Physico-chemical characterization and pharmacological evaluation of sulfated polysaccharides from three species of Mediterranean brown algae of the genus Cystoseira
© Hadj Ammar et al.; licensee BioMed Central. 2015
Received: 1 October 2014
Accepted: 3 January 2015
Published: 13 January 2015
Seaweed polysaccharides are highly active natural substances having valuable applications. The present study was conducted to characterize the physico-chemical properties of sulphated polysaccharides from three Mediterranean brown seaweeds (Cystoseira sedoides, Cystoseira compressa and Cystoseira crinita) and to evaluate their anti-radical, anti-inflammatory and gastroprotective activities.
The different rates of neutral sugars, uronic acids, L-fucose and sulphate content were determined by colorimetric techniques. The different macromolecular characteristics of isolated fucoidans were identified by size exclusion chromatography equipped with a triple detection: multiangle light scattering, viscometer and differential refractive index detectors, (SEC/MALS/VD/DRI). Anti-inflammatory activity was evaluated, using the carrageenan-induced rat paw edema test in comparison to the references drugs Acetylsalicylate of Lysine and Diclofenac. The gastroprotective activity was determined using HCl/EtOH induced gastric ulcers in rats and to examine the antioxidant effect of fucoidans in the three species, the free radical scavenging activity was determined using 1,1-diphenyl-2-picrylhydrazyl.
The pharmacological evaluation of the isolated fucoidans for their anti-inflammatory, and their gastroprotective effect established that these products from C. sedoides, C. compressa and C. crinita exhibited a significant anti-inflammatory activity at a dose of 50 mg/kg, i.p; the percentages of inhibition of the oedema were 51%, 57% and 58% respectively. And, at the same dose, these fucoidans from C. sedoides and C. compressa showed a significant decrease of the intensity of gastric mucosal damages compared to a control group by 68%, whereas, the fucoidan from C. crinita produced a less gastroprotective effect. Furthermore, the isolated fucoidans exhibited a radical scavenging activity.
The comparative study of fucoidans isolated from three species of the genus Cystoseira showed that they have similar chemicals properties and relatives anti-radical, anti-inflammatory and gastroprotective activities which are found to be promising.
KeywordsFucoidans Cystoseiraceae Cystoseira SEC/MALS/VS/DRI Anti-inflammatory activity Gastroprotective activity
Brown seaweeds represent a rich sources of several nutraceuticals components like laminarans, fucoidans, and polyphenols. Among these, fucoidans, a sulphated polysaccharide have been the subject of much interest in recent years, mainly due to their pharmacological and biological potential with anti-viral , anti-cancer , liver protection , anti-inflammatory  and antibacterial  properties and it also can affect the secretion of extracellular matrix proteins  and activate apoptosis . Several studies have attempted to determine the exact structure of fucoidans but only a few examples of regularity in the structure were found. Links, ramifications, the position of the sulphates and other sugars appear to be variables . Fucoidans are generally linear, mainly composed of repeated units of fucoses sulfated at C-2 and/or C-4 with a-(1–3) and/or a-(1–4) linkages . They can also contain uronic acid, optionally acetylated and other neutral sugars such as D-galactose, D-xylose, D-glucose, D-mannose. However, this chemical composition varies depending on the algal specie and it can vary even within the same species. In this paper, we will focus on a comparative study of physico-chemical and biological properties of fucoidans from three species of brown algae of the genus Cystoseira: C. sedoides, C. compressa and C. crinita. Our attention is particularly paid to this genus of algae for its abundance in the Mediterranean area and more specifically on the Tunisian coast sides. Furthermore, the only structural features of sulphated fucans from this genus of brown seaweed Cystoseira indica have been reported by (Mandal et al.) .
Brown seaweeds, (C. crinita, C. compressa, and C. sedoides) were harvested from the Mediterranean sea, from various areas of the coastal region of Monastir and Tabarka (Tunisia), in June 2007, at a depth between 1 and 3 m. These brown algae are of the family of Cystoseiraceae. After collection, the seaweeds were rinsed with fresh water to remove associated debris and epiphytes. The cleaned material was then air dried in the shade at 30°C. The dried samples were finally powdered and stored at – 20°C until use. Identification of specimens was carried out in the National Institute of Marine Sciences and Technologies (Salambôo, Tunisia).
Extraction of crude polysaccharides
The milled sample was soaked in Methanol-Dichloromethane (1:1) at room temperature for 48 h then filtered. This process was repeated three times. A sequential extraction of seaweed’s powders was carried out with petroleum ether then acetone in a soxhlet apparatus to remove lipophilic pigments (such as chlorophylls) and low molecular weight proteins. Depigmented dried seaweeds were treated three times with 2% aqueous solution of CaCl2 during 3 hours, in order to precipitate alginates. After centrifugation, the supernatant enclosing the fucoidans was recovered and then purified by dialysis through tubing of molecular weight cut off 30 KDa and then lyophilized.
