Crude Polysaccharides from Cystoseira species: Extraction, Characterization and in vitro Antioxidant Activity

Introduction

Oxidative stress is an excessive accumulation of free radicals in cells compared with antioxidant defenses. In biological systems, this excess of free radicals results from an imbalance between the production of reactive oxygen species and their neutralization by antioxidant defense systems.^1,2 Uncontrolled reactive oxygen species (ROS) generation induces significant damage to cellular structure and metabolism by damaging various targets such as proteins, lipids, and nucleic acids.³ Oxidative stress is one of the triggering factors of various human diseases, such as cancer, ocular diseases, and neurodegenerative diseases. Moreover, it contributes to the progression of other pathologies by modulating immune and vascular dysfunction.² Additionally, it contributed to the deterioration of sensory and nutritional quality of food.⁴

To eliminate the excess of free radicals, several researches demonstrate that bioactive metabolites derived from natural sources, plant and algae, can be used as natural antioxidants.

Seaweeds, or macroalgae, represent an important source of bioactive secondary metabolites with antioxidant properties, including phenolic compounds,⁵ flavonoids,⁶ carotenoids,⁷ fucoxanthin,⁸ and particularly polysaccharides.⁹

Polysaccharides are the most macromolecular polymers (carbohydrates) that exist in nature.^10,11 Polysaccharides are a class of natural compounds that have been widely investigated and utilized because of their abundance, renewability, biocompatibility, non-toxicity, and biodegradability. Different types of polysaccharides can be extracted from seaweeds, such as alginate, fucoidan, and laminarin from brown algae, and carrageenan, and agar from red algae and ulvan from green algae. Those natural compounds are garnering increasing scientific interest due to their multi-faceted bioactivities and their functional applications in the food, pharmaceutical, cosmetic, and biotechnological industries as emulsifiers, stabilizers, and bioactive agents.¹² Notably, water-soluble polysaccharides exhibit a broad spectrum of biological activities, including antioxidant potential by protecting cells against oxidative stress through the neutralization of free radicals or by enhancing the activity¹³ and expression of antioxidant enzymes, antiinflammatory, anticoagulant, antidiabetic, and antiviral properties.^13,14

Cystoseira is a brown seaweed belong to the Sargassaceae family. This genus contains a variety of bioactive compounds with biological properties.^15,16 Several reports have revealed that Cystoseira polysaccharides, alginates and fucoidans exhibit an important scavenging activity, using various antioxidant tests.^17–20

According to previous research, El Jadida coast is riche in seaweed, the extract from those specices that exhibit an important antioxidant activity.²¹

The present study aims to extract and evaluate, in vitro, the antioxidant activity of crude polysaccharides extracted from three Cystoseira sp., e.g., Cystoseira baccata, Cystoseira mediterranea and Cystoseira myriophylloides collected from Sidi Bouzid. The antioxidant potential was evaluated using five different assays: total antioxidant capacity (TAC), DPPH (2,2-diphenyl 1-picrylhydrazyl) scavenging activity, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonicacid)) scavenging activity, FRAP (Ferric reducing antioxidant power), and hydroxyl-radical scavenging activity.

Experimental

Seaweed pre-treatment – Cystoseira sp. were collected from Sidi Bouzid coast in El Jadida (Atlantic Ocean, Morocco) during the summer of 2021. The identification (macro and microscopic) of three Cystoseira species was carried out by Professor Wafaa Arsalan, Professor at the Faculty of Science, Chouaib Doukkali University, El Jadida (Morocco). The collected algae were transported to the laboratory for further processing.

In the laboratory, the seaweed was cleaned and rinsed several times with running water to remove salt and impurities, then rinsed with distilled water. They were then drained in the open air and shade for 24 h before being oven-dried at 40°C for 12 h. Finally, the dried algae were ground to a fine powder using a mechanical grinder.

Crude polysaccharides extraction – The extraction and purification of the crude polysaccharides was carried out in three steps: the depigmentation to remove pigments and other compound, the extraction using hot water to enhance the solubility and recovery of sulfated polysaccharides, and purification of the polysaccharide using ethanolic precipitation and dialysis (Fig. 1).

