Characterization of sulfated polysaccharide from

International Journal of Biological Macromolecules 126 (2019) 326–336

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Characterization of sulfated polysaccharide from Laurencia obtusa and its apoptotic, gastroprotective and antioxidant activities Sirine Lajili a,c,⁎, Hiba Hadj Ammar b, Zeineb Mzoughi b, Haifa Bel Haj Amor a, Christian D. Muller c, Hatem Majdoub b, Abderrahman Bouraoui a a Laboratoire de Développement Chimique, Galénique et Pharmacologique des Médicaments (LR12ES09), Unité de Pharmacologie Marine, Faculté de Pharmacie de Monastir, Université de Monastir, 5000 Monastir, Tunisia b Laboratoire des Interfaces et des Matériaux Avancés (LIMA), Faculté des Sciences de Monastir, Université de Monastir, Bd. de l'environnement, 5019 Monastir, Tunisia c Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74 route du Rhin, 67401 Illkirch, France

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Article history: Received 12 June 2018 Received in revised form 8 December 2018 Accepted 9 December 2018 Available online 10 December 2018 Keywords: Laurencia obtusa Sulfated polysaccharide Apoptotic activity Gastroprotective activity Antioxidant

a b s t r a c t This study was designed to characterize the physico-chemical properties of the sulfated polysaccharide (SP) isolated from the red alga Laurencia obtusa and to evaluate its apoptotic, gastroprotective and antioxidant activities. The different macromolecular characteristics of SP were determined by size exclusion chromatography combined with multi-angle laser light-scattering detection (SEC-MALLS), Fourier transform infrared spectroscopy (FTIR) analysis and nuclear magnetic resonance spectroscopy (1H NMR and 13C NMR). The native molecular weight of the extracted polysaccharide is high (≥336,900 g·mol−1). It showed high amounts of sulfated groups (28.2%) and low levels of proteins. It was found to be a potent inducer of apoptosis on acute monocytic leukaemia THP-1cell lines with EC50 value of 53 μg·mL−1. Furthermore, a significant gastroprotective effect (p b 0.01) was also observed with a gastric ulcer inhibition of 63.44%, 78.42% and 82.15% at the doses 25, 50 and 100 mg·kg−1, respectively. In addition, SP significantly increased glutathione levels (GSH) and decreased the concentration of thiobarbituric acid-reactive substances (TBARS) in EtOH/HCl-damaged gastric mucosa in rats; it also exhibited an important antioxidant activity in vitro. Therefore, SP, derived from the red alga Laurencia obtusa, may have a potential therapeutic effect against acute myeloid leukaemia and a beneficial potential as gastroprotective and antioxidant natural product. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Marine organisms are found to be very rich sources of foods, medicines and energy. They have also proven to contain structurally diverse bioactive compounds with valuable pharmaceutical and biomedical potentials [1]. Thus, there is a great deal of interest to find novel nature derived bioactive compounds to protect human health, especially those extracted from marine organisms. Algae contain many bioactive primary and secondary metabolites and represent about 9% of marine biomedical compounds [2]. Recently, research interest has focused on sulfated polysaccharides (SPs) from marine algae possessing a broad spectrum of therapeutic properties, which may be useful in complementary or alternative medicine [3].

⁎ Corresponding author at: Laboratoire de Développement Chimique, Galénique et Pharmacologique des Médicaments (LR12ES09), Unité de Pharmacologie Marine, Faculté de Pharmacie de Monastir, Université de Monastir, 5000 Monastir, Tunisia. E-mail addresses: [email protected] (S. Lajili), [email protected] (C.D. Muller), [email protected] (A. Bouraoui).

https://doi.org/10.1016/j.ijbiomac.2018.12.089 0141-8130/© 2018 Elsevier B.V. All rights reserved.

Among the large variety of SPs, those, obtained from red algae, were worldwide used in food applications and were reported to have a large number of medical applications, some of which date from the 1830s. Carrageenan, a sulfated polysaccharide from red algae, is still used in Ireland to make traditional medicinal teas and cough medicines to combat colds, bronchitis and chronic coughs and used to make internal poultices to control stomach ulcers [4]. Furthermore, SPs from red algae, have been shown to exhibit many other biological and pharmacological activities like antitumor [5], antioxidant [6], antiulcer [7], antiinflammatory and immune-modulatory activities [8]. The structure of red seaweed SPs consist of linear chains of alternating 3-linked β-D-galactopyranose units and 4-linked 3,6-anhydro-αgalactopyranose or α-galactopyranose units, having different positions and degrees of sulfation [9]. Cancer is a dreadful human disease, increasing with changing life style, nutrition, and global warming. Acute monocytic leukaemia, a type of acute myeloid leukaemia, is a hematopoietic cancer characterized by a disorder of hematopoietic progenitor cells, which lose their ability for normal differentiation and response to normal regulators of proliferation [10]. Considering the fact that acute myeloid leukaemia

