Marine fucoidans: Structural, extraction, biological activities and their applications in the food industry

. The structural complexity of fucoidans is dependent on the species, source, harvesting time, among other factors. This in turn greatly influences the biological activity of fucoidans, with high degree of sulphation and low molecular weight particularly linked with increased biological activity. Due to the presence of sulphate moieties, fucoidans easily interact with other polymers with far-reaching applications.


Introduction
Fucoidan is a sulphated polysaccharide present in the cell wall of brown seaweed.It is a water-soluble polysaccharide first isolated in 1913 by Kylin from Undaria pinnatifida (Zhao et al., 2018).They are structurally characterized by the presence of an α-fucopyranose backbone with sugar monomers such as glucose, mannose, galactose, and uronic acids (Huang, Kuo, & Chen, 2017;Kopplin et al., 2018).Fucoidans are species-oriented and thus do not possess any specific chemical structure, however, structural properties look similar from specie to specie (Ahmed, Mona, Abdel-Rahim, & Roland, 2020).Many reports showed the biological activities of fucoidans which include anticancer, antioxidant, antiviral, and cardio-protective, among others (Lee & Lee, 2006;Parvez et al., 2019;Raguz & Yague, 2008;Rivas, Gutierrez, Arteaga, Mercado, & Sanchez, 2011).Both high and low molecular weight fucoidans exhibit enhanced apoptosis of cancer cells (Deng et al., 2022;Lu et al., 2018;Park et al., 2017) and anticoagulant activity (Yang et al., 2008;Zhao et al., 2016).Recent in silico and in vitro studies showed high anti-inflammatory properties of fucoidan compared to alginate on THP-1 Cells (Huwait et al., 2022).Research has proven that the biological activities of fucoidans are directly dependent on their unique structural composition, and molecular weight coupled with other factors such as species of origin, harvesting time, extraction method used, and location of seaweed (Zayed et al., 2016).The structural heterogeneity and composition of fucoidans has become a research area of interest in recent years due to their health benefits to human beings (Rioux, Turgeon, & Beaulieu, 2007;Ustyuzhanina et al., 2013).As a result, studies on structural composition has become an area of high exploits.However, several mechanisms underlying their pharmaceutical and biological activities have not been well documented.Additionally, fucoidans have been applied in the pharmaceutical, food, and cosmetic industries and thrived well in these areas due to their array of biological importance.Therefore, this review aims to document the structural compositions, extraction methodologies and their effect on the biological activities of fucoidans, and recent advances in the applications of fucoidans in the food industry.

Table 1a
Extraction and purification of fucoidans.

Species
Extraction method Extraction conditions Purification Yield (%) References

F. evanescens Enzyme-assisted extraction
Cellulase and alginate lyase was added to the seaweed at 40 • C and pH adjusted to 6 for 24 h.

A. nodosum Enzyme-assisted extraction
Cellulase and β-glucanase was added to the seaweed at 50 • C and pH adjusted to 6 for 3 h.
Ultrafiltration using a membrane of 100 kDa.

Extraction and purification of fucoidans
The method of extraction is crucial as it determines yield and purity of fucoidan obtained from seaweeds.Despite the good yield obtained with conventional solvent extraction method, the method is time consuming and the end product require further purification which is not ideal for the food and pharmaceutical industries (Ale, Mikkelsen, & Meyer, 2011;Otero et al., 2021;Yu et al., 2018).Thus, green methods of extracting fucoidan have been developed and these includes microwave-assisted extraction, ultrasound-assisted extraction, enzyme-assisted extraction, pressurized liquid extraction and pulse-electric field extraction method (Hahn, Lang, Ulber, & Kai, 2012).These extraction methods are briefly explained below and further summarized in Table 1a and Fig. 1b.1.1.1.1. Conventional extraction.The conventional method of extracting fucoidan generally employs acids treatment prior to extraction with hot water as the main solvent.Basically, it is a solid (i.e., seaweed) to liquid (i.e., hydrochloric acid, water) extraction technique which involves heating (70-100 • C) with or without agitation in a water bath (Ale, Mikkelsen, & Meyer, 2011;Paz et al., 2021).Extraction of fucoidan usually lasts for several hours to achieve effective extraction (Garcia--Vaquero, Rajauria, & Tiwari, 2020).It is reported that pH, concentration of acid, and temperature determine the yield and process time (Hahn et al., 2012).In some cases, calcium salt (i.e., CaCl 2 ) is added to precipitate alginate (Ammar et al., 2015;Bittkau et al., 2020).For instance, Hanjabam et al. (2019) extracted crude fucoidan from Sargassum wightii by mixing seaweed with acidified distilled water (pH = 2) in a ratio of 1:25 respectively.The suspension was stirred for 15 min and allowed to swell followed by heating to 85 • C with continuous stirring for 2 h.The suspension was neutralized with 1 N NaOH and filtered using a nylon mesh funnel (Hanjabam et al., 2019).The filtrate was subjected to centrifugation at 6500 rpm at room temperature for 15 min.The centrifuged extract was filtered through an 11 μm filter paper and concentrated at 45 • C using a rotary evaporator.Alcohol (70% v/v) was used to precipitate the viscous fucoidan, then filtered with 1 μm nylon filter and freeze-dried.The authors repeated the extraction twice and achieved a yield of 10.59% (Hanjabam et al., 2019).Crude ethanolic extracted fucoidans are purified either by dialysis or ion-exchange chromatography to obtain a pure extract (Hahn et al., 2012).Dinesh et al. (2016) reported 5.96% yield of dry mass fucoidan obtained from S. swartzii using 0.05 M HCl, extraction at room temperature for 24 h.Others have reported higher yield (19% (w/w) of fucoidan extracted from Sargassum species using a conventional method of extraction (Hifney, Fawzy, Abdel-Gawad, & Gomaa, 2016).Despite their effectiveness, there are drawbacks associated with the conventional extraction methods which includes extended extraction times, requires large volumes of solvents, and high degree of heat that can denature and alter the structure of fucoidan (Paz et al., 2021).Furthermore, the relationship between the extraction time and yield is critical, as extended duration at higher temperatures (80 • C and above) may decrease fucoidan yield and influence their biological activity.It is reported that extraction at higher temperatures (80 • C and above) degraded the hydrogens bonds which led to a loss of fucose integrity (Baba, Mustapha, & Joe, 2018;Flórez-Fernández, Balboa, & Domínguez, 2020;Sugiono, Widjanarko, & Soehono, 2014).

