**3. Discussion**

Marine organisms are reported to be immense sources of secondary metabolites that possess novel biological activities. These sources have proven to be useful in the treatment of diseases and hence be utilized prospectively in pharmacology and medicinal sectors [19]. The antioxidant activity of Hawaiian marine algae has been studied by Kelman et al. (2012), providing an extensive report on di fferent marine algal species including red, brown, and green algae [20]. The sulfated polysaccharides from marine algae as a source of antioxidant secondary metabolite were reviewed by Wijesekara et al. (2011) [21]. Brown algae *Ecklonia cava* was reported as a source of antioxidant components further conducting its abilities in vitro scale. The secondary metabolite of interest; phlorotannin derivatives were reported to be exhibiting noteworthy antioxidant potential [22].

*Padina* is a genus of brown algae in which the thallus is calcified. The seaweed is fan shaped, widely distributed in warm tropical waters from lower intertidal to deep subtidal zones [23]. *Padina boryana*, in particular, has previously been studied by Sanjeewa et al. (2019), purifying fucoidan and evaluating its anti-inflammatory properties [24]. The structure of fucoidan was widely evaluated by Usoltseva et al. (2017) [25]. The species form the Maldives has not been deeply studied for its secondary metabolites specifically against antioxidant potentials. Hence, this study aimed to extract water soluble sulfated polysaccharide from the above under-explored brown algae and to investigate its antioxidant properties, specifically on the ethanol precipitation (PBP) which is rich in fucose.

Polysaccharides among other metabolites receive much attention due to their high availability, and diversified structure with vivid functional groups attached to its backbone [26]. Polysaccharides lack structural homogeneity. Fucoidan is one such polysaccharide containing fucose as its main component. Sulfate groups are substituted in the structure of fucose forming ester bonds. Fucoidan is unique due to its higher content of L-fucose and sulfate groups [27]. Di fferent insertions into the backbone of the structure are also possible (mannose, glucose, and galactose). The point of sulfation and the degree of sulfation could be altered form one species to another. The bioactive properties of fucoidans prevail and increase due to the substitution of sulfate groups. One such report emphasized the action of the anionic sulfate group is that it enhances the nonspecific binding of proteins [28]. Hence, the potential of PBP is attributed to the higher degree of polysaccharide content and its sulfate substitution percentage. Another important component of crude polysaccharide is alginate. The chemical composition indicates higher polysaccharide yield in the PBP and sulfate content exhibits a similar trend. The yield of the polysaccharide is justifiable via the activity of celluclast enzyme to breakdown the cell wall. Furthermore, the dielectric constant of the solution is lowered by the addition of ethanol and polysaccharides are precipitated. The structural characterization of the PBP was supported by the FTIR analysis. The FTIR spectral data collectively revealed the correspondence of PBP to commercial fucoidan.

Previous literature reveals the potential of crude polysaccharides to act as antioxidants. The composition including its functional groups and monosaccharides are reported to synergistically induce the free radical scavenging activity [29,30]. Therefore, monosaccharide composition of the samples was analyzed and results indicate an increment in fucose and galactose contents. Higher values of fucose and galactose composition were available in PBP compared to PBE accrediting PBPs' elevated potential to act as an antioxidant.

DCF-DA assay was used in the evaluation of intracellular ROS scavenging activities. The stain is absorbed into the cells through the membrane and converted to DCFH, a non-fluorescent component, via the cellular esterases. Thus, intracellular ROS converts DCFH to fluorescent active DCF and detectable with a fluorimeter. Intracellular ROS levels were observed to be upregulated with H2O2 pre-treatment while treatment with PBP dose-dependently downregulated it. Furthermore, the protective e ffects of PBP against H2O2 were observed by cell viability analysis.