S. Lajili et al. / International Journal of Biological Macromolecules 126 (2019) 326–336

has the lowest survival rate of all leukaemias and that many anticancer drugs used in chemotherapeutic treatments developed resistance and side effects [11], the search of new natural anticancer agents against acute myeloid leukaemia is urgently needed to fight this type of cancer in the future. On the other hand gastric ulcer is a chronic disease, affecting millions of individuals worldwide. Multiple mechanisms are likely to be involved in this pathogenic process, including imbalance between aggressive and protective factors, involving the generation of free radicals and disturbance in nitric oxide (NO) production, reduction of mucus and bicarbonate secretion and inhibition of neutrophil adherence to endothelial cells [12]. Many synthetic drugs are used to treat gastric problems, while they trigger diverse side-effects. Thus, exploring more effective and safer anti-gastric ulcer agents from natural resources is of great importance [13]. Since reactive oxygen species (ROS), in excess of normal needs of the cell, may indiscriminately damage the structural and function integrity of tissues leading to many health disorders including cancer [14] and gastric ulceration [15] and that synthetic antioxidants such as butylated hydroxytoluene, butylated hydroxyanisole, propyl gallate and tertiary butyl hydroquinone present many adverse side effects [16,17], the search of effective and natural antioxidants has become crucial. In recent years, algal SPs, especially those extracted from red algae have been shown to have antitumor and immune-stimulating activities [18] and have been demonstrated to play an important role as freeradical scavengers and antioxidants for the prevention of oxidative damage in living organisms [19]. Due to many advantages such as having low side effects, low cost and also being easily accessible in comparison to common treatment methods, SPs may play an important role in sustaining life. Therefore, as a part of our effort to further explore the therapeutic potential of SPs from seaweeds, we attempted to study one species of red algae growing on the Tunisian coast “Laurencia obtusa” Hudson (Rhodomelaceae). Laurencia polysaccharides have been reported as polymer of galactose (galactan, occasionally substituted by xylose) but more complicated structures than agar and carrageenan [20–22], exhibiting a high molecular mass (≥100 kDa) and high electronegativity, which causes them to interact electrostatically with specific proteins, thereby contributing to different biological actions. This study was designed to describe the isolation and the physicochemical characterization of sulfated polysaccharide (SP) from the Mediterranean red alga Laurencia obtusa and to evaluate its apoptotic, gastroprotective and antioxidant activities.

and lipids. Approximately, 20 g of depigmented dried seaweeds were stirred for 2 h in NaHCO3 solution (0,5 M) (0.1% w/v) at 90 °C, the pH was adjusted to 8.0 with NaOH for slightly alkaline treatment. This is the pH where sulfated galactans are assumed to be stable [24]. The suspension was then filtrated through one-fold gauze. This process was repeated twice on the residue. The two filtrates were mixed and centrifuged at 3000 rpm for 30 min. The resulting supernatant enclosing SP was purified by ultra-filtration using a Minitan Cell equipped with a series of membranes with molecular weight cut off 100 kDa in order to eliminate proteins, low molecular weight compounds and salts. The ultra-filtration was conducted against deionised water (Milli Q process) for about 48 h until the conductivity become as low as that of deionised water [25]. The extensively ultra filtrated SP was finely lyophilized (Fig. 1). 2.3. Physico-chemical characterization 2.3.1. Chemical composition Total carbohydrates were determined by the phenol–sulfuric acid method using galactose as a standard [26]. Uronic acids contents were determined using carbazole method and glucuronic acid as a standard [27]. The sulfate content was determined by the turbidimetric method using sodium sulfate (Na2SO4) as a standard after hydrolyzing the polysaccharide in 2 M HCl at 100 °C for 2 h [28]. Proteins were estimated by the Lowry method, with bovine serum albumin as a reference protein [29]. 2.3.2. FTIR analysis In order to investigate the functional groups of the SP, the IR spectrum was determined using a Fourier transform infrared spectrometer (FTIR) (Perkin Elmer 1600 spectrometer). The SP was ground with spectroscopic grade potassium bromide (KBr) powder (1 mg SP in 100 mg KBr) and then pressed into 1 mm pellets for FTIR measurement in the wave number range of 400 and 4000 cm−1.