Enzyme-assisted extraction.
Enzyme-based extraction (EBE) is a green method that employs the use of digestive enzymes such as proteases (i.e., alcalase) or flavourzyme and carbohydrase (i.e., cellulases or viscozyme) (Heo, Park, Lee, & Jeon, 2005).Enzymatic extraction of fucoidan from seaweed is effective because it degrades cell wall without compromising components inside the seaweed (Hahn et al., 2012).EBE of fucoidan depends on the appropriate enzymes (cellulase, alginate lyase, β-glucanase, etc.), optimum conditions (temperature, pH), and the seaweed utilized (Adadi, Barakova, & Krivoshapkina, 2018;Rhein--Knudsen, Reyes-Weiss, & Horn, 2023).A previous report demonstrated that EBE reduced extraction time; enhanced extractability/yield; conserved biological activity of fucoidans, and minimized or eliminated solvent use.In addition, it is environmentally friendly and does not arouse criticism; renewable, relatively cheaper than organic solvents; and exhibit high specificity (Adadi & Barakova, 2019;Adadi et al., 2018;Rhein-Knudsen et al., 2023).However, one key drawback is the liability of enzymes to denaturing at high temperatures (Abdul Khalil et al., 2018;Adadi et al., 2018).During enzymatic extraction of fucoidans, polyphenols are released as by-products that contaminates the fucans thus requiring extended purification (Ahn, Jeon, Kang, Shin, & Jung, 2004;Park, Shahidi, & Jeon, 2004).Generally, parameters such as temperature, pH, enzyme/sample ratio, and solvent used determine the effectiveness of the process (Abdul Khalil et al., 2018;Adadi & Barakova, 2019;Adadi et al., 2018).Nielsen et al. extracted fucoidan from F. evanescens with cellulase and alginate lyase at 40 • C for 24 h.Heat (90 • C) was used to halt the enzymatic reaction and cooled on ice (Nielsen et al., 2021).After centrifugation, 2% CaCl 2 was added to precipitate alginate and the mixture centrifuged again.72% ethanol was used to precipitate the fucoidan and lyophilized (Nielsen et al., 2021).Similarly, a recent report showed that treating S. latissimi and Alaria esculenta with commercial cellulase blend Cellic® Ctec2 along with endo-(AMOR-PL7) and exo-acting (AMOR-PL17) thermophilic alginate lyases at a ratio of 30/70 resulted in the highest fucoidan purity, with less content of alginate and glucose compared to the convention methods (Rhein-Knudsen et al., 2023).Cellulase and alginate lyase assisted extraction of fucoidan from Sphingomonas sp has also been reported (Dörschmann et al., 2020).Enzyme assisted extraction of fucoidan from F. evanescens and S. latissimi resulted in 40% and 29% yields respectively and had substantial amount of low molecular weight alginate compared to mild acidic extraction (Nguyen et al., 2020).In general, enzyme specificity enhances clean and effective extraction processes resulting in high yields and purified forms of fucoidan.
Table 1a shows previous literature of enzymatic assistant extraction of fucoidan.
1.1.1.5.Purification methods.Purification is the next step after extraction of fucoidans due to contamination with low molecular weight impurities such as polyphenols and proteins (Flórez-Fernández et al., 2020;Otero et al., 2021).To obtain purified fraction of fucoidans, various purification processes such as precipitation, membrane filtration, and chromatographic methods are used (Alboofetileh, Rezaei, & Tabarsa, 2019;Dobrinčić et al., 2021;Nguyen et al., 2020) (Table 2).The precipitation method is widely used due to its simplicity and rapid nature.It involves the addition of either ethanol or CaCl 2 to the crude extract followed by series of centrifugation to obtain purified fucoidan (Flórez-Fernández et al., 2020;Zayed & Ulber, 2020).Report showed that ethanol is preferred in precipitation compared to other solvents due to its lower dielectric constant thus forms ionic bonds with the sulphate groups of fucoidan and the positive ions in solution (Hahn et al., 2012).
In addition, ethanol precipitation of fucoidan decreased concentration of contaminants such as proteins, polyphenols, and ash (Fernando et al., 2020).Precipitation of crude fucoidan extract resulted in 13.9% (Dobrinčić et al., 2021) and 14.04% (Yuan & Macquerie, 2015a) yields of pure fucoidan.Others employed different precipitation methods such as isoelectric point precipitation, salting out, and trichloroacetic acid denaturation to achieve a significantly purified fucoidan with less protein concentrations.However, the presence of some low molecular weight compounds (i.e., polyphenols) persisted (Wang et al., 2021).Salts (i.e., calcium acetate) (Saboural et al., 2014) and cationic surfactants (CTAB) were used to significantly reduce the concentration of proteins and other lower molecular wight compounds thus improved purified fucoidan recovery yield (Bilan et al., 2010).
The membrane filtration, also known as dialysis membrane method,
The chromatographic methods such as the anion-exchange, size exclusion and affinity chromatography are efficient in purifying crude fucoidan (Zayed & Ulber, 2019).The anion-exchange method is based on binding affinity of anionic exchangers for sulphate esters on the fucoidan.
The size exclusion chromatography, on the other hand, is a purification method based on the separation of molecules by their shape and size through a porous matrix (García-Vaquero, Rajauria, O'Doherty, & Sweeney, 2017).The common matrices used are hydrogels, Sephadex®, Superdex®, Sepharose®, among others (Flórez-Fernández et al., 2020;Kaiyun et al., 2018;Zhang & Row, 2015).This method is fast however, the resolution is very low as compared to other techniques, as such others have coupled size exclusion chromatography with dialysis or ion-exchange chromatography to enhace purification (Cutler, 2004).Size-exclusion chromatography with a ÄKTA Pure 25 column and UV-Vis detector was used to separate and detect inpurities (i.e., polyphenols and proteins) in crude fucoidan samples.Subsequently, purified fucoidan was obtained after elution with phosphate buffer (Neupane, Bittkau, & Alban, 2020).The type and flow rate of the mobile phase enhance separation of other molecules from fucoidan (Zhang & Row, 2015).It is reported that low flow rate, enhanced diffusion thus longer retention time and the vice verse.However, high flow rate can influence diffusion of the polysaccharides through the column which can lead to poor purification (Zhang & Row, 2015).
Lastly, the affinity chromatography involves passing crude fucoidan extract through a stationary phase with ligands or affinities that binds with fucoidan (Nagy, Peng, & Pohl, 2017).Lectins and toluidine are used as the affinities for separation of fucoidans where the basis of seprartion or purification is either by ionic or disperse interactions between molecules (Hahn et al., 2016).Affinity chromatography is rapid with high perfomance and resolution.However, it is expensive and lectin affinity can be influenced by the presence of sulphate esters (Zayed & Ulber, 2019).With regards to ionic interactions, the types of affinities for fucoidan purification should be within pH of 1-6 to

Table 2b
Antitumor mechanism of fucoidans.Antiproliferative activity Non-small-cell human bronchopulmonary carcinoma (NSCLC-N6) ehnance its efficacy (Zayed et al., 2016).Report showed that at a lower acidic pH, the affinities formed aggregates and capture fucoidans in solution whilst impurities passed thourgh (Zayed, Dienemann, Giese, Krämer, & Ulber, 2018).Toluidine-blue affinity chitosan microsphere was effective in fucoidan purification due to their swelling nature.The nature of the microsphere allows for the fast transfer of low molecular weight fractions (adsorbates) and their desorption after the process (Qiao & Du, 2021).Fucose-specific lectins was used to purify fucoidan extract by mainly targeting α-fucopyranose chains similar to fucosylated proteins in glycoproteins (Matsumura et al., 2007).Additionally, purification with amino-derivatized Sepabeads® modified with toluidine blue yielded purified fucoidan fraction of ~95% purity (Hahn et al.,

Table 2c
Anticoagulant and antithrombic activity of fucoidan.).These methods provide details regarding the molecular structure, bonding pattern as well as functional groups of components in the purified samples.NMR is a widely used method for the characterization of the structural features of polysaccharides as well as impurities profiling (Zha et al., 2015).By  Where NO: nitric oxide, TNFα: tumor necrosis factor alpha, IL -1β: interleukin 1 beta, LPS: lipopolysaccharide, NF-κB: nuclear factor kappa B, PBMC: peripheral blood mononuclear cell, THP-1: human leukemia monocytic cell.