Fig. 1.

The extraction and purification process used to obtain crude polysaccharides from Cystoseira sp.

Seaweed powder was depigmented using acetone (1:15; w/v) for 24 h (twice). then methanol (1:15; w/v) for 24 h (twice) under agitation. After filtration and drying, the depigmented and defatted powder was suspended in distilled water and heated at water at 80°C for 3 h, then filtered and centrifuged (6000 rpm, 20 min), this process is repeated twice to optimize the extraction. The supernatants were collected and concentrated with a rotary evaporator at 45°C. The soluble polysaccharides were precipitated with 3 volumes of 96% cold ethanol and incubated at 4°C overnight for 3 times. After centrifugation (8000 rpm, 30 min), the precipitate was solubilized in ultrapure water and dialyzed (Snakeskin Dialysis Tubing, 3.5 kDa MWCO, 35 mm dry Martini I.D., 35 feet) for 72 h and then lyophilized during 72 h. The powder obtained was crude polysaccharides. The yield was calculated using the following equation:

Colorimetric assays – Colorimetric assays were used to determine the biochemical composition of the crude polysaccharides obtained.

Total carbohydrate concentration was determined using the phenol and sulfuric acid method and D-glucose as standard.²² Total phenolic compounds content was determined by the Folin-Ciocalteu method using gallic acid as a standard.²³ Protein concentrations were determined using the Coomassie Brilliant Blue G-250 method and bovine serum albumin as a standard.²⁴ The sulfate was evaluated using the turbidimetric method (BaCl₂/gelatin) with K₂SO₄ as a standard.²⁵

HPAEC-PAD analysis of crude polysaccharides – Monosaccharide composition was determined using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection. 10 mg of crude polysaccharides were hydrolysed with 1 mL of TFA 2 M and heated at 100°C for 2 h, then the hydrolysate was neutralized with an ammonia solution and filtered. The samples were analyzed by HPAEC-PAD using a Dionex ICS-3000. A 25 µL aliquot of each sample was injected into a thermostated CarboPac PA1 column (Dionex Corporation, 4 × 250 mm) set at 25°C, with a flow rate of 1 mL/min. The elution process began with an isocratic gradient of NaOH (18 mM) for 30 min, followed by a linear gradient of sodium acetate in NaOH (200 mM) for 20 min. The final gradient was composed of 100% sodium acetate (1 M) in NaOH (200 mM) for 20 min. The system was subsequently washed with NaOH (200 mM) for 15 minutes and reequilibrated with NaOH (18 mM) for 7 minutes. Data analysis was performed using Chromeleon software (version 6.8).

FTIR spectroscopy – Fourier-Transform Infrared (FT-IR) measurements were performed using a Thermo Scientific Nicolet Impact 400D FT-IR instrument (Nicolet Instrument Co., Madison, WI, USA). The spectra were obtained at ambient temperature in the wave-number range of 400 to 4000 cm⁻¹ with an average of 32 scans, and the results obtained are analyzed by OMNIC software.

Total antioxidant capacity – The total antioxidant capacity of the crude polysaccharides was carried out according to a protocol adapted by Prieto et al., (1999).²⁶ A 0.1 mL of sample at different concentrations (0.2–5 mg/mL) was mixed with 1 mL of solution A (ammonium molybdate, monobasic sodium phosphate NaH₂PO₄ and sulfuric acid H₂SO₄ (0.6 M) and then incubated at 95°C for 90 min. After cooling, the absorbance was read at λ = 695 nm. Ascorbic acid was used as a positive standard, and the results were expressed by mg equivalent Ascorbic acid/mg of crude polysaccharides.

DPPH radical-scavenging activity – DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of crude polysaccharides was evaluated using two methods: qualitative assay using a Silica plate and quantitative assay using a spectrophotometric assay.

Evaluation of antioxidant activity by silica plate – The evaluation of antioxidant activity was realized according to the protocol established by Chibi et al., (2019)²¹ with some modifications. 5, 10, and 15 μL of the crude polysaccharides and ascorbic acid were deposited on a silica plate (CCM Sil G25 UV 254 mm-Marcherey-Nagel, 5 × 20 cm, thickness 25 mm), and then the ethanolic solution of DPPH (0.02%) was sprayed uniformly on the plate in shelter from the light. After development, the plates were read after 30 min. The images were made using a scanner (HP Deskjet 2050A). The antioxidant activity of the extracts was estimated by the fading of spots compared to ascorbic acid spots.