2. Materials and methods 2.1. Sample collection The red alga Laurencia obtusa was collected during June 2012, from the coastal region of Bizerte (Tunisia) in the Mediterranean Sea at a depth of 1–2 m. The identification of specimen was carried out by the National Institute of Marine Sciences and Technologies (Salambo, Tunisia). A voucher specimen was deposited in the Department of Pharmacology, Faculty of pharmacy, Monastir University under the following reference (Lo 2865). The collected samples were transported to the laboratory, cleaned by rinsing with sea water and distilled water, to remove associated debris and epiphytes. The seaweeds were then air dried in the shade at 30 °C, powdered using a blender and stored at −20 °C until extraction. 2.2. Extraction of polysaccharide For the extraction of SP, the modified method by Craigie and Leith [23] was used. Powdered algae were treated with petroleum ether then with acetone in a soxhlet apparatus to remove lipophilic pigments

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Fig. 1. Sulfated polysaccharide's extraction from Laurencia obtusa.

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2.3.3. Nuclear magnetic resonance (NMR) The lyophilized SP was kept over P2O5 in vacuum for several days and dissolved in deuterium oxide D2O (99.96%) before NMR analysis. The 1H (500Mz) and 13C (125Mz) NMR spectra were recorded with a Bruker Avance III HD500 spectrometer equipped with a single 5 mm BBFO Broad Band probe (15N-31P/1H/2H) at 80 °C. The trimethylsilylpropionate (TSP) was employed as an internal standard (δ = 0.0 ppm). The 13C NMR spectrum was performed using a Jmodulated spin-echo pulse sequence (JMOD). 2.3.4. Size exclusion chromatography For measuring the weight average molecular weight (Mw), the extracted SP (0.1 wt%) was dissolved overnight in NaNO3 (0.1 M). The eluent and the sample were filtered before use through 0.1 and 1 μm hydrophilic membranes (Millex SV, Millipore), respectively. The sample injected through a 100 μL loop, was eluted on a TSK-GEL GMPWXL column (Tosohaas) with a flow rate of 0.5 mL·min−1. Integration of the refractive index signals showed that residual adsorption on the stationary phase was minimal: N90% of polysaccharide sample was recovered at pH N 6. A MALLS detector (DAWN™ DSP, Wyatt Technology Co), coupled with a refractive index detector (Optilab, Wyatt Technology Co) as concentration detector, was used to obtain on-line determination of the absolute molar mass (M) and the root mean-square radius of gyration ((rg2)0.5) for the elution fraction of about 0.01 mL which allow the molar mass and size distribution to be calculated for the SEC profile using ASTRA software (Wyatt Technology Co) [30]. Well adapted representation of experimental Data, such as (rg2)0.5 versus M for example, give information about polymer contribution in the solution: (rg2)0.5 = KM x The light scattering intensity was detected simultaneously at twelve angles ranging from 27° to 132°. The differential index of refraction dn/dc, in our case is about 0.14 mL·g−1, a typical value for polysaccharides [31]. Zimm plot (first-order fitting) of the data collected for each slice, was observed to be linear over a large range of molar mass and was used to determine M and (rg2)0.5 from extrapolation of the intercept and the initial slope [32]. SEC-MALLS experiments were carried out at 40 °C. 2.4. Apoptosis assay 2.4.1. Cell culture Acute monocytic leukaemia cells (THP-1, ATCC® TIB-202) were maintained in RPMI1640 medium (Life Technologies, Saint Aubin, France) with GlutaMax™, supplemented with 10% (v/v) fetal bovine serum (BioWhittaker, Verviers, Belgium) and 1% (v/v) penicillin streptomycin (10,000 units/mL and 10,000 μg·mL−1, Life Technologies, Saint Aubin, France). Cells were grown in humidified atmosphere with 5% CO2 at 37 °C in 25 cm2 and 75 cm2 flasks up to 70–80% confluency prior to treatment. Cells were replicated every 2–3 days and the medium changed once in-between. 2.4.2. Microcapillary flow cytometry analysis THP-1 apoptosis was quantified by microcapillary flow cytometry using Annexin V-FITC/propidium iodide apoptosis assay. Annexin VFITC (Immuno Tools, Germany) is a fluorescein-labelled lectin which binds to phosphatidylserine (PS) on cell membrane. Externalization of PS from inner to outer leaflet of the cell membrane represents an early indicator of apoptosis since it represents an “eat-me” signal for the phagocytic cells [33]. Propidium iodide (PI) (Miltenyi Biotec Inc., USA) is a fluorescent intercalating agent, which binds to DNA. PI stains only dead cells with damaged cell membranes [34]. Combination of both dyes enables detection and discrimination of live, apoptotic and dead cells. In our case, negative and positive controls were included in all Annexin V-FITC/PI apoptosis assays. Untreated cells in culture medium with DMSO (50 μM) were used as a negative control.

