotrichia, Bactrophycus, Arthrophycus, and Sargassum. This subgenus classification is based on the morphological characteristics of the seaweed thallus. In addition, the distribution pattern can also be used to distinguish between Sargassum subgenera. The most common subgenera found in subtropical/temperate regions are Bactrophycus and Arthrophycus. Subgenus Phyllotrichia is only found in Australia and adjacent areas, while the Sargassum subgenus is widely distributed in tropical areas [16]. Due to the high level of morphological plasticity caused by differences in environmental condition, molecular marker techniques combined with morphological observation are implemented to resolve taxonomic issues [17]. A sequence of the internal transcribed spacer of nuclear ribosomal DNA (ITS) is commonly used to analyze the phylogenetic relationship among Sargassum species. Figure 1 shows the phylogenetic tree of the Sargassum genus based on internal transcribed spacer (ITS)-2 gene sequences. The phylogenetic tree was constructed based on several Sargassum species used in various anti-inflammatory activity studies. The accession number of each gene sequences is obtained from the database of the National Center for Biotechnology Information.

Crude Extracts and Their Partitions
An anti-inflammatory activity summary of Sargassum's crude extracts and their partitions is shown in Table 1. Various types of solvents with different polarities are used for extraction and partitioning of the anti-inflammatory compounds. Mun et al. [18] compared the anti-inflammatory activity of the ethanolic extracts from fermented and non-fermented (fresh) samples of the brown seaweed S. thunbergii in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells.
Kang et al. [22] reported that the dichloromethane fraction of S. fulvellum and ethanol fraction of S. thunbergii had a stronger effect in inhibiting mouse ear edema than the water fraction. Based on the GC-MS analysis result of both mentioned fractions, compounds associated with fatty acids and simple organic compounds, such as hexadecanoic acid, neophytadiene, tetradecanoic, 8-heptadecene, and 3,7,11,15-tetramethyl-2-hexadecen-1-ol, dominated the two fractions content. These compounds were thought to be the responsible anti-inflammatory agent. Molecular docking study conducted by Balachandran et al. [93] found that hexadecanoic acid and (E)-9-octadecenoic ethyl ester contained in S. wightii were effective in inhibiting the COX-2 enzyme activity. Several other compounds that are allegedly responsible for the anti-inflammatory activity of the non-polar fraction include fucoxanthin or its derivatives [33], fucosterol or other steroid compounds [20,23,28,29,31,33,51], and phenolic compounds [18,23,30,31,33]. Although phenolic compounds are commonly known to have hydrophilic natures, their polarities will actually depend on their chemical structures. A phenolic component consists of various groups with a wide range of structural variations [94]. Various types of lipophilic phenolic components can be found in nature in the form of steryl phenolic acid esters, phenolic acid lactones, phenolic fatty acid esters, and others [95].
In addition to the promising potential of lipophilic extracts/fractions, several studies had shown that a water-soluble extract of Sargassum was also effective in suppressing the inflammatory response [35,44,48,71,81]. Kang et al. [71] reported that the S. fusiforme's water extract could suppress the production of TNF-α in the LPS-induced C2C12 myotube cells and increase the production of cytokines associated with increased insulin sensitivity, such as IL-6 and IL-10. The chemical profile of that water extract was not identified, but the phenolic component was thought to be partially responsible for its anti-inflammatory activity. Jaswir et al. [35] showed that water extracts from several species of Sargassum had an NO inhibitory effect in LPS-induced RAW 267.4 cells, even though the anti-inflammatory activity was not as strong as that of other water extracts obtained from Padina australis and Turbinaria turbinata. They found that the anti-inflammatory activity of the observed brown seaweed water extract was positively correlated with fucose and uronic acid content. Another study showed that the polar β-glucan extract obtained from S. crassifolium has a potent anti-inflammatory activity in the rat ear edema model [48].
Sargassum's crude extract not only shows its effectiveness in the acute inflammatory model but also in the chronic inflammatory model, as reported by several studies [28,29,44,50]. The study of Dhas et al. [44] reported that the administration of gold nanoparticles made from a S. swartzii water extract (28 days treatment) improved insulin sensitivity and reduced the serum production of TNF-α, IL-6, and CRP in alloxan-induced diabetic rats. The administration of the methanol extract of S. subrepandum (four months, 100 mg/kg body weight) in rats with dyslipidemia triggered an improvement in their plasma lipid profile and reduced the content of MDA, NO, leptin, and TNF-α in rat serum [50]. Dyslipidemia in the mentioned study was triggered by the atherogenic diet given to Sprague Dawley rats for eight consecutive months. Kwon et al. [28] and Gwon et al. [29] proved that the meroterpenoid-rich fraction obtained from ethanolic extract of S. serratifolium improved the inflammation condition in both high-fat diet-induced and high-cholesterol diet-induced C57BL/6J mice.
Sanjeewa et al. [100] reported that CSP yielded from Celluclast enzyme digestion against S. horneri had the same Fourier transform infrared (FT-IR) spectrum as a commercial fucoidan. Wen et al. [102] found that the purified SP derived from S. horneri (fractionated by column Q Sepharose Fast Flow) contained higher fucose and sulfate ester content than its crude form, and this ester exists along with the elevated anti-inflammatory activity.
The molecular weight (Mw) of SP seems to affect its biological activity. A study of Neelakandan and Venkatesan [101] revealed that a 10 kDa fraction derived from the CSP of S. wightii exhibited stronger anti-inflammatory activity than the fraction with Mw > 10 kDa and was more effective than its crude extract. The anti-inflammatory effect of their study was demonstrated by the inhibition of edema formation in carrageenan-induced rat paw edema, the suppression of leukocyte and neutrophil migration in the peritonitis model, and inhibition of edema formation in the Freund's complete adjuvant-induced arthritis model. The result of their study controverts the findings of Sanjeewa et al. [96], who reported that the SP fraction with Mw > 30 kDa was the most effective in suppressing NO production (IC50 = 87.12 µg/mL) by LPS-induced RAW 264.7 cells among other lower Mw fractions (<5, 5-10, and 10-30 kDa). Wu et al. [103] found that the SP fraction with an Mw of 386.1 kDa had the highest NO inhibitory activity compared to other fractions (1193.2, 864.4, 106.3, 55.9, 15.4, and 1.9 kDa). Fractions with a high Mw (1193.2, 864.4, and 386.1 kDa) tended to have higher anti-inflammatory effects than low Mw fractions (55.9, 15.4, and 1.9 kDa). The effect of SP's molecular weight on anti-inflammatory activity will depend on various factors, i.e., the testing model, sample, extraction method, and SP structural configuration, including its active groups. The effect of sulfation levels on SP's anti-inflammatory activity was also studied by Wu et al. [103], who found that the 386.1 kDa fraction with 9.42% sulfate content showed the highest NO inhibition among other samples with different sulfate content (50.83%, 31.01%, and 0.84%). The SP fraction with 50.83% sulfate content showed the lowest NO inhibitory effect. Although the study of Wen et al. [102] stated that an increase in sulfate esters in the SP coincided with an enhancement of the anti-inflammatory effect, the study of Wu et al. [103] confirmed that the correlation between them is not always linear.
Fernando et al. [4] summarized that fucoidan functionality will depend on the monosaccharide sequences, sulfation levels, and connectivity of sulfate groups. Fucoidan has a similar characteristic to heparan sulfate, which has an antithrombotic effect with a low bleeding effect [114]. Fucoidan has been reported to inhibit the secretion of gelatinase A and stromelysin in IL-1β-induced fibroblasts cells, increase the association of MMPs (matrix metalloproteinases) with their inhibitors (TIMPs), and decrease the activity of leukocyte-secreted elastase. Based on these facts, fucoidan can be used to overcome aberrant extracellular matrix degradation, which usually occurs in inflammatory-related disease. The fucose-containing sulfated polysaccharide (FCSP) term is now considered more relevant than fucoidan because the sugar monomer constituent of common bioactive SP in brown seaweed is not only fucose, but also other sugars such as galactose, rhamnose, mannose, xylose, and glucose [115].