Evaluation of the antioxidant activity by spectrophotometry – The DPPH radical-scavenging capacity of the crude polysaccharides was evaluated by the method of Hentati et al., (2018).¹⁸ In the dark, 0.5 mL of samples at different concentrations of (0.2 – 5 mg/mL) were mixed with 0.5 mL of DPPH solution in ethanol (95%). The mixture was incubated for 30 min at an ambient temperature (25°C). The absorbance was read at λ = 517 nm. Ascorbic acid was used as a positive standard.

DPPH scavenging activity was estimated following the equation:

A control: absorbance of DPPH without sample; A sample: absorbance of sample with DPPH; A0: absorbance of sample without DPPH.

Ferric-reducing power – The Ferric-reducing power of the crude polysaccharides was examined using the method described by Oyaizu (1986).²⁷ 0.5 mL of each sample at different concentrations (0.2–5 mg/mL) were mixed with 1.25 mL of phosphate buffer (0.2 M, pH = 6.6) and 1.25 mL of potassium ferricyanide (1%, w/v). After incubation (30 min, 50°C), the mixture was treated with 1.25 mL of trichloroacetic acid (10%, w/v) and centrifuged for 10 min at 3000 g. 1.25 mL of the supernatant was thoroughly mixed with ultra-pure water (1.25 mL) and 0.25 mL of a 0.1% (w/v) ferric chloride solution. The absorbances were measured at 700 nm after incubation (10 min, 25°C). Ascorbic acid was used as a standard.

ABTS radical-scavenging activity – ABTS (2 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging was carried out according to the method of Wu et al., (2019).²⁸ The ABTS solution (7 mM) was prepared by mixing ABTS and potassium persulfate K₂S₂O₈ for 16 h in the dark. This solution is diluted to obtain an optical density of 0.7 at wavelength 734 nm. 0.2 mL of each sample at different concentrations (0.1–5 mg/mL) was mixed with 0.6 mL of the ABTS solution, then incubated for 7 min. The absorbance was read at λ = 934 nm. Ascorbic acid was used as a standard. ABTS scavenging activity was estimated following the equation:

A control: absorbance of ABTS without sample; A sample: absorbance of sample with ABTS; A0: absorbance of sample without ABTS

Hydroxyl radical scavenging activity – Hydroxyl radical scavenging activity was evaluated according to a protocol adapted by Delattre et al., (2015).²⁹ A 0.2 mL of each sample or ascorbic acid at different concentrations (0.02–1 mg/mL) was mixed with 0.2 mL of FeSO₄ (5 mM). After vortex homogenization, 0.2 mL H₂O₂ (1% w/v) was added. Then the mixture was incubated at room temperature (25°C) for 1 h. The absorbance was read at λ = 510 nm. The scavenging activity was estimated following the equation:

A control: absorbance of H₂O₂ without sample; A sample: absorbance of sample with H₂O₂; A0: absorbance of sample without H₂O₂.

Statistical analysis – All the experiments were conducted in triplicate. The results were expressed as mean ± standard deviations (SD) and error bars in the figures indicate standard deviation. The differences among values were assessed using one-way analysis of variance (ANOVA) in SPSS 26.0 (IBM SPSS, Armonk, NY: IBM Corp., USA). Tukey's multiple range test was employed for multiple comparisons, and the statistical significance was set at a p < 0.05. The 50% inhibitory concentrations (IC₅₀ values) were calculated using the regression equation formula in EXCEL.

Results and Discussion

The crude polysaccharides were extracted and purified from the Moroccan brown alga Cystoseira baccata (CB), Cystoseira mediterranea (CM) and Cystoseira myriophylloides (CMy). The extraction yields of polysaccharides are 13.5, 11.61, and 12.37% for CB, CM, and CMy, respectively.

