**2. Results**

#### *2.1. Yields and Chemical Composition of the Polysaccharide Extracts and Fractions*

Based on the compositional analysis (AOAC methods) *S. polycystum* indicated 2.75 ± 0.08% moisture, 17.62 ± 0.16% ash, 52.46 ± 0.52% carbohydrate, 17.36 ± 0.04% protein, and 1.85 ± 0.06% lipids on a dry basis. The yield of ethanol precipitate was 6.88 ± 0.52%. The ash content of the ethanol precipitate was 0.08 ± 0.01% (relative to the dry weight of raw material).

#### *2.2. Purification of Polysaccharides Molecular Weight Distribution and Monosaccharide Composition*

As indicated in Figure 1a, anion exchange purification resolved the polysaccharide precipitate into five fractions (F1–F5). The recovery yield of F1 and F5 was higher than other fractions. The molecular weight distribution of fractions (F1–F5) indicated a decreasing trend and was estimated to be centered on 77.0, 65.5, 59.5, 60.0, and 39.5 kDa, respectively (Figure 1b). The monosaccharide composition of each fraction is given in Table 1. F1 had a higher galactose and mannose content, whereas the other successive fractions indicated increasing amounts of fucose. Fucose content was highest in F5 (Figure 1c) with galactose and a minor amount of glucose.

**Figure 1.** Purification of precipitated polysaccharides by DEAE-cellulose anion exchange chromatography. (**a**) Separating the collected column eluates into five fractions based on their polysaccharide content, (**b**) molecular weight distribution of the eluted column fractions (F1–F5), and (**c**) the monosaccharide composition analysis of the fraction F5.

**Table 1.** The composition of column fractions.


Chemical composition was calculated based on triplicate determinations. Results are given as the means ± SD.

#### *2.3. Characterization of Polysaccharide Structure (FTIR and NMR Analysis)*

Wavenumber range 400–2000 cm<sup>−</sup><sup>1</sup> of FTIR spectra covering the fingerprint region of polysaccharides were used to characterize the structural properties of polysaccharides. All FTIR spectra, including column fractions and the commercial fucoidan standard, indicated prominent peaks at 845, 1035, 1616 cm<sup>−</sup>1, and a broad peak between 1220–1270 cm<sup>−</sup>1. A clear di fference was seen for the intensity of 1220–1270 cm<sup>−</sup><sup>1</sup> peak between each spectrum, although the intensity of the peak at 1035 cm<sup>−</sup><sup>1</sup> was similar between all. The peak at 1733 cm<sup>−</sup><sup>1</sup> was absent in the commercial fucoidan, whereas a minor peak was recorded for F5. However, 1733 cm<sup>−</sup><sup>1</sup> peak was prominent in all other fractions (F1–F4). 1H and 13C NMR spectra of deuterium exchanged F5 fraction are presented in Figure 2b,C. In the 1H spectrum, solvent peaks were observed at 2.50 ppm for dimethyl sulfoxide and 4.80 ppm for deuterium oxide. Unresolved peaks between 5.1–5.4 ppm could be assigned to protons of <sup>α</sup>-<sup>l</sup>-fucopyranosyl units. Prominent peaks observed at 4.46, 3.90, and 3.71 ppm were respectively assigned to H-1, H-5, and H-3 of D-galactons. The two overlapping peaks at 1.15, and 1.4 ppm were assigned to methyl groups in fucose [1,2]. In the 13C spectrum, prominent peak 101.6 and peaks between 65–80 are arising from the (1–6)-β-<sup>d</sup>-linked galactons [2].

**Figure 2.** Structural characterization of polysaccharide fractions. (**a**) FTIR spectra of the fractions (F1–F5) provided in comparison to the commercial fucoidan, (**b**) 1H NMR spectrum of F5, and (**c**) 13 C NMR spectrum of F5. The NMR spectra were obtained for the deuterium exchanged polysaccharides.