S. hemiphyllum
A combination of oligofucoidan (LMF) and fucoxanthin resulted in enhancement of the intestinal epithelial barrier and immune function against LPS stimulation through suppression of IL-1β and TNF-α production and increased secretion of IL-10 and IFN-γ in CaCo2 cells co-cultured with B. lactis 1 A combination of oligofucoidan (LMF) and fucoxanthin resulted in enhancement of adiponectin production, and decreased production of TNF-α and IL-6 in type II diabetes mouse model 2

S. muticum
Suppression of ROS production in PMA-induced neutrophil and suppression of PGE2, COX-1, and COX-2 expression in A23187-induced erythrocytes. [142] S. vulgare Suppression of NO production in LPS-induced RAW 264.7 and direct scavenging of NO in a cell-free system. [143] Note: References followed by an asterisk (*) used samples from tropical area. The superscripted numbering as listed in reference and modulation of inflammation in the same row are related to one another. This superscripted numbering is discontinous between rows and only applied to the same row.
According to the above summary, there are still many potential anti-inflammatory compounds from the Sargassum species to be unraveled, especially groups of fatty acids and lipid derivatives. Some Sargassum species are reported to be rich in anti-inflammatory fatty acids, primarily EPA (C20:5n3) and SDA (C18:4n3) [14,148]. Chen et al. [148] revealed that some Sargassum species often used in traditional Chinese medicine have satisfactory ratios of polyunsaturated fatty acid (PUFA) to saturated fatty acid (SFA; >0.45) and n-6/n-3 (<10), which can improve pathological conditions in cardiovascular diseases, which are strongly related to inflammatory disorders. Some essential PUFAs in Sargassum, especially EPA and SDA, are incorporated into glycolipid structures [149][150][151]. PUFA-rich polar lipids in Sargassum, including the glycolipid group, are reported to have biological activities that benefit human health, such as fibrinolytic and anti-cancer activities [149,152]. The NMI fatty acid (non-methylene interrupted, C20:2n11,16) found in S. marginatum is thought to correlate with apoptotic activity in human blood cancer cells (HL-60) [149].

Terpenoids
Terpenoids are a large group of secondary metabolites. This compound is composed of isoprene units (2-methyl-1,3 butadiene, C 5 H 8 ) and produced by the biosynthesis process with mevalonate as a parent [153]. Sterol is a triterpenoid derivative containing six units of isoprene, and fucosterol is a dominant sterol commonly found in brown seaweed [4]. Several mentioned compounds such as tuberatolide B, sargachromenol, sargachromanol D, sargaquinoic acid, sargahydroquinoic acid, and isonahocol E3 are included in the meroterpenoid group. The group of meroterpenoid is characterized to possess a polyprenyl structure bound to hydroquinone or similar aromatic rings [12]. Isoketochabrolic acid (IKCA) belongs to the C18 terpenoid-related carboxylic acids group [154]. Loliolide or 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (HTT) is included to the monoterpenoid hydroxylactone group [155]. Various terpenoid compounds have been shown to play an important role as modulators in the NF-kB signaling pathway, so this group of compounds is known as a potent anti-inflammatory agent [153]. The non-polar characteristic of terpenoids will make them diffuse into inflamed cells easily, and may produce a rapid effect in modulating the inflammatory response. Chemical structures of several terpenoid compounds can be seen in Figure 2.
to the monoterpenoid hydroxylactone group [155]. Various terpenoid compounds have been shown to play an important role as modulators in the NF-kB signaling pathway, so this group of compounds is known as a potent anti-inflammatory agent [153]. The non-polar characteristic of terpenoids will make them diffuse into inflamed cells easily, and may produce a rapid effect in modulating the inflammatory response. Chemical structures of several terpenoid compounds can be seen in Figure  2.