Table 2f
Antibacterial activity of fucoidan.(Du et al., 2022).In addition, de-shielding of the anomeric proton at 5.1-5.8ppm or anomeric carbon at 98-103 ppm confirms the presence of α-linked sugar monomers (Yao et al., 2021).In general, 1 H NMR spectroscopy is a more sensitive method than 13 C NMR, however, the overlap of signals is less an issue in 13 C NMR (Korva, Kärkkäinen, Lappalainen, & Lajunen, 2016).FT-IR is another widely used method in polysaccharides characterization and purity evaluation.It is also used to elucidate the position of sulphate ester groups and glycosidic bonds (Zayed et al., 2020).Sulphated polysaccharides like fucoidan exhibited a broad band around 1220-1260 cm − 1 , ascribed to the presence of sulphate ester groups (S--O) (Gómez-Ordóñez & Rupérez, 2011).Additionally, bands around 845 cm − 1 correspond to C-O-S, secondary axial sulphate, might indicate that the sulphate group is located at C-4 of the fucopyranosyl residue (Chale-Dzul, Moo-Puc, Robledo, & Freile-Pelegrín, 2015).The peak centering 1035 cm − 1 represents the characteristic FTIR band of C-O-C stretching vibrations of the glycosidic bridges (Du et al., 2022).Also, fucoidans exhibit two bands in the 3600-2000 cm − 1 region corresponding to OH stretching vibration, and CH stretching in pyranoid ring, and C-6 groups of fucose units.Besides, bands around 1032 and 1043 cm − 1 arise from guluronic and mannuronic acid residue, which is characteristic of residual alginate (Chale-Dzul et al., 2015).Furthermore, the matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry is one of the frequently used MS methods for analysis of fucoidans and other polysaccharides.It is used to distinguish polysaccharides based on their oligosaccharide profile, taking into account monosaccharide constituents and sequence, linkage type, branch features as well as functional groups (Guo, Ai, & Cui, 2018).To achieve this fucoidan sample is partially hydrolyzed, derivatized and analyzed with tandem MS.In negative ion mode, fragment ion at m/z 225 representing [FucSO3-H2O] can be detected using MALDI-TOF-MS.Also, several fucooligosaccharides of different length and degree of sulfation of both derivatized and underivatized fragments can be observed as well, such as [FucSO 3 ] − at m/z 243.02; [Fuc 2 SO 3 ] − at 389.08; [Fuc 3 (SO 3 Na) 3 -Na] − at 739.1 (Zayed et al., 2020).Anastyuk, Shevchenko, and Gorbach (2015) provide more extensive information on the characterization of fucoidans using MALDI-TOF-MS.The electrospray-ionization (ESI) tandem MS is also another widely preferred MS method for characterization of fucoidans.ESI differs from MALDI largely in ionization strategy.Comparatively, MALDI-TOF has higher sensitivity for glycans, provides good ionization even at higher mass range, and it can tolerate contaminants (Guo et al., 2018).
Besides molecular/structural characterization, comprehensive monosaccharide profiling and linkage analysis can be useful for differentiating fucoidans from other polysaccharide contaminants (Fitton, Stringer, & Karpiniec, 2015).This usually involves complete hydrolysis, derivatization (when necessary) and then analysis using gas chromatography (GC) or high-pressure liquid chromatography coupled (HPLC) with MS (Li et al., 2022).Fucoidans are generally expected to possess higher fucose content relative to other sugars (Du et al., 2022).Moreover, the ratio of fucose to other monosaccharides should improve in purified samples as carbohydrate contaminants are removed during purification.Additionally, glycosyl linkage analysis is a widely used method for characterization of oligo-and polysaccharides.It involves derivatization of the sugars monomers of a polysaccharide to partially methylated alditol acetates (PMAAs) which are then analyzed with GC-MS (Sims, Carnachan, Bell, & Hinkley, 2018).Fucoidans typically contain α-(1 → 3)-L-fucose or alternating α-(1 → 3) and α-(1 → 4)-linked L-fucose.In addition, the purity of fucoidans can be assessed by directly identifying and quantifying common contaminants like protein and phenolic compounds.Colorimetric assays such the Lowry, Bradford, and BCA assays can be used to quantify total protein content in fucoidan extract (Olson, 2016).Ideally, purified fucoidan samples should have no proteins.
The challenge in assessing and validating the purity, however, is the chemical heterogeneity of fucoidans.At present, it is generally accepted that some sugar monomers and other functional groups are not universally present in all fucoidans.As such it is difficult to judge whether observed structural characteristics of non-conventional monomers or functional groups are impurities or components of the fucoidans themselves.A single method is, thus, not sufficient to fully characterize the purity of fucoidans.Nevertheless, these techniques can differentiate fucoidans from common contaminants such as proteins and other polysaccharides.

Chemical composition of fucoidans
Fucoidans are anionic polysaccharides composed of fucose, sulphate, and other monosaccharides (Table 1b).The net negative charge of fucoidans is due to the presence of the sulphate groups (Li et al., 2008).The position of these sulphate groups on fucoidan differs between species (Fig. 1a).In brown seaweeds such as A. nodosum or F. vesiculosus, the sulphate groups are situated at C 2 and/or C 4 and rarely at C 3 (Wang, Jayawardena, et al., 2020).However, fucoidans in C. filum possess sulphates linked at either C 2 or C 4 (Bilan & Usov, 2008).The presence of these sulphate groups in fucoidans has been linked to various biological activities and the ability to form nanoparticles, nanoemulsions and nanogels via electrostatic interaction with positively charged polymers (Bilan & Usov, 2008).Table 1b shows the chemical composition of fucoidan extracted from various seaweeds.The position of these sulphate groups on fucoidan are influenced by the location, season, extraction process, and harvesting time (Mak et al., 2013).

Antioxidant activity
In cells or cellular processes (such as cellular respiration), oxidative stress resulting from the production of free radicals can lead to regenerative diseases (i.e., inflammations, cancer, macular degeneration, among others).Free radicals are reactive oxygen species (ROS) such as hydroxyl radical, hydrogen peroxide, superoxide anion, singlet oxygen and nitric oxide (Lee & Lee, 2006).Generally, low ROS concentrations are beneficial as they regulate biochemical processes in cells (i.e., cell division) whereas high levels cause disruption of cellular homeostasis thus triggering various diseases (i.e., insulin resistance, atherosclerosis, heart failure, etc.) (Kang et al., 2015).Antioxidants are substances that scavenge free radicals from the body to prevent diseases (Soubra, Sarkis, Hilan, & Verger, 2007).
The antioxidant activity of fucoidans depends on several factors such as, concentration, molecular weight, degree of sulphation, type of sugar monomers, substituents, and their positions (Jin, Ren, Liu, Zhang, & Zhong, 2018;Palanisamy et al., 2017).The concentration of fucoidans needed to scavenge free radicals is very important; since high concentrations caused adverse reactions (Wang, Liu, et al., 2009;Wang, Zhang, Zhang, Zhang, & Li, 2009).Also, the antioxidant activity of fucoidans is influenced by their molecular weight.High molecular weight fucoidans couldn't effectively cross the lipid bilayer to exert full antioxidant activity thus a lower molecular weight fucoidan with potent crossing ability is preferred due to its high scavenging activity (Zhang et al., 2010).Another report showed low molecular weight fucoidans from L. japonica had significant effect on the oxidation of low-density lipoprotein (LDL) compared to high molecular weight fractions (Zhao, Wang, & Xue, 2011).The authors indicated that the presence and position of sulphate esters on the fucose sugar enhanced the scavenging activity (Zhao et al., 2011).