The yields of polysaccharides extracted from the three Cystoseira using hot water extraction, cold ethanol precipitation, and dialysis are important compared to the yields of polysaccharides extracted from the red alga Gracilaria lemaneiformis using three extraction procedure: hot water (9.14%), ultrasound-Assisted (8.52%) and microwave-Assisted (9.16%).⁹ Additionally, the yield from Cystoseira sp. was higher than that obtained from the brown algae Cystoseira barbata (6.59%),³⁰ Sargassum angustifolium³¹ and comparable to the polysaccharides extracted from Dendrobium denneanum (15%) using water and ethanol precipitation.³²

Polysaccharides yield is affected by various factors, including the environmental conditions (such as water salinity, temperature, and the season of harvest), as well as the purification and the extraction protocols. Norouzi et al., (2023) demonstrate that extraction parameters such as water-to-solid ratio and extraction time, have a direct impact on the yield of polysaccharides extracted from Sargassum angustifolium. Using 80° to extract the polysaccharides provides a high yield. Previous researches demonstrate that an increase in temperature raises the internal energy of the molecules, enhances the solubility of the substances, and promotes the breaking of the internal and external hydrogen bonds of the biopolymer.³³

The biochemical composition of the three polysaccharides is presented in Table 1. The crude polysaccharides are mainly composed of total carbohydrates, with smaller amounts of proteins and phenolic compounds. The highest concentration of total carbohydrates was observed in CPM (48%), followed by CPMy (43%) and CPB (37%). The protein content was 8% for CPB and 5% for both CPM and CPMy. As for phenolic compound, the content was 0.5, 1 and 2% for CPB, CPM, CPMy, respectively.

Table 1.

Global composition of crude polysaccharides from Cystoseira sp.

The relatively low percentages of proteins and phenolic compounds in the crude polysaccharides suggest that most of these components were effectively removed through acetone, methanol treatment, ethanol precipitation, and dialysis processes. The total carbohydrate content of the crude polysaccharides is close to that found in Gracilaria lemaneiformis polysaccharides, and higher than those of Sarcodia ceylonensis (22.91%), Ulva Lactuca (23.71%),⁹ and Sargassum vulgare polysaccharidic fractions.³⁴ However, there are lower compered to polysaccharides obtained from Sargassum angustifolium (73.8%).³¹ The glucose found in most likely derived from laminarans.³⁵

The protein contain is compared to that in Sargassum angustifolium polysaccharides (4.47%),³¹ and low compared to Cystoseira barbata (9.86%).³⁰ The protein content in three crude polysaccharides is higher than that in the polysaccharidic fractions from Sargassum vulgare,³⁴ and fractions from Chondrus crispu (1.4–4.4%), Ahnfeltiopsis devoniensis (1.1–4.9%), Sarcodiotheca gaudichaudii (3.1–10.6%) and Palmaria palmata (0.4–2.3%).³⁶ The phenolic and sulfate contain are lower than those in polysaccharides extracted from Cystoseira barbata (4.98%)³⁰ and Gracilaria fisheri (7.19% ).³³

The purity of polysaccharides is influenced by the extraction protocol. Premarathna et al., (2024) demonstrated that the composition and molecular weight of polysaccharide fractions from red seaweed C. crispu, A. devoniensis, S. gaudichaudii and P. palmata change depending on the extraction parameters.³⁶ Additionally, the extraction temperature significantly affects the sulfate content of the crude polysaccharides, the highest sulfate content was obtained at high temperature compared to those obtained at low extraction temperature.³⁷ The structural difference might be attributed to the fractionation process in addition to the different algal species.

The crude polysaccharides are mainly composed of neutral monosaccharides (Table 2). CPB, CPM, and CPMy composed of fucose (8.4, 10.19 and 9.65 mg/g), glucose (2.62, 2.92 and 9.84 mg/g), galactose (2.41, 3.05 and 2.67 mg/g) and xylose (1.69, 2.51 and 1.25 mg/g) for CPB, CPM, and CPMy, respectively. Those results suggest that the three crude polysaccharides were heteropolysaccharides, with the presence of fucoidans and laminarins, characteristic of brown algae polysaccharides.³⁴

Table 2.

Monosaccharides compositions of crude polysaccharides extracted from Cystoseira sp.