Fucoxanthin and Its Derivatives
Fucoxanthin is the dominant carotenoid in brown seaweed, which can absorb the light spectrum in the range of 450-540 nm. Fucoxanthin has a unique structure, which includes allenic bonds, conjugated carbonyl, epoxides, and acetyl groups. Carotenoids are usually found in nature as trans isomers. However, cis-isomers can still be found in small amounts due to the isomerization process. Several studies have shown that the cis-trans configuration of a carotenoid isomer affects its bioactivity [117,163,164]. Heo et al. [117] showed that all-trans-(6'R) fucoxanthin isomer more effectively inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 cells than its cis isomers (9'-cis-(6'R) fucoxanthin and a complex of 13-cis (6'R) fucoxanthin and 13'-cis-(6'R) fucoxanthin). A complex of the 13-cis and 13'-cis-(6'R) fucoxanthin isomer showed significant cytotoxicity in cells, so the anti-inflammatory effect can be associated with its cytotoxicity. Apo-9fucoxanthinone, a product of fucoxanthin degradation, is also reported to have promising antiinflammatory activity [27,[130][131][132][133]. Chemical structures of fucoxanthin and its derivatives are shown in Figure 3.

Fucoxanthin and Its Derivatives
Fucoxanthin is the dominant carotenoid in brown seaweed, which can absorb the light spectrum in the range of 450-540 nm. Fucoxanthin has a unique structure, which includes allenic bonds, conjugated carbonyl, epoxides, and acetyl groups. Carotenoids are usually found in nature as trans isomers. However, cis-isomers can still be found in small amounts due to the isomerization process. Several studies have shown that the cis-trans configuration of a carotenoid isomer affects its bioactivity [117,163,164]. Heo et al. [117] showed that all-trans-(6'R) fucoxanthin isomer more effectively inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 cells than its cis isomers (9'-cis-(6'R) fucoxanthin and a complex of 13-cis (6'R) fucoxanthin and 13'-cis-(6'R) fucoxanthin). A complex of the 13-cis and 13'-cis-(6'R) fucoxanthin isomer showed significant cytotoxicity in cells, so the anti-inflammatory effect can be associated with its cytotoxicity. Apo-9-fucoxanthinone, a product of fucoxanthin degradation, is also reported to have promising anti-inflammatory activity [27,[130][131][132][133]. Chemical structures of fucoxanthin and its derivatives are shown in Figure 3.

Other Lipid-Soluble Compounds
The other types of lipid-soluble anti-inflammatory compounds that have been reported are polyketide macrolactone and grasshopper ketone. Chemical structures of those compounds can be seen in Figure 4. Polyketide is a secondary metabolite compound characterized by the presence of alternating carbonyl and methylene groups and is generally produced by fungi [167]. Maneesh et al. [144] succeeded in isolating two types of aryl-polyketide lactone compounds from S. wightii. These compounds had strong anti-inflammatory and antioxidant activity. The two isolated compounds are 4-(8-ethyl-tetrahydro-7-oxo-2H-pyran-5-yl)-propyl-4′-methyl benzoate (compound 1) and methyl-2-(12-oxo-7-phenyl-8-vinyl-1-oxa-4,9-cyclododecadien-3-yl)-acetate (compound 2). The study found that the isolated aryl polyketide lactones had a more effective 5-LOX and COX-2 inhibitory activity than standard synthetic drugs, such as ibuprofen and sodium salicylate, and had higher COX-2/COX-1 selectivity than aspirin and ibuprofen. The inhibitory activity of the aforementioned compounds against 5-LOX, COX-2, and COX-1 enzymes is strongly influenced by their electronic and hydrophobic characteristics. Electron-rich centers, such as the cyclic esters and vinyl or aryl substituents contained in aryl polyketide lactones, act as unsaturation centers determining the inhibition effect on pro-inflammatory enzymes.

Other Lipid-Soluble Compounds
The other types of lipid-soluble anti-inflammatory compounds that have been reported are polyketide macrolactone and grasshopper ketone. Chemical structures of those compounds can be seen in Figure 4. Polyketide is a secondary metabolite compound characterized by the presence of alternating carbonyl and methylene groups and is generally produced by fungi [167]. Maneesh et al. [144] succeeded in isolating two types of aryl-polyketide lactone compounds from S. wightii. These compounds had strong anti-inflammatory and antioxidant activity. The two isolated compounds are 4-(8-ethyl-tetrahydro-7-oxo-2H-pyran-5-yl)-propyl-4 -methyl benzoate (compound 1) and methyl-2-(12-oxo-7-phenyl-8-vinyl-1-oxa-4,9-cyclododecadien-3-yl)-acetate (compound 2). The study found that the isolated aryl polyketide lactones had a more effective 5-LOX and COX-2 inhibitory activity than standard synthetic drugs, such as ibuprofen and sodium salicylate, and had higher COX-2/COX-1 selectivity than aspirin and ibuprofen. The inhibitory activity of the aforementioned compounds against 5-LOX, COX-2, and COX-1 enzymes is strongly influenced by their electronic and hydrophobic characteristics. Electron-rich centers, such as the cyclic esters and vinyl or aryl substituents contained in aryl polyketide lactones, act as unsaturation centers determining the inhibition effect on pro-inflammatory enzymes.