Antitumor activity
Cancer is a deadly disease whose mortality and morbidity rate has increased over the years.Currently, chemotherapy is widely used for cancer treatment but comes with significant adverse effects such as hair loss, bone marrow malfunction, among others (Wang, Probin, & Zhou, 2006).Due to this, various bioactive polymers (i.e., fucoidans) have been explored as natural alternatives with less or no adverse effects.Fucoidans isolated from seaweed exhibited anticancer activity against breast adenocarcinoma cell line MC7F-7, lung carcinoma cell line A-549, colon adenocarcinoma cell line WiDr (Li et al., 2017).In addition, fucoidan isolated from F. vesiculosus and U. pinnatifida exhibited antiproliferative activity against prostate and hepatocellular cancer cells (Mak et al., 2013).Research has shown the inhibitory activity of fucoidans against various cell lines associated with colon cancer like human colorectal adenocarcinoma HT-29, DLD-1, and HCT-116 (Vishchuk, Ermakova, & Zvyagintseva, 2011).Others reported that various fucoidan concentrations inhibited MCF-7, HepG2, NCI-H460 and HeLa cancer cell lines in a dose-dependent manner (Hoang et al., 2022).Similarly, fucoidan showed effective inhibition against MCF-7 compared to HepG2, NCI-H460 and HeLa cancer cell lines (Hoang et al., 2022).
The mechanism of fucoidan antitumor activity is summarized in Fig. 2b and Table 2b.The antitumor potency of fucoidans is correlated with the carbon backbone, degree of sulphation and the molecular weight (Ale, Mikkelsen, & Meyer, 2011).Fucoidan has been reported to exhibit its antitumor or anticancer activity by inducing both intrinsic and extrinsic pathways of apoptosis.Also, fucoidan induced the activation of the caspase genes (i.e., caspase 9, 8, and 3) with a significant downregulation of apoptotic proteins inhibitors (Yang, wang, Wang, Teng, Liu, Yang, Hou, & Zou, 2013).Fucoidan-mediated inhibitory mechanisms and reactions on relevant receptors related to cancer and apoptosis have been depicted in Fig. 2b.Fucoidans can also trigger apoptosis via ROS-mediated mitochondrial pathway by increasing the production of ROS leading to the induction of mitochondrial oxidative damage and subsequent cell death (Wang, Li, White, & Lu, 2014).Fucoidan induced autophagy in stomach cancer cells leading to cell death thus decreased tumor growth (Park, Kim, Nam, Kim, & Choi, 2011).
The antitumor activity of fucoidans is highly associated with the chemical structure and molecular weight (Cumashi et al., 2007).Low molecular weight fucoidans (LMWF) showed more potent antitumor activity than high molecular weight fucoidans.LMWF induced apoptosis of human colon cancer cells through p53-independent mechanism (Park et al., 2017).Also, fucoidan isolated from brown algae with high degree of sulphated groups exhibited potent antitumor activity against cancer cells than those with low sulphated groups (Ermakova et al., 2011).Contrarily, fucoidans isolated from S. hornery that contain no sulphated group showed antitumor activity, this was attributed to its high purity (Ermakova et al., 2011).The purity of fucoidan and its concentration determines the antitumor activity in various tumor model studies.

Anticoagulant and antithrombic activity
Disorder of blood coagulation is a very serious health condition caused by various diseases which can lead to death.Increased blood Fig. 2b.Clinical applications for fucoidan that interacts with the receptors-mediated anti-cancer pathways and reactions.Adapted from Hsu and Hwang (2019).Copyright@2019, Springer Nature.clotting (thrombosis) as well as its absence which causes excessive bleeding (hemorrhage) may lead to mortality (Rivas et al., 2011).Heparin (sulphated polysaccharide) extracted from mammalian tissues is commercially available and widely used as an anticoagulant.However, it has been criticized due to their adverse effects such as thrombocytopenia, hemorrhagic activity and among others (Suleria et al., 2017).Thus, bioactive polysaccharides extracted from brown algae has been experimented and shown strong antithrombic activity without any side effects (Sudharsan et al., 2015).Hoang et al. showed that fucoidan (1000 ppm) demonstrated potential anticoagulant activity but lower than the conventional anticoagulant Heparin (Hoang et al., 2022).
Fucoidan has a potent anti-thrombotic activity and acts as an Xainhibitor to prevent blood clots in the blood vessels therefore decreasing the probability of strokes, brain disorders and hypertension developments (Lapikova et al., 2008).Fucoidans prolonged prothrombin time (Ustyuzhanina et al., 2013).Others have demonstrated that fucoidans increased the levels of tissue-plasminogen activator (Ti-PA) which prevented the coagulation of blood in the blood vessels (Cui et al., 2014).Brown seaweeds (i.e., F. vesiculosus, F. serratus, N. decipiens, S. latissimi, U. pinnatifida) have been reported to exhibit strong antithrombic activity (Sun, Zhang, & Miao, 2018).The Anticoagulant and antithrombic activity of fucoidan has been corroborated by several researchers as shown in Table 2c.
Like other biological activities, the antithrombin activity of fucoidans has been reported to be dependent on the chemical composition (such as sulphation) and molecular weight (Wang, Zhang, Zhang, Hou, & Zhang, 2011).Yang et al., revealed that, fucoidans exhibited potent anticoagulant activity at a molecular weight of about 10 kDa-300 kDa (Yang et al., 2008).Others reported that low molecular weight fucoidan (M w = 7.6 kDa) isolated from L. japonica was bio-accessible thus absorbed and exhibited antithrombic activity than high molecular weight fucoidan (Zhao et al., 2016).Further reports showed that fucoidan with high sulphate groups positively influenced the anticoagulant activity compared to other fractions with less content (Chevolot, Mulloy, Ratiskol, Foucault, & Colliec-Jouault, 2001;Wang et al., 2011;Wang et al., 2010).

Antiviral activity
Recent health and research data demonstrates how susceptible humans are to various viral infections which causes serious ailments and even death (Parvez et al., 2019).Antiviral drugs have been developed to combat these viruses.These synthesized antiviral drugs are unfortunately, not without drawbacks including organ toxicity, viral resistance, narrow spectrum of activity (Wang, Wang, & Guan, 2012).Thus, research has been focused on developing bioactive polysaccharides as antiviral agents with less or no side effects (Ventola, 2015).Moreover, in vitro and in vivo trials with fucoidan showed a broad antiviral spectrum, thus may be used in conjunction with griffithsin protein and antiherpetic drugs to prevent viral diseases (Lomartire & Gonçalves, 2022).Various fucoidan concentrations inhibited viral infections including hepatitis, influenza virus, human immunodeficiency virus (HIV), bovine viral diarrhea, herpes simplex virus (HSV) and human papilloma virus (Dinesh et al., 2016).Table 2d summarizes the antiviral activity of fucoidan against viruses.Concentration of 0.5 and 1 μg/mL fucoidan extracted from brown seaweed showed 84.0 ± 4.3 and 84.6 ± 7% inhibitory effects against HIV-1 reverse transcriptase (Queiroz et al., 2008).Also, in vitro study showed that fucoidan isolated from C. okamuranus decreased the infectivity of Newcastle disease virus with low toxicity compared to Ribavirin (Lizondo-Gonzalez & Cruz-Suarez, 2012).Crude fucoidan zinc oxide (ZnO) nanoparticles (46.88 μg/mL) showed 99.09% inhibitory effects against dengue 2 viruses than crude fucoidan a positive control (Kothai, Arul, & Anbazghan, 2022).Furthermore, an in vivo study demonstrated potent inhibitory activity of fucoidans against the influenza virus (H1N1) which decreased gross lung pathology in a mouse model (Richards et al., 2020).
The antiviral activity of fucoidans has highly been associated with degree of sulphation, molecular weight, species, and the sugar backbone (Sun et al., 2018).Highly sulphated fucoidans exhibited high antiviral activity compared to low sulphated ones.Also, fucoidans extracted from the genus Fucus spp exerted more potent virucidal activity than those from S. scroederi and D. mertensii (Queiroz et al., 2008).Most importantly, LMWF was more effective and efficient in treating viral infections than HMWF.Additionally, LMWF isolated from L. japonica elevated the immune system as well as the spleen and thymus index (Adhikari et al., 2006).The interaction between negatively charged sulphate groups of fucoidan and the positively charged glycoproteins of the virus cell envelope inhibits the attachment of the virus to the host cell (Jiao et al., 2012) which prevent virus from replicating (Tan et al., 2022).Fucoidan from F. vesiculosus significantly upregulated 834 genes to produce innate immunity against human norovirus (hNoV) when injected in a zebrafish larva.The authors also revealed the inhibitory activity of fucoidan against Tulane virus (TV) in an in vitro study which significantly blocked its replication (Tan et al., 2022).These studies demonstrate promising results regarding the antiviral activities of fucoidans, for pharmaceutical and food industries to explore.