CPB, CPM, and CPMy shows a similar infrared spectral (Fig. 2). FTIR Analyze of the three crude polysaccharides show the presence of typical peaks of polysaccharides.⁹ The absorption bands at 3000–3600 cm^-1, concentrated at 3265, 3267 and 3264 cm^-1 represents O–H vibrations, the peaks at 1417 cm^-1 and 1589 cm^-1 represents asymmetric and symmetric stretching of C=O,⁹ band of peaks at 1217–1225 cm^-1 represents S=O stretching, the peaks at 1030 cm^-1 could be assigned to the stretching modes of pseudosymmetric sulfate groups (O=S=O). Peaks around 817 cm^-1 were observed, which showed that the sulfate groups were in equatorial positions.³⁸ The absorbance peaks in the region 800-1250 cm^-1 correspond to sulfate groups.¹⁸ Polysaccharides of macroalgae exhibit different structural characteristics and interesting biological functions. In this study, the different antioxidant mechanisms of the crude polysaccharides extracted were evaluated using various antioxidant assays, including TAC, DPPH, FRAP, ABTS, and OH assays.

Fig. 2.

FTIR spectrum of Cystoseira sp. Crude polysaccharides; CPB: Crude polysaccharides from C. baccata, CPM: Crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides. The results are presented as mean and standard deviation (n = 3).

The aim of total antioxidant capacity (TAC) or phosphomolybdate assay is to evaluate the capacity to donate electrons, which is indicated by the dark green color. The results of the total antioxidant capacity are presented in Fig. 3. The crude polysaccharides extracts were expressed as equivalents of mg L-ascorbic acid. The results show that the total capacity was concentration dependent. The total antioxidant capacities of the three crude polysaccharides are 114, 154 and 323 µg equivalent of ascorbic acid /mg extract for C. baccata, C. mediterranea and C. myriophylloides, respectively. CPMy exhibited significantly higher (p < 0.05) DPPH radical-scavenging activity compared to CPB and CPM. CPB, CPM, and CPMy exhibited significant antioxidant capacities comparable to those of Dictyota dichtoma var. velutricata (129.45 and 325.24 mg ascorbic acid equivalents/g extract at the concentration of 1 and 3 mg/mL, respectively) and Padina pavonia (127.94 mg ascorbic acid equivalents/g extract at the concentration of 3 mg/mL).³⁹ Therefore, GB can be considered as a good electron donor.

Fig. 3.

Total antioxidant activity of crude polysaccharides from Cystoseira sp.; CPB: crude polysaccharides from C. baccata, CPM: crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides. The results are presented as mean and standard deviation (n = 3).

DPPH radical scavenging activity assay is a widely used method to evaluate the free radical scavenging ability of natural compounds. The scavenging activity is measured by the discoloration of the DPPH solution in the presence of the antioxidant.⁴⁰ The three polysaccharides tested react positively with DPPH, indicating their antioxidant activity (Fig. 4). The discoloration of DPPH in the spots of deposits indicates that the crude polysaccharides have an antioxidant activity. The more deposits are discolored, the more antioxidant activity is important.

Fig. 4.

Silica plate revealing decolorized spots due to the reaction of Cystoseira sp. crude polysaccharides with DPPH after 120 minutes; AsAc: Ascorbic acid, CPB: crude polysaccharides from C. baccata, CPM: crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides

DPPH radical-scavenging activity of the three crude polysaccharides was tested by spectrophotometry also. The scavenging rate of the DPPH radical of CPB, CPM and CPMy, at concentrations of 0.2 to 5 mg/mL (Fig. 5). The scavenging activity was concentration-dependent anti-radical activities in both standards and the crude polysaccharides. At 5 mg/mL, CPM and CPMy possessed significantly higher (p < 0.05) DPPH radical-scavenging activity (100%) compared to CPB (69.44%). The IC₅₀ were 2.033, 0.620, and 0.306 mg/mL for CPB, CPM, and CPMy, respectively. The lower IC₅₀ values suggests that CPMy and CPM could donate more hydrogen atoms than CPB, also their stronger antioxidant potential. CPB, CPM and CPMy possessed high DPPH radical scavenging activity compared to laminarin extracted from Cystoseira barbrata¹⁹ and Sargassum thunbergii (IC₅₀ between 3.13 and 4.43 mg/mL).⁴¹ CPMy also shows an important inhibition close to crude polysaccharides from Sargassum angustifolium with IC₅₀ = 0.32 mg/mL.³¹

Fig. 5.