Total carbohydrates were determined for all the extracted polysaccharides by the phenol – H2SO4 method  using galactose as a standard. Whereas, uronic acids were determined using carbazole method  and glucuronic acid as a standard. The sulphate content of the polysaccharides was determined by the turbidimetric method using sodium sulphate (Na2SO4) as a standard after hydrolyzing the polysaccharides in 2 M HCl at 100°C for 2 h . The content of L-fucose units in fucoidans was estimated by a colorimetric assay with L-cysteine . FTIR were performed in KBr pellets (1 mg polysaccharide in 100 mg KBr). The spectra were recorded on a Perkin Elmer 1600 FTIR spectrometer from 400 to 4000 cm−1.
Molecular weight determination
Analysis of various samples was performed using size exclusion chromatography (SEC) equipped with a triple detection: multi-angle light scattering (MALS) (Down HELEOS II, Wyatt Technology, Ca, USA), viscometer detector (VD) (Viscostar II, Wyatt Technology, Ca, USA) and differential refractive index (DRI) (RID 10 A Shimadzu, Japan). The SEC system consists of a pump (LC10 Ai Shimadzu, Japan) at a flow rate 0.5 mL/min and two columns OHPAK SB 804 and 806 HQ.
The samples were dissolved in the eluent (LiNO3 0.1 mol/L) at 2 g/L. The dissolution was carried out by stirring at 380 rpm for 24 h at room temperature. 3 mL solutions were filtered through membrane 0.45 microns (regenerated cellulose) before injection.
The analyzes were performed by a data processing Zimm  “order 1” using angles from (from 34.8° to 142.8°). The corresponding value of dn/dc, in our case is about 0.15 mL/g, the typical value for a polysaccharide . The Astra 22.214.171.124 software package is set to collect and extrapolate data with the aim to obtain for each elution volume the molecular weight and the gyration radius. With an integration of the peak, we calculated the number (Mn) and weight (Mw) average molecular weight and the z-average gyration radius.
where NA is Avogadro’s number, M is the molar mass, [η] is the intrinsic viscosity (g mL−1), and ν is a conformational parameter that takes the value of 2.5 in the case of a spherical conformation.
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity
Where the A control is the absorbance of the control (DPPH solution without sample), the Asample is the absorbance of the test sample (DPPH solution plus test sample), and the Asample blank is the absorbance of the sample only (sample without DPPH solution).
All experiments were performed according to the guidelines established by the European Union on Animal Care (CCE Council 86/609). Wistar rats (150 – 200 g) of both sexes purchased from Pasteur Institute (Tunis, Tunisia) were used. They were housed in groups of eight to ten animals in plastic cages at 20-25°C and maintained on a standard pellet diet with free access to water. Animals were fasted for 24 h before the experiments.
The anti-inflammatory activity of isolated fucoidans was evaluated using the carrageenan induced rat paw oedema test. Wistar rats were divided into groups of six animals and the oedema was induced by injecting 0.05 ml of 1% carrageenan subcutaneously into the sub-plantar region of the left hind paw . Isolated fucoidans from C. crinita, C. sedoides and C. compressa (25 or 50 mg/kg) and reference drugs were administered intraperitoneally (i.p.) 30 min before the injection of carrageenan. The control group received the vehicle (Saline water 2.5 ml/kg, i.p.). The reference groups received Diclofenac (10 mg/kg, i.p) and (ASL, 300 mg/Kg, i.p.).
Where, Vt is the average volume for each group and Vo is the average volume obtained for each group before any treatment .
The gastroprotective activity of fucoidans from three species of genus Cystoseira was studied in HCl/EtOH induced gastric ulcer . Rats were divided into different groups, fasted for 24 h prior receiving an intraperitoneal injection of the isolated fucoidans (25 or 50 mg/kg). Two other groups received Ranitidine (60 mg/kg, i.p.) and Omeprazole (30 mg/kg, i.p.) as reference drugs. After 30 min, all groups were orally treated with 1 ml/100 g of 150 mM HCl/EtOH (40:60, v/v) solution for gastric ulcer induction. Animals were sacrificed 1 h after the administration of ulcerogenic agent; their stomach were excised and opened along the great curvature, washed and stretched on cork plates. The surface was examined to detect the presence of lesions and to measure their extent. The summative length of the lesions along the stomach was recorded (mm) as lesion index.
Results were analyzed using One Way ANOVA (Fisher LSD post hoc test) and expressed as mean ± s.e.m, using SPSS Statistics Software (SPSS for Windows software release 18.0). Difference between means of treated and control groups were considered significant at P < 0.05.
Results and discussion
Extraction and chemical analysis
The main concern in the isolation procedures of fucoidans was to avoid their contamination with other polysaccharides, like laminaran and especially alginic acid. The hot extraction, in the presence of CaCl2 was allowed to separate the insoluble calcium alginate from the soluble fraction. This fraction is rich in fucoidan and laminaran. To eliminate this latter we had to recourse to dialysis.