Anti-inflammatory activity
Inflammation is a defense mechanism that is stimulated by the immune system as a line of action against infections or other harmful stimuli.When controlled correctly inflammation helps the body's resistance to disease, however, hyperinflammation leads to tissue damage.Uncontrolled inflammation leads to dysregulated function which causes diseases such as arthritis, periodontitis, allergies, Crohn's disease and among others (Megha, Joseph, Akhil, & Mohanan, 2021).Bioactive polysaccharides have been reported to exhibit anti-inflammatory potency in several models (Phull, Majid, Haq, Khan, & Kim, 2017).
Using denaturation of egg albumin as a model showed that purified fucoidan elicited potent anti-inflammatory effects mediated by the inhibition of protein denaturation.In addition, the anti-inflammatory effects were dose dependent and positively correlated with the content of fucose and sulphate (Obluchinskaya, Pozharitskaya, & Shikov, 2022).Similarly, rats fed with 400 mg/rat fucoidan-based cream showed considerable anti-inflammatory effect compared to 100 mg/rat diclofenac gel 1% (positive control) (Obluchinskaya, Pozharitskaya, Flisyuk, & Shikov, 2021).
In vitro study showed that 250 mg/kg fucoidan exerted potent antiinflammatory effects compared to 10 and 50 mg/kg fucoidan treated mice but not the positive control (diclofenac sodium) (Manggau et al., 2022).Also, fucoidans regulates the inflammatory response, mainly intestinal inflammatory response (Park et al., 2017).In addition, purified fucoidans inhibited inflammatory enzymes, such as nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and other inflammatory mediators such as nitric oxide (NO) and pro-inflammatory cytokines (Jayawardena et al., 2020;Wang, Jayawardena, et al., 2020).Another study revealed that fucoidan extracted from T. decurrens suppressed the expression of COX-2, interleukin 1β and matrix metallopeptidase 9 (Dinesh et al., 2016;Manikandan et al., 2020).The anti-inflammatory activity of fucoidans has been shown in several other studies (Table 2e).The anti-inflammatory activity of the fucoidan was associated with the degree of sulphation, monosaccharide composition, and the species from which it is extracted.It was reported that, fucoidans with high sulphate content inhibited production and release of inflammatory mediators compared to low content ones (Jayawardena et al., 2020).Also, fucoidans with high fucose backbone showed high anti-inflammatory activity than those of low fucose residues (Sanjeewa et al., 2018).

Antibacterial and antifungal activities
Bacterial species such as Clostridium tetani, Neisseria gonorrhoeae, Mycobacterium tuberculosis, Vibrio cholerae cause life-threatening diseases such as tetanus, gonorrhoeae, tuberculosis, and cholera respectively.Antibiotics are used to cure bacterial infections however, the fight against these bacterial infections has reached a plateau where new bacterial species have developed resistance against these antibiotics (O'neill, 2014).The menace of antibiotic-resistance causes approximately 700,000 deaths yearly which is estimated to increase (World Health Organization, 2020).Many researchers have evaluated potent bioactive compounds with less toxicity to combat bacterial infections and fucoidan is a promising candidate (Yu et al., 2018).
Fucoidans exhibit antibacterial potency by binding to the membrane proteins of bacteria and induce expression of apoptotic pathways that disrupts the bacterial membrane (Ibtissam et al., 2009) (Fig. 2cA-B).The mechanism of action is attributed to the presence of sulphate group, glucuronic acid, and the position of sugar backbone (Ibtissam et al., 2009;Yu et al., 2018) (Table 2f).Research has showed that Gram-positive bacteria are more susceptible to inhibition by fucoidan as compared to Gram-negative bacteria because the lipopolysaccharides (LPS) present on Gram-negative cell walls confer additional strength to the bacteria (Nagayama, Iwamura, Shibata, Hirayama, & Nakamura, 2002).Other have reported inhibitory activity of fucoidans against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus licheniformis and the result showed that all tested strains were susceptible to fucoidan (Ayrapetyan et al., 2021).Moreover, Gram-negative bacteria (E.coli) were more susceptible to inhibition by fucoidan compared to Gram-negative ones partly because it binds to the bacteria membrane and altered the lipopolysaccharide resulting in leaching of intracellular metabolite (Ayrapetyan et al., 2021).
Fucoidan reportedly caused distortion and destructive breakage in the cell membrane of the Candida spp and inhibited DNA and protein synthesis (Alghuthaymi et al., 2021).

Industrial application of fucoidans
Currently, new, and significant developments have evolved in the  pharmaceutical, food, cosmetics, and the nutraceutical industries due to consumer awareness and demand for healthier products.Thus, producers have adopted new means to produce novel products from natural functional ingredients (Wijesinghe & Jeon, 2012).Fucoidans are an excellent candidate to develop these products due to their potent biological activities which confer health benefits to humans.Below are some applications of fucoidans in the food industry (Fig. 3a).

Application of fucoidan in the food industry
In the food industry, fucoidans have been successfully used to develop delivery vehicles for bioactive compounds, food packages, designing functional foods, and as stabilizers.

Fucoidan-based nutraceuticals
Nutraceuticals are food or parts of food that provide medical or health benefits, including the prevention and treatment of disease (Defelice, 1995).Various fucoidan-based delivery systems are summarized in Table 3a and Fig. 3b.Generally, fucoidans are used as delivery systems such as nanoparticles, nanoemulsions, nanocapsules, ands hydrogels to encapsulate bioactive compounds (i.e., quercetin, anthocyanin, etc.) for control release, enhance bioavailability and targeted delivery in the body (Zhang, Wei, & Xue, 2021).Fucoidan is used to formulate nutraceuticals due to their high stability, bioavailability, controlled release and among others (Venkatesan, Murugan, & Seong, 2022).In vitro digestion of zein-fucoidan encapsulated resveratrol showed a controlled release of bioactive compound to target cell compared to free resveratrol control (Liu, Qin, Jiang, Chen, & Zhang, 2022).While the release of free resveratrol 50% in 2 h, the release from zein-fucoidan encapsulated resveratrol only reached 50% after 2.5 h.In addition, the cumulative release of zein-fucoidan encapsulated resveratrol and free resveratrol plateaued at 4 and 6 h after digestion.The delay enhanced adsorption and bioavailability of resveratrol for intended biological activities such as anti-aging, anti-cancer effects, etc (Liu et al., 2022).Pterostilbene is heavily unstable bioactive compounds, thus limits its application to design nutraceuticals.Fabricated zein/fucoidan nanocomposite effectively shielded pterostilbene from lights with control release compared to zein nanocomposite under simulated gastrointestinal conditions (Liu et al., 2022).Combined fucoidan-chitosan protected the core material (insulin) from degradation with control release under simulated gastric fluid (SGF, pH 2.0), simulated intestinal fluid (SIF, pH 6.8), and simulated body fluid (SBF, pH 7.4) thus enhance its bioavailability for managing diabetic patients compared to free insulin (Tsai, Chen, Lin, Ho, & Mi, 2019).Similarly, carboxymethyl cellulose nanofibril loaded in fucoidan/alginate promotes apoptosis with concomitant wound healing potential (Shanmugapriya, Kim, & Kan, 2020).Also, fucoxanthin loaded in fucoidan-based nanoemulsion showed high antiobesity activity with decrease biochemical parameters (i.e., fasting blood glucose, triglycerides, total cholesterol, high-density lipid, and low-density lipid) and liver enzymes (alanine transaminase, aspartate transaminase, alkaline phosphatase) in