Polysaccharides
The most abundant active polysaccharides derived from brown seaweed are FCSPs and alginic acid. Their chemical structures can be seen in Figure 5. The main constituent of FCSPs is Lfucopyranose, which may be substituted with sulfate or acetate and/or have side branches containing fucopyranoses or other glycosyl units [169]. Alginic acid is anionic polysaccharide consisting of β-Dmannuronate and α-L-guluronate as basic monomers. Alginic acid is reported to exhibit potential anti-inflammatory activity in the rat arthritis model induced by type-2 collagen and Freund's complete adjuvant [138,139].
The majority of exploratory studies of pure compounds from Sargassum (Table 3) use single components as the only responsible agent in improving inflammatory conditions, except for the studies by Hwang et al. [137] and Lin et al. [141].Hwang et al. [137] found that the combination of LMF (low molecular weight fucoidan; with a Mw around 0.8 kDa) and HS-Fucox (high stability fucoxanthin) derived from S. hemiphyllum effectively suppressed inflammatory response in LPSinduced CaCo2 cells co-cultured with Bifidobacterium lactis. This was indicated by a decreased production of TNF-α and IL-1β, as well as increased production of anti-inflammatory cytokines such as IL-10 and IFN-γ. In addition, the treatment of LMF-HS Fucox also helped maintain intestinal epithelial cell integrity, which was indicated by an increase in the mRNA expression encoding the tight junction protein (occludin, claudin-1, and claudin-2). LMF can promote the growth of probiotic B. Lactis, thereby helping to maintain the structural integrity of colon cells. Park et al. [170] reported that LMF (Mw ± 1 kDa) has a stronger NO inhibition effect than the high molecular weight fucoidan/HMF (Mw ± 100 kDa) in the LPS-induced RAW 264.7 cells. In the model of type 2 collageninduced arthritis, oral administration of HMF (300 mg/kg, 49 days) exacerbated the severity of arthritis and inflammatory conditions in joint cartilage, whereas LMF treatment reduced arthritis severity and the levels of Th1-dependent collagen-specific IgG2a. This contradicts the findings of Sanjeewa et al. [96] and Wu et al. [103], as discussed in the previous section. These two studies also observed the NO inhibition level in a similar cell culture model. Sanjeewa et al. [96] obtained an FCSPrich fraction through filtration techniques, while Wu et al. [103] obtained crude FCSPs with different molecular weights using the acid hydrolysis technique (also as conducted by Park et al. [170]). This contradiction could not be further explained because the chemical characteristic information for each FCSP fractions is limited, especially for the sulfation level which may greatly determine the bioactivity of FCSPs [103,171].

Polysaccharides
The most abundant active polysaccharides derived from brown seaweed are FCSPs and alginic acid. Their chemical structures can be seen in Figure 5. The main constituent of FCSPs is L-fucopyranose, which may be substituted with sulfate or acetate and/or have side branches containing fucopyranoses or other glycosyl units [169]. Alginic acid is anionic polysaccharide consisting of β-D-mannuronate and α-L-guluronate as basic monomers. Alginic acid is reported to exhibit potential anti-inflammatory activity in the rat arthritis model induced by type-2 collagen and Freund's complete adjuvant [138,139].
The majority of exploratory studies of pure compounds from Sargassum (Table 3) use single components as the only responsible agent in improving inflammatory conditions, except for the studies by Hwang et al. [137] and Lin et al. [141].Hwang et al. [137] found that the combination of LMF (low molecular weight fucoidan; with a Mw around 0.8 kDa) and HS-Fucox (high stability fucoxanthin) derived from S. hemiphyllum effectively suppressed inflammatory response in LPS-induced CaCo2 cells co-cultured with Bifidobacterium lactis. This was indicated by a decreased production of TNF-α and IL-1β, as well as increased production of anti-inflammatory cytokines such as IL-10 and IFN-γ. In addition, the treatment of LMF-HS Fucox also helped maintain intestinal epithelial cell integrity, which was indicated by an increase in the mRNA expression encoding the tight junction protein (occludin, claudin-1, and claudin-2). LMF can promote the growth of probiotic B. Lactis, thereby helping to maintain the structural integrity of colon cells. Park et al. [170] reported that LMF (Mw ± 1 kDa) has a stronger NO inhibition effect than the high molecular weight fucoidan/HMF (Mw ± 100 kDa) in the LPS-induced RAW 264.7 cells. In the model of type 2 collagen-induced arthritis, oral administration of HMF (300 mg/kg, 49 days) exacerbated the severity of arthritis and inflammatory conditions in joint cartilage, whereas LMF treatment reduced arthritis severity and the levels of Th1-dependent collagen-specific IgG 2a . This contradicts the findings of Sanjeewa et al. [96] and Wu et al. [103], as discussed in the previous section. These two studies also observed the NO inhibition level in a similar cell culture model. Sanjeewa et al. [96] obtained an FCSP-rich fraction through filtration techniques, while Wu et al. [103] obtained crude FCSPs with different molecular weights using the acid hydrolysis technique (also as conducted by Park et al. [170]). This contradiction could not be further explained because the chemical characteristic information for each FCSP fractions is limited, especially for the sulfation level which may greatly determine the bioactivity of FCSPs [103,171].

Phenolic Compounds
Phlorotannin is a typical phenolic group of brown seaweed. These compounds have a broad molecular size range (from 126 Da to 650 kDa) and are divided into six main classes, fucols, phloretols, fucophlorethols, fuhalols, isofuhalols, and eckols. Phlorotannin is composed of phloroglucinol units (1,3,5-trihydroxybenzene) with different degrees of polymerization. The classification of phlorotannin is based on the different bond types between their constituent units [143]. Lopes et al. [143] reported that the phlorotannin content in some types of seaweed belonging to the Sargassaceae family ranged from 74.96 to 815.82 mg phloroglucinol/kg. The main phlorotannin classes found in Sargassum are fuhalols, phlorethols, and fucophlorethols [173][174][175]. Li et al. [173] reported that the phlorotannin in S. fusiforme was dominated by a fuhalol group with DP of 2-12. Lopes et al. [143] proved that phlorotannin affected the NO levels in LPS-induced RAW 264.7 through NO direct scavenging and/or the modulation of inflammatory signals in cells. The anti-inflammatory activity of phlorotannin is strongly influenced by its qualitative composition. The chemical structures of phloroglucinol and examples of several phlorotannin classes commonly found in Sargassum are shown in Figure 6.

