**3. Discussion**

Cancer is one of the leading causes of death throughout the world, which is predicted to increase in the future. The need for anticancer agents is an ever-increasing necessity, as many anticancer drugs cause serious side e ffects related to their inherent toxic e ffects. The literature suggests that fucoidan from brown algae could be a potential anticancer agen<sup>t</sup> with promising bioactive e ffects and biocompatible properties [15]. A recent clinical study suggests that the administration of low molecular weight fucoidans to patients undergoing chemotherapy for metastatic colorectal cancer significantly improved the disease control rate compared to the control group [16]. The bioactive properties of fucoidan largely depend upon its structural characteristics. The structure of fucoidan is highly heterogeneous and indicates variation based on its source. In a previous study, fucoidan obtained from *S. polycystum* by hot water extraction has shown moderate antioxidant activity for DPPH scavenging, total antioxidant and reducing power assays, and anticancer activity in MCF-7 cells with an IC50 of 50 μg mL−<sup>1</sup> [13]. Current investigations focus on the evaluation of antiproliferative properties of polysaccharides, and a purified fucoidan fraction from the Celluclast assisted extraction of *S. polycystum*. The selection of enzyme-assisted extraction supposedly gives better extraction yields compared to the conventional water extraction of polysaccharides. Here Celluclast, a food-grade cellulase, assists in hydrolyzing cellulose in cell walls [17]. Hexane washing removes the lipids and nonpolar pigments, whereas ethanol assists in removing un-bound phenolic compounds. Formaldehyde pretreatment causes polymerization of phenolic compounds that remain bound to the polysaccharide matrix and with other polar molecules via strong intermolecular interactions [1]. This prevents the water-based extract from getting contaminated by phenolic compounds. pH value is another factor that determines the solubility of anionic and cationic polysaccharides. Brown algae are rich in anionic polyelectrolyte alginic acid (pKa around 3.4–4.4) unlike other than fucoidan (pKa around 1.0–2.5) [18]. The optimum extraction conditions of Celluclast (pH 4.5) hence minimize the solubility of high molecular weight alginic acid, thus preventing the contamination of intended fucoidan extraction. However, CaCl2 was incorporated into the extract at a later stage to facilitate the complexation and precipitation of any remaining alginic acid as calcium alginate [1]. Proteins are among the compounds soluble in water, which ge<sup>t</sup> precipitated with the addition of ethanol. To prevent the contamination of the final ethanol precipitate with proteins, we incorporated a commercial food grade protease (Alcalase) to the extraction mixture to facilitate the hydrolysis of proteins. Finally, ethanol was added to the neutralized and concentrated extraction mixture to lower its dielectric constant and precipitate polysaccharides. Essentially, the above extraction method, except for some minor modifications, was optimized during our previous studies to be e fficient in purifying fucoidans [1]. The precipitated polysaccharides were subjected to anion exchange chromatography, where five fractions were obtained. Elution of the DEAE-Sepharose column with increasing Cl- concentrations enabled the successive elution of less negative to highly negatively charged polysaccharides. The chemical composition of the fractions indicates increasing amounts of sulfate through the successive column fractions agreeable with the increasing negative charge of the polysaccharides. The total polyphenol and protein contents were negligible in the fractions.

The molecular weight of sulfated polysaccharides is a major deterministic factor of its bioactivity [1]. The fractions indicated that di fferent molecular weights ranged between 39–77 kDa. The lowest molecular weight was observed in F5, which indicated the best antiproliferative properties. Similar observations have reported that low molecular weight fucoidans are very e ffective as antiproliferative agents [5]. The composition of monosaccharides explicated substantial deviation from each other. An increase of the fucose content was observed with successive fractions, the highest being 71.96% in F5 with 12.31% of galactose (a galactofucan). L-fucose is the major monosaccharide constituent in fucoidans, which reports associating with sulfate groups. This agrees with the relatively high sulfate content in F5 compared to other fractions. Much evidence is, therefore, in agreemen<sup>t</sup> with F5 being a fucoidan. Crude fucoidan obtained from *S. polycystum* by Palanisamy et al. (2017) reported 46.8% fucose content with 22.35 ± 0.23% sulfate with minor amounts of other monosaccharides, galactose

(14.3%), glucose (11.5%), and xylose (13.2%) [13]. Bilan et al. (2013) have described the purification (anion-exchange chromatography) and structural characterization of two highly sulfated galactofucan fractions from *S. polycystum* following desulfation, methylation analysis, Smith degradation, and partial acid hydrolysis following mass-spectrometric and NMR monitoring [4]. Their fine structure report consists of mainly 3-linked α-l-fucopyranose units with C4-sulfate substitution, which is common to fucoidans and short sequences of above structures interspersed by residues of single 2-linked α-d-galactopyranose with C4 sulfation which is rather an unusual configuration.