Table 3a
Fucoidan-based nutraceutical delivery systems.Thus, fucoidan has great potential as delivery vehicles for hydrophobic bioactive compounds for personalized health needs.

Nanoparticles from fucoidan
Due to the presence of sulphate groups fucoidans can bind cationic polymers to form nanoparticles (Luo, Teng, & Wang, 2012).Fucoidan can form various nano-sized delivery systems such as polysaccharide-fucoidan composite nanoparticles, protein-fucoidan composite nanoparticle and bead complexes.These nanoparticles are formed by the electrostatic interaction between fucoidans and their corresponding cationic polymers for successful encapsulation of an active ingredient (Zhang, Wei, & Xue, 2021).
Polysaccharide-fucoidan composite nanoparticles are formed due to the electrostatic interaction between fucoidan and oppositely charged polysaccharide.The most popular cationic polysaccharide used to form fucoidan composite nanoparticles is chitosan (Luo, Du, Wang, & Wang, 2014).Fucoidan/Chitosan nanoparticles are effective delivery carriers (Fig. 3c) due to the electrostatic interaction between the sulphate groups in fucoidan and the amine groups on the chitosan (Liu et al., 2020).Additionally, the presence of carboxylic groups in uronic acids in some fucoidans facilitates the formation of fucoidan/chitosan nanoparticles.Nanoparticles from fucoidan and chitosan are pH sensitive and play a key role in the release of encapsulated substance (Huang, Chen, & Lam, 2014;Tran, Duan, & Tran, 2020).Several parameters such as the size, pH, and cross-linked ionic interactions are important factors to adhere to when developing fucoidan nanoparticles (Lee et al., 2020).
Previous report showed that fucoidan/chitosan nanoparticles were stable in an acidic pH where sulphate groups on fucoidan and amine on chitosan undergo ionization to maintain their shape (Lee & Huang, 2019).However, pH above 6.5 altered the shape of nanoparticle due to the deprotonation of the amino group leading to rupture and release of the bioactive compound (Lee & Huang, 2019).Formulation of fucoidan (F)-chitosan(C) nanoparticle of an average size 316 ± 21 (d.nm) at ratios 1F:1C, 3F:1C and 5F:1C showed that 5F:1C was stable at pH 3 (Coutinho et al., 2020).Further pH responsive analysis revealed that an increase in pH leads to an increase in size of fucoidan/chitosan  nanoparticle (i.e., 296 ± 6 to 515 ± 18).Conclusively, 5F:1C was selected for effective delivery of methotrexate which enhanced its oral bioavailability (Coutinho et al., 2020).A stabilized fucoidan-based nanoparticle with size of 180 nm has also been reported (Lu et al., 2017).pH profile analysis showed that the release of bioactive compound was effective at a pH 4.5 where the average size of the nanoparticle was 500 nm (Lu et al., 2017).
In an attempt to increase the oral bioavailability of quercetin and prevent degradation, a fucoidan/chitosan nanoparticle was developed using polyelectrolyte self-assembly method (Barbosa et al., 2019).The obtained nanoparticles with particle sizes ranging from 300 to 400 nm were physiochemically stable in an acidic pH.Evaluation of different formulations (1F:1C, 3F:1C, and 5F:1C) showed that 3F:1C and 5F:1C were effectively stable as pH value increased (Barbosa et al., 2019).Silver-nanoparticles with chitosan-fucoidan coating had no aggregation indicating a good electrostatic interaction between fucoidan and chitosan (Venkatesan, Singh, Anil, & Kim, 2018).
In addition, protein-fucoidan composite nanoparticles have been reported mainly using zein as the cationic biopolymer.Zein, a protein from maize is highly hydrophobic; it contains non-polar amino acids like leucine, proline, and polar amino acids like glutamine (Kasaai, 2018).They can easily self-assemble to form nanoparticles due to their amphiphilic character, however, the major drawback is the unstable nature of zein nanoparticles in pH around the isoelectric point, high temperature and salt concentrations (Liu et al., 2020).Fucoidans, being an anionic polysaccharide, reacts with the cationic amino group of zein to form stable nanoparticles by forming complexes.This complex is mainly due to hydrogen bonds and electrostatic forces between zein and fucoidan (Cheng & Jones, 2017).
Different sizes of zein(Z)/fucoidan(F)-based composite nanoparticle have been formulated (10Z:1F, 5Z:1F and 2Z:F1) to encapsulate pterostilbene (Liu et al., 2020).The results showed that, high amount of zein in the formulation decreased the interaction with fucoidan while an increase in fucoidan to zein in composition increased the interaction and fabrication of the nanoparticles.The latter resulted in an increase in the net surface charge creating an electrostatic repulsion between the nanoparticles.In addition, the formulation with high fucoidan to zein ratio showed a high degree of interconnectedness indicating a good fabrication among other formulations (Fig. 3d) (Liu et al., 2020).

Fucoidan based emulsions
Emulsions refers to a mixture of immiscible liquids containing an oil and water phase used to deliver both hydrophobic and hydrophilic bioactive compounds (Mwangi, Lim, Low, Tey, & Chan, 2020).A typical emulsion contains oil, water, and a surfactant.To design fucoidan-based emulsions, the fucoidan serves as the surfactant or the emulsifier.Various studies proved the emulsifying ability of fucoidans in emulsions (Chang, 2015;Xu, Liu, Luo, Liu, & McClements, 2017;Zhang, Wei, & Xue, 2021).Fucoidans are used in oil-water, water-oil, water-oil-water, and oil-water-oil emulsions (Xu et al., 2017) which acted as surfactant by forming complexes between positively charged polymers in solution.
In fucoidan-based emulsions, the interfacial stability is ensured by the complex formation between fucoidan and other biopolymers, mainly, proteins which is dependent on the electrostatic deposition of the negative charges on fucoidan and the surface charges of proteincoated emulsions (Chang, 2015;Zhang, Wei, & Xue, 2021).The interfacial deposition of fucoidan on caseinate-coated lipid droplets showed a good and stable emulsions of fish oil (Chang, 2015).The authors explained that the stability of the fish-oil emulsion was based on equal concentration of fucoidan and the lipid droplets.A transmission electron microscopy showed a thin layer around the caseinate-coated lipid droplets which was attributed to the electrostatic deposition of the fucoidans (Chang, 2015).
Analysis of different polysaccharide-based nanoemulsions prepared from levan, fucoidan, alginate, guar gum and, κ-carrageenan with olive and castor oil as carrier oils to encapsulate curcumin showed that fucoidan nanoemulsions had the smallest size compared to the other nanoemulsions.All the polysaccharides exhibited comparable encapsulation efficiency (>61%) compared to Tween 20 as a standard emulsifier (Richa, 2020).Fucoidan and chitin were used as emulsifiers to design oil/water Pickering emulsion and properties (stability, antioxidant activity) evaluated at different environmental stresses (Hu et al., 2022).The emulsions showed higher antioxidant activity and stability in neutral, alkaline, acid conditions compared to isoelectric point.In addition, high salt concentration (500 mM) decreased antioxidant activity and stability of the emulsions (Hu et al., 2022).