Yields of extraction and carbohydrates analysis
Total sugar (%)
Uronic acid (%)
Sulfate (% SO 3 Na)
The different colorimetric assays confirm that isolated polysaccharides are fucoidans, mainly composed by fucose (43 to 61%). The amount of sulphates was determinated for the isolated fucoidans. There was no difference found between the three species; an average of 16% was observed for all. Besides, the isolated fucoidans were moderately sulphated compared to those of Cystoseira indica (11.5%) , Saccharina longicruris (12%) , Fucus vesiculosus (12%)  and Sargassum stenophyllum (28%) .
The most diagnostic peaks in the IR spectra of extracted polysaccharides
O-H assoc. stretching vibration
C-H stretching vibration
C = O stretching vibration
asymmetrical bending vibration of CH3
S O stretching vibration
C-O-C stretching vibration
Average macromolecular characteristics of fucoidans isolated from C. crinita, C. compressa, and C. sedoides ) determined by SEC/MALS/VD/DRI (0.1 mol/L LiNO 3 )
[η] (mL.g −1 )
R H (nm)
The knowledge of molecular weight and intrinsic viscosity for each elution volume can be used to determine the Mark-Houwink exponent. Values between 0.7-0.8 are in agreement with random coil conformation .
However, it’s important to note that chemical composition, molecular weight and structure varies depending on the source of fucoidans, the harvest period and the extraction methods.
DPPH radical scavenging activity
EC50 values of fucoidans extracted from C. crinita, C. compressa, and C. sedoides in radical scavenging activity
0.13 ± 0.01
0.96 ± 0.01
0.84 ± 0.06
0.76 ± 0.04
Effect of the administration of fucoïdanes isolated from C. sedoides, C. compressa and C. crinita and reference drugs in carrageenan induced rat paw edema
Edema (mL × 10 −2 )
Edema inhibition (%)*
26 ± 2.75
57.5 ± 1.51
59 ± 3.27
17.66 ± 1.63
24.83 ± 1.72
29.33 ± 1.21
15.66 ± 5.12
25.83 ± 2.40
28.66 ± 3.61
13.66 ± 3.20***
29.66 ± 5.20***
40.16 ± 4.70***
10.66 ± 4.08***
28.16 ± 3.38***
41.66 ± 1.21***
12.66 ± 2.73***
26.83 ± 1.04***
29.83 ± 2.40***
11.83 ± 4.09***
24.83 ± 2.48***
28.00 ± 2.36***
12.66 ± 3.43***
26.50 ± 3.39***
19.83 ± 1.94***
13.20 ± 2.68***
24.16 ± 2.56***
28.50 ± 2.50***
Results of antiulcerogenic activity of fucoïdanes isolated from C. sedoides, C. compressa and C. crinita on gastric ulcer induced by HCl/ethanol solution
Average lesion (mm)
Ulcer inhibition (%)*
50.33 ± 5.50
43.38 ± 4.35
17.50 ± 1.38
20.83 ± 2.56***
58.57 ± 5.09a
16.00 ± 2.19***
68.19 ± 4.35b
22.83 ± 3.70***
54.60 ± 7.37a
15.83 ± 4.60***
68.51 ± 5.24b
28.33 ± 4.09***
43.66 ± 5.13a
20.17 ± 6.17***
59.90 ± 7.26b
HCl-ethanol induced gastric mucosal lesions may be multifactorial, with static blood flow contributing significantly to the hemorrhagic as well as the necrotic aspects of the tissue injury . The decrease in the number of lesions may be due the reduction in the levels of gastric secretion . The alteration in the acidity/volume of the gastric juice is due to the production of HCl, which may increase the permeability of the mucosal membrane . Thus, the overall protection by fucoidans against HCl-ethanol induced gastric ulceration in experimental rats suggest that it contains some anti-ulcer agents that may hasten the decomposition of free radicals generated, thereby strengthening the gastric mucosal antioxidant defense system suggesting an gastroprotective effect of fucoidans from brown algae.
The comparative study of fucoidans isolated from three species of the genus Cystoseira showed that they have similar properties regarding the percentage in sulphates, L-fucose content and their molecular weight. However this requires an advanced structural study to determine the length of the general chain and the branching of fucoidans.
These similarities are reflected on pharmacological activities; in fact the different isolated fucoidans have similar anti-radical, anti-inflammatory and gastroprotective activities which are found to be promising.
Acetylsalicylic of lysine
- C. sedoides:
- C. compressa:
- C. crinita:
Average molecular number
Average molecular weight
Average hydrodynamic diameter
We gratefully acknowledge the financial support of the Doctorale School of Materials, Devices and Microsystems of Monastir, further special thanks goes to Mr. Christophe Rihouey for the technical support regarding SEC/MALS/VD/DRI analyses.
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