#### *2.4. Variation of Antiproliferative Activity of the Column Fractions*

As shown in Figure 3, F4 and F5 promptly reduced the viability of both HL-60 and MCF-7 cells compared to other fractions. The antiproliferative e ffects of F5 were stronger than F4. The IC50 values related to F5 treatment was 84.63 ± 0.08 μg mL−<sup>1</sup> and 93.62 ± 3.53 μg mL−<sup>1</sup> respectively on HL-60 and MCF-7 cells. The viability of Vero cells (Figure 3c) was simultaneously evaluated to compare the cytotoxic e ffects of these polysaccharides on non-cancerous cells. The viability of Vero cells was 89.83 ± 1.76% at 100 μg mL−<sup>1</sup> concentration.

**Figure 3.** Antiproliferative activity of polysaccharide fractions as a measure of cell viability. (**a**) HL-60, (**b**) MCF-7, and (**c**) Vero cells. Cells were pre-seeded in 96 well plates for 24 h and incubated with samples for another 24 h. Cell viability was measured by MTT assay. Results are given as the means ± SD (n = 3). Significant differences from the control were identified at \* *p* < 0.05 and \*\* *p* < 0.001.

#### *2.5. E*ff*ect of F5 upon Apoptotic Body Formation in HL-60 and MCF-7 Cells and Pathway Studies by Western Blot Analysis*

As shown in Figure 4a, under the Hoechst 33342 staining, HL-60 and MCF-7 cells indicated increased nuclear fragmentation and condensation (intensified spots) for increasing F5 concentrations. Nuclear double staining method (Figure 4c) assists in distinguishing early and late apoptosis or necrosis. Green fragmented nuclei (early apoptosis) were seen under 25 μg mL−<sup>1</sup> of F5 treatment after a 24 h incubation period, which increased with increasing F5 concentrations. The presence of orange color spots together with green color fragmented nuclei (late apoptosis) were detected under 50 μg mL−<sup>1</sup> concentration. Increased late apoptosis events were observed with 100 μg mL−<sup>1</sup> of F5 concentration, whereas a few necrotic HL-60 cells were also detected. Western blot results indicated increased production of Bax, caspases, and p53 (only MCF-7) levels with increasing F5 concentrations together with increased PARP cleavage. Alternatively, reducing levels of Bcl-xL were also detected with increasing F5 concentrations.

#### *2.6. F5 Increased the DNA Damage in HL-60 and MCF-7 Cells and the Population of Sub-G1 Hyperploid Cells*

Comet assay serves as a tool in identifying cellular DNA damage. As depicted in Figure 5a,b, the length of the comet tail increased in both HL-60 and MCF-7 cells with increasing F5 concentrations. Flow cytometric analysis with propidium iodide (PI) is widely employed in identifying the accumulation of cells in different phases of cell cycle based on their DNA content [14]. According to Figure 5c,d, the proportion of Sub-G1 apoptotic cells increased with increasing F5 concentrations.

**Figure 4.** Effects of F5 in inducing apoptotic body formation in HL-60 and MCF-7 cells and analysis of the levels of molecular mediators. (**a**) Cells under Hoechst 33342 staining, (**b**) Western blot analysis of the levels of apoptosis-related molecular mediators in HL-60 cells, (**c**) cells under nuclear double staining, and (**d**) Western blot analysis of the levels of apoptosis-related molecular mediators in MCF-7 cells. Experiments were repeated three times to confirm the reproducibility.

**Figure 5.** Effects of F5 in inducing single-cell DNA damage in HL-60 and MCF-7 cells and cell cycle analysis. Comet assay (**a**) HL-60 cells, (**b**) MCF-7 cells. Cell cycle analysis of (**c**) HL-60 cells, and (**d**) MCF-7 cells. Pre-seeded cells were exposed to different concentrations of F5 for 24 h. Experiments were repeated three times to confirm the reproducibility.