Fucoidan in functional foods
A functional food is any food that exerts health benefits beyond the basic nutritional needs of human (Zhao et al., 2018).The foods we eat mainly provide us with energy and support our growth.However, if the right quantity and type of food is not eaten, it can cause diseases.Due to this, consumers therefore opt for healthy foods rather than tasty but unhealthy ones.As a result, functional foods have become an ideal solution to problems related to foods since they provide contents that help in disease prevention coupled with nutritional benefits (Hasler, 2002).The design of functional foods mostly involves the incorporation of bioactive ingredients such as polymers into food matrix to elicit their full biological activities (Crawford, 2000).
Fucoidans as a bioactive polysaccharide is endowed with a lot of biological activities and have been successfully integrated into food matrix to provide health benefits (Zhao et al., 2018).A summary of various functional foods from fucoidan is described in Table 3b.Foods such as yoghurt, beverages, pasta, etc., have been fortified with fucoidan to improve the quality and sensory properties as well as bioavailability of nutrients.Lim, Mustapha, & Maskat. (2017) reported that the addition of fucoidan tea significantly increased the antioxidant activities of seaweed tea compared to the control.(Lim, Mustapha, & Maskat, 2017).Ribeiro and colleagues prepared fucoidan fortified pasta by substituting durum wheat semolina with milled F. vesiculosus flour (<0.25 mm) at 1, 5.5 and 10 g/100 g including a control without fucoidan (Ribeiro et al., 2022).The results showed no significant difference (p > 0.05) between the treated and control pasta with regards to moisture and protein content.However, 10 g/100 g treated pasta had significantly higher fiber and ash content compared to the other treatments including the control group (Ribeiro et al., 2022).Nevertheless, sensory panelists preferred the control and pasta fortified with low concentration of fucoidan (1 and 5.5 g/mL) compared to 10 g/mL treated pasta (Ribeiro et al., 2022).Bread fortified with fucoidan was observed to have large volumes, softer crumbs and retained biological (antioxidant & anticancer) activity after baking compared to the control group (Koh, Lim, Lu, & Zhou, 2020).In addition, bread fortified with fucoidan showed a decrease in the glycemic index compared to the control bread (Koh, Chong, Lu, & Zhou, 2022).In vitro analysis showed that fucoidan in bread was highly bioaccessible (i.e., from 77.1 to 79.8%) within the digestive tract (Koh et al., 2022).It is reported that fucoidan supported survival of probiotics in the gastrointestinal tract (Zaporozhets et al., 2014).Oral administration of fucoidan at 250 mg/kg significantly (p < 0.05) enriched the microbiota diversity of the GIT compared to alginate and prednisolone (Hu et al., 2022).Rats fed with A. nodosum and L. japonica extracted fucoidans showed a decrease in opportunistic pathogenic bacteria (peptococcus) whilst beneficial bacteria (lactobacillus and ruminococcaceae) increased.In addition, antigen load and the inflammatory response were significantly reduced (Shang et al., 2016).Others have reported that fucoidan functional food facilitates growth of probiotics Bifidobacterium adolescentis, Lactobacillus acidophilus, and L. casei (Liu et al., 2018).Thus, fucoidan base beverages could be used to modulate gut microbiota to improve health.Report shows that food processing decreases the concentration of fucoidan in the final product (Koh et al., 2020).Interestingly higher fucoidan concentrations were recovered in digesta and sediment (~89.2-93.8%)but not dialysate of the fucoidan fortified bread (Koh et al., 2022).
Fucoidan has proven to be a promising candidate for designing functional foods.However, the production and application of fucoidan at the industrial level is still limited due to low yield ⁓3-30% per dry weight (January, Naidoo, Kirby-McCullough, & Bauer, 2019).Thus, huge seaweed biomass is required to meet the demands of fucoidan for the food and pharmaceutical industries.In this regard.improve seaweed cultivation, and optimized fucoidan extraction methods (green method) (Anisha, Padmakumari, Patel, Pandey, & Singhania, 2022;Hifney et al., 2016) are highly critical to feed the food industries with the raw materials needed to meet demand of consumers.Another important factor is the purity of fucoidan as it is a criteria to consider when designing functional food (Liu et al., 2018;Shanthi et al., 2021).

Fucoidans in edible films
Edible films and coatings generally refer to any material, usually thin, used to wrap or coat a product with the aim of extending shelf-life and can be eaten together with the product.These edible films are biopolymer based and has become promising substitutes for the conventional packaging.Recently, edible films are preferred to synthetic ones because they are biodegradable, natural, renewable, and nontoxic (Gomaa, Hifney, Fawzy, & Abdel-Gawad, 2018).In addition, edible films incorporate some biological activities into their products and effectively protects the product from going bad.By design, these biological activities are released at a controlled rate in the product.In the formulation of edible films, bioactive polymers such as pectin, alginate, whey, and fucoidans, are used (Gomaa, Fawzy, Hifney, & Abdel-Gawad, 2018;Zhang, Wei, & Xue, 2021).
Fucoidans, as we have described above, are endowed with numerous biological activities, making them an attractive candidate for developing biofilms.In the development of edible films or coatings, fucoidans are mostly joined to other polymers since they are unable to form gels on their own (Venkatesan, Bhatnagar, & Kim, 2014).As such in the formulation of edible films with fucoidans, they are mostly joined to a cationic polymer and a cross-linking agent or a cation (Gomaa, Fawzy, et al., 2018;Venkatesan et al., 2014).This was evident in a different study where edible films were designed using fucoidan and gelatin with sorbitol as the plasticizer (Govindaswamy, Robinson, Geevaretnam, & Pandurengan, 2018).The fucoidan edible film showed a good mechanical and barrier integrity compared to control films.The fucoidan-gelatin edible film showed a potent antioxidative property that inhibited microbial growth when used as a packaging film.Additionally, the fucoidan biofilms had good thickness of 61.60-66.25 μm (Govindaswamy et al., 2018).
Another study reported the development of a fucoidan-based edible film with the incorporation of chitosan and calcium ion (Ca 2+ ) (Gomaa, Fawzy, et al., 2018).The addition of fucoidan increased the film thickness, water vapor permeability, opacity, coupled with reduced water solubility and enhanced antioxidant activity compared to biofilms with only chitosan and alginate.In addition, fucoidan helped release the encapsulated polyphenolic compounds in a controlled manner (Gomaa, Fawzy, et al., 2018).Furthermore, the formulation of alginate-fucoidan based edible films showed good barrier qualities against UV light thus suitable as packaging material due to their potential to delay or prevent oxidative deterioration, discoloration, flavor loss, as well as improve and maintain the quality of packaged food products (Gomaa, Fawzy, et al., 2018).Highest dose (5%) fucoidan coatings extended mongo shield life for 35 days by decreasing respiration, nutrient loss, and weight loss compared to lower doses (1 and 3%) and control at room temperature (Xu & Wu, 2021).Sensory experts preferred fucoidan treated mangoes compared to the control after 35 days storage.In addition, treated mangoes retained more ascorbic acid compared to the control group (Xu & Wu, 2021).Fucoidan-coating was reported to have significantly retained the antioxidant capacity of strawberries and the total polyphenol content after 5 days storage (Luo, Li, Liu, Yang, & Duan, 2020).The addition of essentials oils (i.e., cinnamon oil) may improve the mechanical and barrier properties as well as biofuntional activity (biocidal activity, antioxidant capacity, etc) of the biofilms (Pan et al., 2023).The above studies demonstrate that fucoidan-based edible films can be used to extend shield life of perishable produce like vegetables and fruits.Fucoidan-based edible films inhibit microbial growth, decrease lipid peroxidation, total volatile nitrogen, and aroma loss, thus can be use in meat, fish, and animal-based produce.