Celastrol (SurroMED, USA) (50 μM) was used as a positive control since it induces apoptosis in mostly all mammalian cancer cell lines [35]. THP-1 cells were seeded in 96-well plate with cell concentration 1 × 105 cells/mL. Increasing concentrations of SP (10, 20, 50, 100 and 200 μg·mL−1) in culture medium were freshly prepared from stock solution prior to each experiment. To each well containing 100 μL of cells in culture medium, 100 μL of compound in culture medium was added. 96-well plates were then incubated in CO2 incubator for 24 h. Next day, annexin V-FITC (3 μL) and PI (3 μL) were added to each well and the plate was incubated at room temperature in the dark for 10 min. Finally, fluorescence measurements were performed using microcapillary flow cytometer. Experiments were performed and interpreted as follows: cells that were Annexin V (−)/PI (−) (lower left quadrant) were considered as living cells, the Annexin V (+)/PI (−) cells (lower right quadrant) as apoptotic cells, Annexin V (+)/PI (+) (upper right quadrant) as necrotic or advanced apoptotic cells and Annexin V (−)/PI (+) (upper left quadrant) may be bare nuclei, cells in late necrosis or cellular debris. Dose response curve and EC50 value, which represents the concentration of SP that induces apoptosis in 50% of all cells after 24 h incubation, were evaluated under the InCyte software 2.6 (Guava/Millipore/ merck). 2.5. Gastroprotective activity 2.5.1. Animals Wistar rats (150–200 g) of both sexes purchased from Pasteur Institute (Tunis, Tunisia) were used. They were housed in groups of eight animals in plastic cages at 20–25 °C and maintained on a standard pellet diet with free access to water. Animals were fasted for 16 h before the experiments. Housing conditions and in vivo experiments were approved according to the guidelines established by the European Union on Animal Care (CCE Council 86/609). 2.5.2. Acute toxicity Animals were divided into five groups (n = 6) and fed orally with the SP at doses of 50, 100, 200, 400 and 500 mg·kg−1 body weight in water. The animals were monitored carefully for 24, 48, 72 h after SP administration and then for the next 7 days. At the end of this experimental period, the rats were observed for signs of toxicity, morphological behavior and mortality. 2.5.3. Gastric lesions induced by EtOH/HCl The gastroprotective activity of SP was assessed using the EtOH/HClinduced gastric ulcer on rat [36]. Rats were divided into groups of six animals and fasted for 16 h prior test. Control group were treated orally with saline (0.9% NaCl, 2.5 mL·kg−1, p.o.); test groups received by the same route SP (25, 50 and 100 mg·kg−1) and standard groups received sucralfate (500 mg·kg−1, p.o.), ranitidine (60 mg·kg−1, p.o.) and omeprazole (30 mg·kg−1, p.o.) 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 the ulcerogenic agent; stomachs were excised and opened along the great curvature, rinsed under a stream of water and pinned flat on a cork board, the surface of each stomach was examined for the presence of lesions and the extent of the lesions was measured. The summative length of the lesions along the stomach was recorded (mm) as lesion index. The mean ulcer index was determined by dividing the total ulcer indices in a group by the total number of animals in that group. The percentage severity of ulceration was determined by dividing the scores of ulcers of each group by the total number of scores in the control group and the result multiplied by 100. 2.5.4. Lipid peroxidation analysis The measurement of thiobarbituric acid reactive substances (TBARS) level in gastric tissue is commonly used to evaluate lipid peroxidation