Phenolic Compounds
Phlorotannin is a typical phenolic group of brown seaweed. These compounds have a broad molecular size range (from 126 Da to 650 kDa) and are divided into six main classes, fucols, phloretols, fucophlorethols, fuhalols, isofuhalols, and eckols. Phlorotannin is composed of phloroglucinol units (1,3,5-trihydroxybenzene) with different degrees of polymerization. The classification of phlorotannin is based on the different bond types between their constituent units [143]. Lopes et al. [143] reported that the phlorotannin content in some types of seaweed belonging to the Sargassaceae family ranged from 74.96 to 815.82 mg phloroglucinol/kg. The main phlorotannin classes found in Sargassum are fuhalols, phlorethols, and fucophlorethols [173][174][175]. Li et al. [173] reported that the phlorotannin in S. fusiforme was dominated by a fuhalol group with DP of 2-12. Lopes et al. [143] proved that phlorotannin affected the NO levels in LPS-induced RAW 264.7 through NO direct scavenging and/or the modulation of inflammatory signals in cells. The anti-inflammatory activity of phlorotannin is strongly influenced by its qualitative composition. The chemical structures of phloroglucinol and examples of several phlorotannin classes commonly found in Sargassum are shown in Figure 6.
There are few researchers still using a protein denaturation inhibition model to screen antiinflammatory activity in this new prostaglandin era. The reason behind choosing this model is based on the parallelism between two physio-pathological phenomena, namely inflammation and protein denaturation, both of which can be caused by the same stimulants (e.g., heat, radiation, organic
There are few researchers still using a protein denaturation inhibition model to screen anti-inflammatory activity in this new prostaglandin era. The reason behind choosing this model is based on the parallelism between two physio-pathological phenomena, namely inflammation and protein denaturation, both of which can be caused by the same stimulants (e.g., heat, radiation, organic solvents, etc.) [176]. Various types of NSAIDs (non-steroidal anti-inflammatory drugs) are reported to inhibit the in vitro denaturation of biologically active plasma proteins [177]. Protein denaturation can be the initial stage of further protein modification-for instance, protein glycosylation. Abnormal protein glycosylation phenomena occur frequently in chronic inflammatory diseases [178]. Nevertheless, the protein denaturation model is less convincing for most researchers today because the protein denaturation experiment is applied by mild heating (47-50 • C), which is not physiological. The implementation of the RBC membrane stabilization model for anti-inflammatory screening is based on the structural similarity between RBC and lysosome membranes. Lysosomes are important organelles that elicit the effectors mechanism in a late inflammatory response through releasing bactericidal enzymes and various proteinases, thereby maintaining their structural integrity is important for suppressing the inflammatory process [43].
Ten out of 18 studies on the anti-inflammatory activity of Sargassum's CSP utilized tropical species [97,99,101,105,[107][108][109][111][112][113]. Preetha and Devaraj [99] reported that subcutaneous administration of S. wightii CSP improved high cholesterol diet-induced hypercholesterolemia in rats, manifested by the improvement of the plasma lipid profile, decreased serum TNF-α, CRP, and plasma fibrinogen, the decreased cardiac mRNA expression of iNOS and COX-2, and the suppression of lysosomal enzymes and cardiac NO production. Fernando et al. [108] studied the effects of CSP from S. polycystum (SPF) in LPS-induced RAW 264.7 cells. They found that SPF treatment (25-100 µg/mL) reduced NO production, suppressed the expression of iNOS and COX-2 proteins, and increased cell viability. Furthermore, SPF reduced the production of PGE 2 and some pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. Lavanya et al. [105] observed the effect of CSP from S. ilicifolium (SIF) in TPA-induced polymorphonuclear leukocytes (PMNL). They reported that the SIF treatment increased the viability of PMNL cells and reduced the production of cathepsin D (lysosomal enzyme), nitrite, and TNF-α. Information on the effects of SP from tropical Sargassum on inflammatory conditions is more profound than that in the previous section (the crude extract). This is because some researchers have tried to uncover the anti-inflammatory effects on cell units, and have used diverse inflammatory parameters to explain the effects between parameters in improving inflammatory conditions.

Potential Anti-Inflammatory Compounds of Tropical Sargassum
Based on the literature summaries in Tables 1-4, polar and non-polar compounds of both tropical and subtropical Sargassum showed promising anti-inflammatory activity in various screening models. Various non-polar compounds from brown seaweed, which are thought to be responsible anti-inflammatory agents include sterol/terpenoids, omega-3 PUFAs, fucoxanthin and its derivatives, grasshopper ketone, and the polyketide group, while the water-soluble anti-inflammatory agents from Sargassum are sulfated polysaccharides, alginic acid, β-glucan, and phenolic components.