Health concerns of fucoidan
Generally, many studies about fucoidans mainly focused on their biological activities and ignoring important aspects of health concerns or adverse effects.Despite their health-related benefits, very little is known with regards to the toxicity of fucoidans in the human system.Many in vitro cytotoxicity studies showed no adverse effects of fucoidan at various concentrations on cell lines (Lim et al., 2016;Ramu, Anita, Geetha, & Jarayaman, 2020;Kim, Lee, Lee, & Lee, 2010;Usoltseva et al., 2022).Others have reported that high dose of fucoidan (900-2500 mg/kg) from Laminaria japonica prolonged blood clotting and caused kidney failure (Li, Zhang, & Song, 2005).The activity of liver enzymes (alanine transaminase) and lipoprotein metabolism were altered when 2000 mg/kg fucoidan was administered to Sprague-Dawley (SD) rats (Chung et al., 2010).In addition, SD rats showed a slight increase in serum urea nitrogen when fed with 1350 mg/kg fucoidan (Kim, Lee, et al., 2010).Furthermore, nanoparticles from fucoidan-poly (lactic-co-glycolic acid) (PLGA) showed some extent of cytotoxicity on breast cancer cells (Lai, Chiang, Hsu, Cheng, & Chen, 2020).Also, different concentrations of resveratrol-loaded zein fucoidan particles showed negligible cytotoxicity against HIEC-6 cells with good biocompatibility (Liu et al., 2022).Similarly, high concentration of fucoidan (1000 μg/mL) exerted low cytotoxicity (<10%) on Vero cells with no visible destruction of cell morphology (Pliego-Cortés et al., 2022).Despite the possibility of some extent of toxicity of fucoidan, only a few studies reported on Fucoidan based delivery systems are promising candidates in nutraceutical and pharmaceutical industries to deliver active ingredients to target cells with minimal or no effects compared to conventional drugs.Therefore, further investigations are required to decipher the concentration of fucoidan that may pose a potential adverse effect on humans.

Closing remarks
Fucoidan is considered as a bioactive compound that can confer health benefits to humans with habitual consumption.Partly because in vitro and in vivo studies have extensively demonstrated the dosedependent antioxidant, antiinflammatory, antimicrobial, antithrombic and immunomodulatory activities of this sulphated polysaccharides (Hoang et al., 2022;Hsu & Hwang, 2019;Obluchinskaya et al., 2022;Qi et al., 2022;Tan et al., 2022).To maximize the biological effects on cells, pure fucoidan without traces of extracting solvents and other contaminants i.e., proteins, polyphenols should be used.Contaminants may lead to over or underestimation of fucoidan concentration, variation in fucoidan yield and subsequent bioactivity.Recent report showed that enzyme assisted extraction of fucoidan from seaweed resulted in pure fucoidan without the need for further purifications steps (Rhein- Knudsen et al., 2023).Thus, thorough investigation regarding the use of innovative methods of extracting fucoidan is required.Reports further showed that, depending on the molecular weight, the biological activity of fucoidan varied (Sakai et al., 2019;Takahashi et al., 2018;Tsai, Tai, Huang, Chang, & Wang, 2017) thus thorough investigations are required to completely map the range of molecular weight that exerts potent biological function and to what extent.The structural variation of fucoidan due to different degrees of sulfation, acetylation and branching further hinders methods for purity quantification.The lack of standardized purification methods poses a threat of obtaining purified fucoidan for industrial application as different methods with varied results have been documented.Methods for quality assurance of fucoidan has been extensively discussed in section 2.1.1.6.As research advances, new innovative methods of checking fucoidan quality assurance will emerge to augment the existing methods.This would help advance the application in the industry.
Going forward, it is paramount for regulatory bodies in various jurisdictions to come to consensus regarding quality parameters as well as Case study 1-10; NR-not reported; HMW-high molecular weight; LMW-low molecular weight; LMF-low molecular weight fucoidan; HMWF-high molecular weight fucoidan.
E.O.Mensah et al. standardized methods for assessing and validating the purity of fucoidans.Robust and high-throughput quality assurance techniques would further advance the industrial application of fucoidans.
Owing to their health benefits, fucoidan has been extensively exploited in the food, pharmaceutical and cosmetic industries.However, there are limited clinical trials (Table 3e) regarding their effects on humans.This is an issue which needs international attention as the in vitro and in vivo findings may not necessarily be translated on to human beings.Recent report showed that preclinical findings do not envisage efficacy on humans (Koushki, Yekta, & Amiri-Dashatan, 2023).Therefore, more in vivo studies are required to decipher the right fucoidan concentration and molecular weight that elicit biological effects on humans.In fact, in vivo studies with closely related species to humans is highly recommended as this gives a clearer picture of the mechanistic action compared to in vitro and mice models.

Conclusion and future prospects
Fucoidan is undoubtedly endowed with several biological activities of therapeutic significance including antioxidant, anti-inflammatory, antimicrobial, antithrombic and immunomodulatory activities.These have been corroborated by many studies both in vitro and in vivo using animal models.Generally, these biological activities are dependent on the structural complexity and compositional heterogenicity of fucoidans i.e., degree of sulphation, monosaccharide composition as well as type and position of substituents which in turn are dictated by the species and cultivation parameters including location and season.The presence of reactive groups such as sulphate groups on sugar monomers and carboxylic acid groups in uronic acid promotes the interaction of fucoidans with other polymers.Over the years, fucoidans have been shown to be promising candidates in the food and nutraceutical industries particularly for developing delivery vehicles such nanoparticles, nanoemulsions hydrogels.Moreover, fucoidans possess great stabilizing properties.Together, these biological and physical properties of fucoidans have urged their application in designing functional foods.To increase the usability of fucoidans in the food industry green extraction technologies are increasingly being explored.This notwithstanding, little to no information is available about the tolerance and toxicity of fucoidans in humans and thus more research is required.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 2c .
Fig. 2c.The antibacterial effects of C. submersum extracted fucoidan against E. coli (black bars) and S. aureus (grey bars).The inhibition diameter (A) and zone (B) of different fucoidan concentrations 250, 500, 750, 1000, and 1250 ppm and ampicillin 10 mcg.The antifungal activity of C. submersum extracted against A. flavus.The inhibition diameter (C) and zone (D) of different fucoidan concentrations including a control.Adapted from Hoang et al. (2022).Copyright © The Polymer Society of Korea and Springer.
E.O.Mensah et al.

Table 1b
Chemical composition of fucoidans.

Table 2d
Antiviral activity of fucoidans and their mechanism.
E.O.Mensah et al.

Table 3e
Clinical application of fucoidan.