Oxidative stress is caused due to the presence of reactive oxygen species (ROS). Cellular metabolism and environmental factors contribute to the production of ROS. At moderate concentrations, ROS plays an important role in the function of physiological cell processes while high concentrations lead to adverse e ffects [31]. The highly reactive molecules damage cell structures including macromolecules thus alter the physiological functions. Moreover, the imbalance between the ROS and its counterpart antioxidants creates oxidative stress. Cell viability, proliferation rate, and further functions are a ffected by oxidative stress. Even though aerobic organisms possess integrated antioxidant systems, under pathological conditions these systems can be overwhelmed [3]. H2O2 is a distinct ROS species with physiological significance among others such as superoxide anion (O2 -•) and hydroxyl radical ( •OH). H2O2 is produced in the cells upon phagocytosis due to the superoxide burst and converted. Xanthine oxidase, NAD(P)H oxidase, and amino acid oxidase contribute in the production of hydrogen peroxide [32]. In the presence of transition metal ions, H2O2 break down and result in OH- and •OH via the Fenton reaction [33]. Therefore, this study focuses on the stimulation via H2O2 under both in vivo and in vitro conditions.

The genetic material is modified by the ROS via di fferent mechanisms involving DNA strand breakage, sugar moiety modifications, base unit degradation, deletions, and translocations [34]. Possible DNA modifications lead to carcinogenesis, aging, and numerous diseases. The e ffect was evaluated in the study via nuclear staining methods. It was evident the e ffect of H2O2 on the condensation and fragmentation of the nuclear material which was dose dependent and down regulated via the treatment of PBP.

Superoxide anion radicals are scavenged via SOD, while CAT plays an important role in the detoxification of the H2O2. Hence, these could eliminate free radicals and help the conversion of reactive toxic components to non-toxic elements and protect the live organelles from oxidative damage. The antioxidant enzymes were initially hampered by the excess ROS created via the e ffect of H2O2, though the potential of PBP recovered the enzyme expression hence reducing ROS production. Keap1 is an inhibitor protein, a cysteine-rich protein that is anchored to the actin cytoskeleton. It is responsible for the cytosolic sequestration of Nrf2 under physiological conditions. Keap1 promotes ubiquitination and degradation of Nrf2 under normal physiological conditions. Under stressful conditions in which the Nrf2-dependent cellular mechanism is active (electrophiles and oxidants are rich in this stage), the Nrf2 is rapidly released from Keap1. Dissociated Nrf2 is translocated to the nucleus and binds to antioxidant response element (ARE). Keap1 also receives redox information or environmental cues via its highly reactive cysteine residues and referred to as the sensor of the Nrf2-Keap1 system [35]. The dissociation of the system is a relatively rapid event. The breakdown of the system leads to Keap1 stabilization. Nrf2 also increases its half-life [36]. This allows successful nuclear translocation and cytoprotective gene transcription. A similar e ffect of antioxidant polysaccharides was reported by Zhou et al. (2019) [37].

The membrane lipid bilayer is disrupted via ROS stimulated lipid peroxidation thus membrane bound receptor activities are altered leading to increased tissue permeability [38]. Lipid peroxidation results in unsaturated aldehydes which are potent inactivators of cellular proteins through the formation of cross linkages [3]. The e ffect of ROS was evaluated in vivo scale using zebrafish. The ROS production levels examined via DCFDA fluorescent staining initially indicated the decline of intensities revealing the protective e ffect of PBP. Furthermore, DPPP staining exhibited the lipid peroxidation intensities to be upregulated under H2O2 stimulation and significant decline under PBP pre-treatment. The results confirmed the antioxidant potential of PBP against H2O2 induced oxidative stress. Thus, zebrafish cell death was successfully downregulated dose-dependently as indicated. The results obtained in the study well aligns with an earlier report on polysaccharide extracts from *Hizikia fusiforme* [39]. Furthermore, fucoidan purified from brown algae has incorporated zebrafish studies as an in vivo model [12,40].

Zebrafish involvement in developmental biology and drug discovery has been recognized and the implementation was advanced throughout the years. The size, husbandry, and early morphology is a distinct advantage of the usage of zebrafish over other vertebrate species. This provides researchers to minimize the costs in maintenance as well as in quantities of dosing samples, further in histological assessments [41]. High fertility including transparent embryos makes the species a valuable source [42]. Fluorescent staining methods implemented in the present study also support the above fact.