Lipid-Soluble Bioactive Compounds
The total lipid content in brown seaweed (the Sargassaceae family) is commonly higher in subtropical species (−5% db) than in tropical species (0.8%-1.9% db) [15]. However, high lipid levels (−8% db) were found in tropical S. wightii harvested from Tamil Nadu, Gulf of Mannar, India [179]. The PUFA content and n3:n6 ratio were also found to be higher in subtropical Sargassum [14,150,180]. This could be related to environmental temperature differences. Low-temperature conditions in the subtropical region, especially in winter and fall, can cause an increase of PUFA concentrations in cell membranes, thereby implying the continuity of metabolic processes [181]. Although the PUFA content and n3:n6 ratio in tropical species are lower than those in subtropical ones, the n6:n3 ratio of tropical Sargassum still meets World Health Organization (WHO) recommendations (i.e., below 10). Maintaining its ratio in the recommended range can lower the human body's risk of inflammation-associated diseases, such as cardiovascular and neurological diseases.
Susanto et al. [14] showed that the fucoxanthin level of tropical Sargassum (1.64 ± 0.49 mg/g db) was not significantly different from that of subtropical Sargassum (1.99-2.12 mg/g db), although there was a higher amount in subtropical Sargassum. The fucoxanthin content of seaweed is strongly influenced by species, season, and maturity level [32,182,183]. Lann et al. [184] hypothesized that fucoxanthin in tropical Sargassaceae not only acts as an accessory light-harvesting pigment but also as a photoprotectant. High UV intensity in the tropical area will trigger the formation of free radicals in plant cells, so a certain defense mechanism is required to maintain cell structure integrity.
There is a lack of information on the effects of different latitudes (for tropical and subtropical areas) on the sterol content of Sargassum. Terasaki et al. [181] reported that S. horneri fucosterol content was higher in winter and in deep water seaweed. This phenomenon is related to the seaweed's response in preserving cell membrane integrity at low temperatures and in suboptimal photosynthetic conditions. Fucosterol plays an important role in supporting liquid-ordered membrane phases and determining membrane integrity by balancing the liquid and non-liquid phases in plant cell membranes [185]. The content of fucosterol in the S. horneri harvested from some Japanese waters was in the range of 1.21%-3.21% db [181], while Fleury et al. [186] showed that the S. furcatum fucosterol content harvested from the water area of Rio De Janeiro, Brazil only reached 0.08% wt.

Water-Soluble Bioactive Compounds
Polyphenol content is generally higher in subtropical seaweeds than in tropical ones [184]. However, some studies have proven the opposite [187,188]. Bolser and Hay [189] revealed that tropical seaweed is likely to form stronger chemical defenses to withstand the intense grazing activity of tropical herbivores. Tropical herbivores are known to be more active and diverse. Bolser and Hay [189] hypothesized that the strong chemical defense of seaweed would be manifested by a high content of secondary metabolites, including those of the polyphenol group. Based on these facts, latitude difference is not the only predictor that determines seaweed's phenolic content. Various factors affecting the phenolic content in seaweed are age and maturity, the surrounding ecosystem conditions, latitude, geomorphology, depth, irradiation, hydrodynamic conditions, seasons, and water nutrient levels [184]. Tropical Sargassum species, especially those growing on the water surface, are rich in low Mw phenolic content (<2 kDa), which is attributed to their role as photoprotectants in plant cells [184]. Audibert et al. [190] showed that the phenolic fraction of brown seaweed Ascophyllum nodosum with Mw < 2 kDa had the most potent radical scavenging (ABTS assay) activity compared to the crude phenolic extract and other fractions with higher Mw, although its TPC (total phenolic content) was the lowest among the other fractions. This result indicates that the qualitative composition of the fraction determines its antioxidant activity. Audibert et al. [190] added that the Trolox equivalent antioxidant capacity/TEAC (mmol/l) of the fraction <2 kDa is 3.5 times more active than phloroglucinol and twice more active than quercetin. The antioxidant activity of an active component is often reported to be positively correlated with its anti-inflammatory activity [191][192][193][194].
The biological activity of sulfated polysaccharides (SP) from tropical Sargassum has been discussed more frequently than the activity of other compounds. This is due to the high yield potency of SP, which is essential for its commercial development. The FCSP yield of tropical Sargassum ranges from 2.16% to 7.15% db [195][196][197]. This reported yield is higher than that of other anti-inflammatory compounds, such as sterols, fucoxanthin, omega-3 PUFAs, and phenolic components. Sulfated polysaccharides are also recognized as potential antioxidants [136,195,198]. Several factors affecting the FCSP content in seaweed include mechanical stress (the level of ocean current and waves), the level of maturity, season, salinity, and part of the thallus [199]. In addition to FCSPs, alginic acid has been reported to show promising anti-inflammatory activity [138,139]. Kokilam et al. [200] reported that the yield of alginic acid in S. wightii harvested from Gulf of Mannar, India (tropical) reached 21.71%. Moreover, Aponte de Otaola et al. [201] found that the alginic acid content of several tropical Sargassum species (whole plant) originating from Puerto Rico area were in the range of 17.9%-20.3%.
FCSPs and alginates are important components of the wall structures of algae cells. Both of these components play an important role in attaining the flexibility requirements for intertidal seaweed [201,202]. So et al. [203] compared the protective activity of fucoidan and alginic acid against oxidative stress, both in vitro and cellular systems. The results revealed that fucoidan showed stronger scavenging activity for NO and superoxide anions than that of alginic acid. Fucoidan was also reported to be more effective in inhibiting lipid peroxidation in APPH (2,2 -azobis(2-amidinopropane) dihydrochloride)-induced LLK-PK1 cells.