Vibrational spectroscopic data were used in the structural characterization of the polysaccharides as they provide information about functional groups. The prominent peaks at 845–1035 cm<sup>−</sup><sup>1</sup> and the broad peak between 1220–1270 cm<sup>−</sup><sup>1</sup> respectively, representing the bending vibration of C-O-S, stretching vibration of glycosidic (C-O-C) bridge (typical to all polysaccharides), and the stretching vibrations of S=O bond in sulfate groups [1]. Interestingly the broadened peak between 1220 and 1270 cm<sup>−</sup><sup>1</sup> indicated a higher intensity in both commercial fucoidan and F5 fraction, suggesting a higher sulfate substitution. The intensity of that peak increased with successive fractions agreeing with the increased sulfate content observed. The intense peak at 1616 cm<sup>−</sup><sup>1</sup> is resulting from the asymmetric carboxylate O-C-O vibration [19].

Based on MTT cell viability assay, F5 indicated cytotoxicity on HL-60 and MCF-7 cancer cells with IC50 values of 84.63 ± 0.08 μg mL−<sup>1</sup> and 93.62 ± 3.53 μg mL−<sup>1</sup> respectively. Nonmalignant monkey kidney epithelial cells (Vero) were the least sensitive. Previous studies report substantially different IC50 values for the anticancer activity of fucoidans. According to Isnansetyo et al. (2017), fucoidans purified (anion exchange chromatography) from three tropical algae *Sargassum cristaefolium*, *Turbinaria conoides,* and *Padina fraseri* have shown substantially different IC50 values for cytotoxicity on MCF-7 and WiDr cell lines [19]. Herein *P. fraseri* fucoidan has shown IC50 of 144 and 118 μg mL−1, respectively, on MCF-7 and WiDr cells, whereas the IC50 has ranged between 461 and 663 μg mL−<sup>1</sup> for the fucoidans of *S. cristaefolium*, and *T. conoides*.

There could be a number of possibilities for the observed antiproliferative effects such as apoptosis, necrosis, cell cycle arrest, or a combination of two or more of the above effects. Evaluation of nuclear morphology revealed that F5 treatment increased the apoptotic body formation with an increased number of cells in late apoptosis. Further, the accumulation of cells in the Sub-G1 phase of the cell cycle agrees with the observed formation of apoptotic bodies. As evident from comet assay, DNA fragmentation is a characteristic feature observed in cells undergoing apoptosis. Apoptosis is a cellular homeostatic process that eliminates damaged cells without causing damage to neighboring tissues [20]. Hence substances that could induce apoptosis in cancer cells receive greater attention in the discovery of anticancer drugs.

Apoptosis could be triggered by two main pathways, either the mitochondria-mediated intrinsic pathway or cell surface receptor-mediated extrinsic pathway. Sensitivity to a vast number of death stimuli makes mitochondria-mediated apoptosis the most frequently studied death mechanism. This mechanism is inactivated in many cancer cells. Thus, its activation ramifies the therapeutic perspectives of treating several malignant diseases [20]. The mitochondria-mediated apoptotic pathway is controlled via a complex web of signaling cascade. Some of the major signaling molecules involved are Bax, Bcl-xL, PARP, p53, and caspase-9 and -3. The pro-apoptotic protein, Bax and the anti-apoptotic protein, Bcl-xL, belong to Bcl-2 family proteins. The Bax/Bcl-xL ratio is considered a major prognostic factor of apoptosis. Activation of Bax causes disruptions in the voltage-dependent anion channels in mitochondria releasing pro-apoptotic factors and cytochrome c that propagate apoptosis. Bcl-xL, which resides on the outer mitochondrial membrane, inhibits the activation of pro-apoptotic proteins such as Bax, thereby inhibiting the release of pro-apoptotic factors and cytochrome c [21]. In the present study, treatment of F5 increased the levels of Bax while reducing Bcl-xL. Caspases are next in line where their activation is triggered by cytochrome c, initiator caspases such as caspase-8 and -9, or by autocatalytic processes. The activated effector caspases (caspase-3, -6, and -7) catalyze the cleavage of critical regulatory proteins, such as PARP, which is involved in maintaining genomic

stability by regulating base-excision DNA repair. F5 markedly increased the levels of caspase-9 in both HL-60 and MCF-7 cells. Although caspase-3 is produced in HL-60 cells, MCF-7 cells cannot synthesize caspase-3 due to a deletion in the CASP-3 gene [22]. However, PARP cleavage was observed in both cell lines suggesting that it could have proceeded via activation of another e ffector caspase in MCF-7 cells. These evidences conclude that the mitochondria-mediated pathway is a possible route of apoptosis in HL-60 and MCF-7 cells upon treatment with fucoidan purified from *S. polycystum*. While caspases are considered key biomarkers of apoptosis, numerous other apoptogenic molecular mediators, which do not require caspase activation, have been discovered as potential targets for anticancer drugs. Mitogen-activated protein kinases are a major grouping of such proteins involved in mediating apoptosis, which could be targeted by fucoidans [23]. Hence further analysis of F5 could widen its potential use in anticancer drug research. Structure-activity relationships of fucoidan are still a controversy where fucoidan is highly heterogeneous in its structure. A large number of studies on this topic sugges<sup>t</sup> that sulfate content and molecular weight of polymer are major factors related to the antioxidant, anticancer, and immunomodulatory properties of fucoidan [24]. Present evidence suggests that fucoidans from *S. polycystum* could provide health benefits and could be utilized as candidates in cancer chemotherapy.
