**2. Results and Discussion**

#### *2.1. Chemical Composition of Crude Fucoidans*

The abundance of the two brown algae *B. bifurcata* and *F. spiralis* on the Moroccan Atlantic coast was the reason for the choice of these two species. The yield as well as the chemical composition of the fucoidans extracted from these species are shown in Table 1. FSF and BBF yields were around 8 and 2%, respectively, based on algae dry weight.


**Table 1.** Chemical composition and yield of *F. spiralis* (FSF) and *B. bifurcata* (BBF) crude fucoidans.

**a**Expressed on the weight of dry depigmented algae. **b** Expressed on the weight of dry fucoidans. **c** Molecular weight by High Performance Size Exclusion Chromatography (HPSEC) analysis.

Colorimetric assays show that FSF and BBF contained principally neutral sugar from 45.23 to 51.16 for *B. bifurcata* (BBF) and of *F. spiralis* (FSF), respectively, those extracted fucoidans were also highly sulfated (FSF, 49.53% and BBF, 45.49%).

The main neutral sugars which constitute FSF and BBF was determined by gas chromatography– mass spectrometry (GC-MS) analysis and the result reported in Table 2 shows that was the L-fucose with 63.98 and 90.68%, respectively, based on dry weight of sulfated polysaccharides (FSF and BBF).

The results obtained (Table 1) are di fferent from those demonstrated for fucoidans of *B. bifurcata* from Britain with a higher extraction yield (17% *w*/*w*), 40–42% of carbohydrate with 22.2% of sulfates [23]. Compared to fucoidans extracted from other green algae, the BBF extraction yield remained much lower than that registered of fucoidans of *Cystoseira compressa* (5.2% *w*/*w*) [24] and *Cystoseira barbata* (5.45% *w*/*w*) [8], whereas, it is close to 2.8% of fucoidans purified from *C. crinite* and to 2.2% extracted from *Dictyota dichotoma* [25,26], while the FSF yield was more important than that reported for the algae mentioned above. On the other hand, the sulfates concentration of extracted polysaccharides (FSF and

BBF) was much higher than those reported for fucoidans extracted from *Cystoseira* and *Sargassum* species [8,24,27]. Elsewhere, fucoidans from *Alaria* sp. and *Saccharina japonica* at spore production was highly sulfated than fucoidans obtained at vegetative status of these brown algae species [28]. Thus, fucoidans' yield and their overall chemical composition could be influenced by the procreating status of seaweed [29]. L-fucose was the principal constitutive monosaccharide of FSF and BBF, with a Fucp/Galp ratio of 3.2 and 14.7, respectively (Table 2), indicating a predominate amount of L-fucose than galactose. This composition was similar to that found in fucoidans of *Saccharina cichorioides* (Fucp/Galp ratio of 13.84) related to 88.6% mol of L-fucose and 6.4% mol of galactose [27]. The ratio found in this work for BBF (14.7) appeared higher than that recorded for fucoidans of *S. japonica* with Fucp/Galp of 1.13 [27], and the ratio obtained from *Undaria pinnatifida* fucoidans which was equal to 1.39 [29]. More studies carried out on fucoidans reported the lower Fucp/Galp ratio for fucoidans of *C. compressa* (2.57) [24], *C. barbata* (1.3) [8], *Agarum cribrosum* (2.63) [30], *Lachemilla angustata* (3.93) [31] and *Fucus evanescens* (8.2) [27]. The monosaccharide composition obtained for FSF and BBF reported the presence of more than 50% of L-fucose; this can explain the higher sulfate concentration in FSF and BBF. It was reported that concentration of sulfated residues depend on the nature of fucoidans monosaccharide composition [27,31].

**Table 2.** Monosaccharide composition of *F. spiralis* (FSF) and *B. bifurcata* (BBF) crude fucoidans.


**a** Monosaccharides composition by GC-MS analysis, expressed as molar % of the total identified peaks based on the weight of dry fucoidans.

#### *2.2. Proton Nuclear Magnetic Resonan ce (1H-NMR) and Infrared (ATR-FTIR) Spectroscopies*

To better characterize the fucoidans, an 1H NMR analysis was carried out. FSF and BBF spectra are presented in Figure 1. The two spectra exhibit five regions characteristic of fucoidans. The intense peaks at 1.34 and at 1.22 ppm are from the H6 methyl protons of L-fucopyranose [32]. Signal at 2.14 ppm refer to the methyl protons of the O-acetyl groups [32]. The spectrum between 4.1 and 3.7 ppm corresponds to the protons of the ring (H2-H5) [8]. The signal around 4.3 ppm, is related to the protons of the 4-O-sulfated monosaccharides [33,34]. It is more intense in the case of FSF than BBF, which corroborates the slight difference in sulfates proportions between the two samples (Table 1). Finally the signals region between 5.3 and 5.03 ppm, are attributed to the C-H proton of substituted O=C and to proton H1 of monosaccharides-α-L-fucopyranose [32]. The spectra obtained for FSF and BBF are very similar to those obtained for fucoidans of *Fucus vesiculosus* and *Ascophyllum nodosum* [33], *C. barbata* [8] and *C. compressa* [24].

In parallel, an infrared analysis was carried out. The Attenuated Total Reflectance ATR-FTIR spectra of BBF and FSF were represented in Figure 2. The two spectra showed characteristics bands at 3406–3403, 2941, 1635–1605, 1423–1420, 1222–1223,1027–1013, 836–833, 577–574 and 479–748 cm<sup>−</sup><sup>1</sup> (Figure 2). The absorption peaks around 3406–3403 and 2941 cm<sup>−</sup><sup>1</sup> are attributed to the elongation of (O-H) and asymmetric vibrations of (C-H), respectively [35]. The signals around 1635 cm<sup>−</sup><sup>1</sup> were attributed to the elongation vibrations of (C=O) in uronic monosaccharides [36]. Asymmetric vibrations of elongation within O-S-O were revealed at 1222 and 1223 cm<sup>−</sup><sup>1</sup> indicating the presence of sulfate esters [37], whilst the elongation of sulfur dioxide (O=S=O) could be indicated by the signals at 1027 and at 1013 cm<sup>−</sup><sup>1</sup> for BBF (Figure 2A) and FSF (Figure 2B), respectively [37]. In addition, sulfate groups linked to C4 of fucosyl units seem to be revealed at 836 and 833 cm<sup>−</sup><sup>1</sup> characteristics bands of (C4-O-S) elongation [38]. However, the binding of sulfate groups with galactose residues was indicated by the absorption bands at 577 and 479 cm<sup>−</sup><sup>1</sup> [37].

**Figure 1.** 1H NMR spectra of sulfated polysaccharides from (**A**) *B. bifurcata* (BBF) and (**B**) *F. spiralis* (FSF) at 60 ◦C in D2O solution.

#### *2.3. E*ff*ect of Fucoidans (FSF and BBF) on the Natural Defence of Date Palm Roots*