Anti-Inflammatory Mechanisms of Bioactive Compounds of Sargassum
Inflammation is characterized by some general parameters, such as the overproduction of pro-inflammatory cytokines (generally TNF-α, IL-1β, and IL6), NO, PGE 2 , and ROS. Increased NO production occurs along with the increased activity of the iNOS enzyme. This enzyme converts L-arginine to NO [11], while PGE 2 is produced through cyclooxygenase-2 (COX-2) enzyme activity, using arachidonic acid as a substrate. Arachidonic acid (AA) is a dominant constituent of the phospholipid bilayer in cell membranes. AA is produced from phospholipase A2 s activity against phospholipids. In addition to prostaglandin, the AA substrate can also be converted into pro-inflammatory leukotrienes by the lipoxygenase enzyme (LOX) [31]. Sargassum bioactive compounds have been reported to modulate inflammatory responses via inhibition of NF-kB transactivation, inhibition of the MAPK group's phosphorylation (which impacts the blocking of AP-1 protein translocation), direct NO scavenging, and direct inhibition of important pro-inflammatory enzymes, such as iNOS, COX-2, 5-LOX, and PLA2.
As discussed in the previous section, NF-kB and AP-1 are important proteins that regulate the transcription of various inflammatory-related genes. The inhibition of their activation suppresses inflammatory response. The downstream responses due to NF-kB and AP-1 transactivation are depicted in Figure 7. Adhesion molecules, such as ICAM-1 and VCAM-1, are typical markers of the vascular system's inflammation. These compounds help monocytes attach to vascular endothelial cells and promote the initial phase of atherogenesis. Chemoattractant compounds, such as MCP-1, are produced by inflamed cells to recruit various immune cells such as monocytes, macrophages, neutrophils, and eosinophils to the inflammation site [123]. The matrix metalloproteinase (MMP) plays a role in the regulation of transmigration, extravasation, and monocyte infiltration into inflammatory sites through extracellular matrix degradation [204]. The risk of cardiovascular disease could also be characterized by the increased production of fibrinogen. Fibrinogen is known as a coagulation factor protein and is involved in atherosclerosis processes [99]. Sarithakumari et al. [138] used various parameters to describe the inflammatory response in arthritic rats, and one of these parameters was the xanthine oxidase (XO) enzyme. XO activity is associated with increased oxidative stress in cells. This enzyme is responsible for producing superoxide anion radicals and increasing lipid peroxidation [205]. The inhibition of NF-kB activation by Sargassum bioactive compounds (not only purified compounds, but also crude extracts and CSP) is proven by decreased phosphorylated NF-kB protein expression (p65 and/or p50), both in the nucleus and in cytoplasmic cell extracts [26,37,75,123,132,135], as well as inhibition of phosphorylation and degradation of the IkB protein in the cytoplasmic extracts [96,100,116,123,125,126,129,147]. The Western blotting and ELISA methods are commonly used to see the expression of several of the aforementioned proteins. Confocal microscopy can also be used to visualize the expression of p-NF-kB through the immunofluorescence technique [129]. An NF-kB-dependent luciferase activity assay is able to monitor NF-kB binding into the DNA binding site [83,132]. Activation of the MAPK protein is usually indicated by levels of the phosphorylated MAPK protein (p38, JNK, and/or ERK1/2) in the cytoplasmic cell extract [26,69,73,75,100,103,116,118,[127][128][129]132,135,147] or by an AP-1-dependent luciferase activity assay [132]. Besides inhibiting NF-kB and MAPK group activation, the Sargassum bioactive compound (ethanolic extract of S. serratifolium) is reported to suppress PI3K (phosphatidylinositol 3 kinase) and Akt (protein kinase B) phosphorylation in IL-1β-treated SW1353 chondrocytes. This phenomenon also relates to the improved inflammatory condition [68].
The modulation mechanism of the NF-kB signaling pathway used by Sargassum bioactive compounds is not yet clear. The presence of α-β-unsaturated carbonyl groups in terpenoids is thought to be responsible, in part, for NF-kB inhibition because the methylene group can react with the sulfhydryl cysteine groups (Cys 38 and Cys 120 ) located in the DNA binding domain of the p65 monomer (subunit NF-kB) [206]. Heras and Hortelano [153] summarized some specific mechanisms used in modulating the NF-kB signaling pathway by various terpenoid compounds: (1) Inhibition of IkB kinase (IKK) complex activation, (2) inhibition of proteasomal degradation on the IkB protein, and (3) blocking NF-kB nuclear translocation and/or NF-kB binding to the kB site. The most effective approach in NF-kB modulation is inhibition of the IKK complex's activation.
Some studies have revealed that Sargassum bioactive compounds can directly inhibit some proinflammatory enzymes, such as COX-2, COX-1, 5-LOX, PLA2, and hyaluronidase, via in vitro techniques [39,136,144]. The existence of electronegative functional groups, such as hydroxyl and The inhibition of NF-kB activation by Sargassum bioactive compounds (not only purified compounds, but also crude extracts and CSP) is proven by decreased phosphorylated NF-kB protein expression (p65 and/or p50), both in the nucleus and in cytoplasmic cell extracts [26,37,75,123,132,135], as well as inhibition of phosphorylation and degradation of the IkB protein in the cytoplasmic extracts [96,100,116,123,125,126,129,147]. The Western blotting and ELISA methods are commonly used to see the expression of several of the aforementioned proteins. Confocal microscopy can also be used to visualize the expression of p-NF-kB through the immunofluorescence technique [129]. An NF-kB-dependent luciferase activity assay is able to monitor NF-kB binding into the DNA binding site [83,132]. Activation of the MAPK protein is usually indicated by levels of the phosphorylated MAPK protein (p38, JNK, and/or ERK1/2) in the cytoplasmic cell extract [26,69,73,75,100,103,116,118,[127][128][129]132,135,147] or by an AP-1-dependent luciferase activity assay [132]. Besides inhibiting NF-kB and MAPK group activation, the Sargassum bioactive compound (ethanolic extract of S. serratifolium) is reported to suppress PI3K (phosphatidylinositol 3 kinase) and Akt (protein kinase B) phosphorylation in IL-1β-treated SW1353 chondrocytes. This phenomenon also relates to the improved inflammatory condition [68].