DPPH radical-scavenging activity of crude polysaccharides from Cystoseira sp.; AsAc: Ascorbic acid, CPB: crude polysaccharides from C. baccata, CPM: crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides. The results are presented as mean and standard deviation (n = 3).

The observed differences in DPPH scavenging activity among the extracted polysaccharides may be attributed to several factors, including the purity of the samples (the presence of co-extracted molecules such as phenolic compounds), the sulfate groups,³³ and some specific oses such as mannose, rhamnose, mannose and fucose contenat.^18,42

The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. In the presence of antioxidants, the reaction solution changes color from yellow to blue, indicating the reduction of ferric ions (Fe³⁺) to ferrous ions (Fe²⁺) in an acidic environment.³⁹ The reducing power of the three crude polysaccharides was assessed by monitoring the formation of the ferrous complex at a wavelength of 700 nm (Fig. 6). At 5 mg/mL, the ODs (λ = 700) obtained are 0.898, 0.978, and 1.11 for CPB, CPM and CPMy, respectively. CPMy exhibited significantly higher (p < 0.05) reducing power followed by CPM, and CPB. All samples were weaker than ascorbic acid and FeSO₄.

Fig. 6.

Reducing power of crude polysaccharides from Cystoseira sp.; AsAc: Ascorbic acid FeSO4: Ferrous Sulfate, CPB: crude polysaccharides from C. baccata, CPM: crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides. The results are presented as mean and standard deviation (n = 3). Different letters in the same row mean significantly different by Tukey’s multiple range test (p < 0.05).

However, the absorbance values of three crude polysaccharides are higher than those reported for Durvillaea antarctica (OD = 0.317), Gracilaria lemaneiformis (OD = 0.113),⁸ and laminarin from Cystoseira barbata (OD = 1.37 at 20 mg/mL).¹⁹ CPMy may donate electrons to free radicals, converting them into more stable, non-toxic diamagnetic molecules, thereby contributing to its antioxidant activity.⁴³

FRAP value may be correlated with the molecular weight and the total sulfate content of the polysaccharides. Premarathna et al., (2024) demonstrated that polysaccharide fractions from C. crispus exhibited significantly higher DPPH and FRAP radical scavenging abilities, which may be attributed to their molecular weight and sulfate content, as well as their phenolic content,³⁶ and also may be related to the phenolic contain.⁹

ABTS radical-scavenging activity is evaluated by measuring the discoloration of the ABTS solution, which changes from blue-green to colorless in the presence of the crude polysaccharides CPB, CPM, and CPMy. The ABTS radical-scavenging activities are presented in Fig. 7.

Fig. 7.

ABTS radical-scavenging activity of crude polysaccharides from Cystoseira sp.; AsAc: Ascorbic acid, CPB: crude polysaccharides from C. baccata, CPM: crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides. The results are presented as mean and standard deviation (n = 4).

The results indicate that the scavenging effect on the ABTS radical increases significantly (p < 0.05) with an increase in the concentration of the crude polysaccharides. At a concentration of 5 mg/mL, the maximum scavenging rates of CPB, CPM, and CPMy are 76.15%, 71.44%, and 100%, respectively. Among all the extracts, CPMy exhibited the highest antioxidant capacity with an IC₅₀ value of 459 μg. The inhibition of ABTS radical by CPB, CPM, and CPMy is important compared to polysaccharides extracted from Durvillaea antarctica (IC₅₀ = 3.75 mg/mL), Sarcodia ceylonensis (IC₅₀ = 3.99 mg/mL) and Ulva Lactuca (IC₅₀ = 3.59 mg/mL).⁹ At 2 mg/mL, the three crude polysaccharides show an important ABTS radical scavenging compared to polysaccharides from Dendrobium denneanum (19.6%).³² The antioxidant activity of CPB, CPM and CPMy are lower than three polysaccharide fractions obtained from Sargassum angustifolium were the capability to scavenge the ABTS at 0.5 mg/mL was 75.76%, 91.85%, and 96.07% depending on the fraction.³¹