The modulation mechanism of the NF-kB signaling pathway used by Sargassum bioactive compounds is not yet clear. The presence of α-β-unsaturated carbonyl groups in terpenoids is thought to be responsible, in part, for NF-kB inhibition because the methylene group can react with the sulfhydryl cysteine groups (Cys 38 and Cys 120 ) located in the DNA binding domain of the p65 monomer (subunit NF-kB) [206]. Heras and Hortelano [153] summarized some specific mechanisms used in modulating the NF-kB signaling pathway by various terpenoid compounds: (1) Inhibition of IkB kinase (IKK) complex activation, (2) inhibition of proteasomal degradation on the IkB protein, and (3) blocking NF-kB nuclear translocation and/or NF-kB binding to the kB site. The most effective approach in NF-kB modulation is inhibition of the IKK complex's activation.
Some studies have revealed that Sargassum bioactive compounds can directly inhibit some pro-inflammatory enzymes, such as COX-2, COX-1, 5-LOX, PLA2, and hyaluronidase, via in vitro techniques [39,136,144]. The existence of electronegative functional groups, such as hydroxyl and methoxy, contained in Sargassum bioactive compounds is thought to be responsible for preventing the hydrogen abstraction from arachidonic acid in COX enzymes. Electronegative functionality also has the potential to coordinate the active site of COX/LOX enzymes through ion pairing, thereby preventing the biosynthesis of eicosanoid derivatives [39,136]. Electron-rich structures found in the macrolactone group, such as cyclic esters, the methyl acetate side chain, and vinyl or aryl substituents in the lactone ring, are reported to be positively correlated with the inhibition capacity on the COX-1, COX-2, and 5-LOX enzymes [144]. The reduction of NO concentration in inflamed cells or organisms could be caused by modulation of the upstream NO signaling pathway or direct NO scavenging by antioxidant compounds. Lopes et al. [143] reported that phlorotannin isolated from various species of brown seaweed, including S. vulgare, showed strong direct scavenging activity of Sodium nitroprusside (SNP)-generated NO in a cell-free system. This fact might contribute to the overall anti-inflammatory effect of phlorotannin in LPS-induced RAW 264.7 cells. Besides several aforementioned modulation mechanisms for Sargassum bioactive compounds, Chen et al. [207] proposed that maintaining the equilibrium state of the histone acetylation process as one type of post-translational modification can improve inflammation conditions. They reported that the Sargassum sp. polysaccharide extract treatment significantly inhibited the production of inflammatory cytokines and HAT (histone acetyltransferase) activity, but increased HDAC (histone deacetylase) activity and the mRNA expression of HDAC1 in porcine circovirus type 2 (PCV2)-infected RAW 264.7 cells.
Several other manifestations of the Sargassum's bioactive compounds against inflammatory conditions are include elevated intrinsic antioxidant activity (catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) [24,123,138], decreased production of ROS [24,33,142], decreased pro-inflammatory cell population (T-helper, T-cytotoxic, granulocytes, eosinophils, and monocytes) [76], stabilization of the lysosomal membrane (which inhibits lysosomal enzymes, such as cathepsin D, glucosaminidase, glucuronidase, etc.) [99,105,139], decreased edema volume and neutrophil infiltration in rat edema model [104], decreased production of osteoclastogenic factors (such as RANKL and osteoprotegerin in the IL-1β-induced MG-63 osteoblast cells) [147], improvement of dyslipidemia conditions [50,99], and amelioration of insulin resistance [33,44]. Increased intrinsic antioxidant enzyme activity is associated with the increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factors, the responsible protein in cell defense mechanisms [19,20,77,208]. Lee et al. [209] found that the oral administration of fucosterol (30 mg/kg/ day for seven days) in CCl 4 -induced rat hepatotoxicity was able to improve the nuclear translocation of Nrf2. Related to the improvement of dyslipidemia, Preetha and Devaraj [99] hypothesized that CSP treatment in hypercholesterol diet-induced rats could delay cholesterol absorption in the intestine and might accelerate cholesterol excretion by modulating cholesterol-7-alpha-hydroxylase, a key enzyme for the conversion of cholesterol into bile acids.
Another mechanism that may be ascribed to the anti-inflammatory activity of Sargassum bioactive compounds is the interaction between active compounds and inflammatory-associated receptors, such as TLR4, CD14, CR-3, and SR-A. This interaction can interfere with the recognition of strong inflammatory stimuli by cells, e.g., LPS. The presence of sulfate groups in FCSPs is thought to affect its binding to inflammatory receptors [169,210]. Omega-3 fatty acids (especially SDA and EPA) in Sargassum may undergo incorporation into cell membranes and affect eicosanoid metabolism in cells. EPA and DHA can interfere with the activity of the COX-2 and 5-LOX enzymes against AA, so the production of strong pro-inflammatory PGE 2 and LT 4 can be inhibited [211]. Tian et al. [212] found that RAW 264.7 cell incubation with EPA, DHA, or DPA (docosapentaenoic acid) for 72 h suppressed the inflammatory response after LPS stimulation.

Conclusions
Sargassum is well-known to have beneficial effects on health and is traditionally utilized by the East Asian community to treat various inflammatory-related diseases. Various bioactive compounds of Sargassum show potential anti-inflammatory activity, both in acute and chronic conditions. The potency of tropical Sargassum as a source of the anti-inflammatory agent is not fully explored yet. Sulfated polysaccharides from tropical Sargassum are the most studied compounds for their anti-inflammatory activity. However, there are various other potentially bioactive compounds, such as non-polar compounds (fatty acids and derivatives, carotenoids and derivatives, steroids, and polar lipids), grasshopper ketone, polyketide macrolactone, and phenolic components. Bioactive compounds of Sargassum are reported to modulate the inflammatory response by inhibiting NF-kB and MAPK activation, direct inhibition of inflammation-associated enzymes, and direct scavenging of radical species. The information presented in this paper is expected to provide input for Sargassum development, especially its tropical species, as a source of anti-inflammatory agents.
Funding: This research received no external funding.