The scavenging activity of the crude polysaccharides is related to their composition. Previous studies have shown that the sulfate content affects ABTS scavenging activity in other seaweed samples.^9,18 The crude polysaccharides scavenge the ABTS radical through various mechanisms at different concentrations. The ABTS radical cation extracts an electron or hydrogen atom from the polysaccharides, forming a semiquinone radical. This semiquinone radical can then react with another ABTS radical cation.^43,44 Additionally, the color change of the ABTS solution depends on factors such as reaction duration, intrinsic antioxidant activity, and sample concentration.⁴⁰

This assay was used to evaluate the inhibition of hydroxyl radical formation. It considers OH• scavenging and iron chelation activity of polysaccharides on the same reaction.⁴⁵ This methodology is based on OH• generation by the reduction of Fe³⁺ to Fe²⁺ in the presence of OH⁻salicylic acid adduct, as well as on the hydroxylation of salicylic acid. The hydroxyl radical scavenging effect of the three crude polysaccharides was compared with ascorbic acid (Fig. 8). The results indicated that the scavenging effects on hydroxyl radicals were concentration-dependent (p < 0.05). At 1 mg/mL, the scavenging abilities were 68.17%, 91.37%, and 98.27% for CPB, CPM, and CPMy, respectively. Among all the extracts, CPMy exhibited significantly higher antioxidant capacity (p < 0.05), although all samples demonstrated weaker activity than ascorbic acid. The crude polysaccharides show considerable hydroxyl radical-scavenging activity at low concentrations (1 mg/mL), when compared to polysaccharides extracted from Dendrobium denneanum (78.9%),³² alginates (82%),¹³ and laminarin isolated from Cystoseira barbata (62%)¹⁹ which were evaluated at higher concentrations (2 mg/mL, 5 mg/mL, and 20 mg/mL, respectively).

Fig. 8.

Hydroxyl radical-scavenging activity of crude polysaccharides from Cystoseira sp.; AsAc: Ascorbic acid FeSO4: Ferrous Sulfate, CPB: crude polysaccharides from C. baccata, CPM: crude polysaccharides from C. mediterranea and CPMy: crude polysaccharides from C. myriophylloides. The results are presented as mean and standard deviation (n = 4).

The application of various methods to assess the potential antioxidant activity of the crude polysaccharides demonstrates their versatility as antioxidants across different cellular sites, including the mitochondria, cytoplasm, and nucleus.⁴⁶ The crude polysaccharides show considerable antioxidant potential using different radical scavenging assays. This activity is influenced by factors such as polysaccharides purety, solubility, sugar composition, linkage types, sulfate content, and molecular weight.^{18,19,36,45,47} Additionally, literature suggests that polyphenols bound to polysaccharides might also contribute to the observed antioxidant activity of these crude polysaccharides.³⁰

In this study, crude polysaccharides were extracted from three Cystoseira species, C. baccata, C. mediterranea, and C. myriophylloides with yields of 13.5, 11.61 and 12.37%, respectively. Those polysaccharidic extracts were mainly composed of fucose (10.19, 9.65 and 8.4 mg/g) and glucose (9.84, 2.92 and 2.62 mg/g), along with low percentages of phenolic compounds and proteins. The antioxidant assays demonstrate the ability of the three samples to donate electrons / hydrogen in different chemical environments, especially CPMy with a high activity. This study provides valuable insights into the potential application of Cystoseira sp. polysaccharides as natural compounds with strong antioxidant properties, suitable for the development of functional food products and medicinal applications.

Acknowledgments

This research was financially supported by CAMPUS FRANCE (PHC TOUBKAL 21/117 (French-Morocco bilateral program) Grant Number:45883YJ).

We would like to express our sincere gratitude to Professor Wafaa Arsalan, algologist and professor at the Faculty of Science in El Jadida (Morocco), for her expert guidance and invaluable help in identifying the collected algae samples.

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