and indirectly oxidative stress in vivo. TBARS were determined by the measurement of the concentration of malondialdehyde (MDA) in the homogenate from each gastric sample according to Ohkawa et al., [37]. Fragments of the gastric mucosa were homogenized with icecold phosphate buffer (50 mM, pH 7.4) to give a 10% homogenate. 250 mL of each homogenate was added to the reaction mixture containing 500 μL of thiobarbituric acid (0.6%) and 500 mL of acetic acid (pH 3.4). Then, the mixture was centrifuged at 15,000 ×g for 10 min and the supernatant was heated in a boiling water bath for 30 min. Finally the absorbance was read at 532 nm. Results were interpolated with a standard curve of MDA and expressed in nmol·g−1 of tissue. 2.5.5. Glutathione levels in the gastric tissue Glutathione (GSH) levels in gastric mucosa were measured using the method of Sedlak and Lindsay [38]. After EtOH/HCl induction of gastric lesions, a part of the stomach was weighed and homogenized with ice cold potassium phosphate buffer (200 mM, pH 6.5). To the homogenates 12.5% trichloroacetic acid was added and the suspension was vigorously shaken and centrifuged for 15 min at 3000 rpm at 4 °C. The supernatant of each sample was mixed with Tris–HCl buffer (400 mM, pH 8.9) and 10 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in a 96-well plates. The solution was kept at room temperature for 5 min, and the absorbance was then read at 412 nm. The obtained values were interpolated with a standard curve of GSH and the results were expressed as μg·g−1 of tissue. 2.6. Antioxidant activity assays 2.6.1. DPPH radical-scavenging activity The free radical-scavenging activity of SP was evaluated using the stable radical DPPH, according to the method of Kim et al. [39]. A dilution series of the extracted sample was prepared (0.03–1 mg·mL−1). 1 mL of each sample concentration was mixed with 1 mL of 30 mmol·L−1 DPPH-ethanol solution. The reaction mixture was then stirred vigorously for 10 s using the vortex. Color was allowed to develop in the dark for 30 min. The absorbance is measured at 517 nm against the blank. The radical-scavenging activity, expressed as percentage inhibition of DPPH, was calculated according to the formula: %inhibition ¼

Acontrol −Asample =Acontrol 100

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where the Acontrol is the absorbance of the control (DPPH solution without sample), the Asample is the absorbance of the test sample (DPPH solution plus test sample). The concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage plotted against test samples concentration. DPPH radical-scavenging activity of SP was compared with ascorbic acid and quercetine used as standards. 2.6.2. Ferric reducing antioxidant power (FRAP) The ferric reducing power (FRAP) was evaluated using the method described by Oyaizu [40]. Briefly, 1 mL of diluted SP (0.03–1 mg·mL−1) was mixed with 2.5 mL of potassium phosphate buffer (0.1 M, pH 6.6) and 2.5 mL of 1% (w/v) potassium ferricyanide. The mixture was incubated at 50 °C for 20 min, then, 2.5 mL of 10% (w/v) trichloroacetic acid was added and subsequently centrifuged at 1000 ×g for 10 min. 2.5 mL of the supernatant was mixed with equal volume of water and 0.5 mL of 0.1% (w/v) ferric chloride. The solution was incubated at ambient temperature for 30 min for color development. The absorbance was then, measured at 700 nm and compared with ascorbic acid and quercetine used as standards. 2.6.3. Hydroxyl radical scavenging activity The scavenging activity of SP against the hydroxyl radical was investigated using Fenton's reaction (Fe2+ + H2O2 → Fe3+ + OH− + •OH). The results were expressed as an inhibition rate. Hydroxyl radicals exhibit a small diffusion capacity and are most reactive in the induction of injuries to cellular molecules and, accordingly, deserve special attention. Hydroxyl radicals were generated using the modified method of Smirnoff and Cumbes [41]: in 3 mL sodium phosphate buffer (150 mM, pH 7.4), which contained 10 mM FeSO4.7H2O, 10 mM EDTA, 2 mM sodium salicylate, 30% H2O2 (200 mL) and varying concentrations of SP (0.03–1 mg·mL−1). In the control sample, sodium phosphate buffer replaced H2O2. The solutions were incubated at 37 °C for 1 h, and the presence of hydroxyl radical was detected by monitoring absorbance at 510 nm. 2.7. Statistical analysis Results were analyzed using One Way ANOVA (Fisher LSD post hoc test) and expressed as mean ± s.e.m., using SPSS Statistics Software

Fig. 2. Infrared spectrum of SP extracted from the red alga Laurencia obtusa.

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(SPSS for Windows software release18.0). Difference between means of treated and control groups were considered significant at p b 0.05. 3. Results and discussion 3.1. Characterization and identification The SP was extracted from the red alga Laurencia obtusa with an acceptable yield of 17.90%. According to Canelon et al. [42], yields of sulfated galactans isolated from Laurencia obtusa and Laurencia filiformis

were 12.8% and 16.2%, respectively, after hot water extraction. Yields of sulfated polysaccharides isolated from Laurencia papillosa, Laurencia cruciata, Laurencia pedicularioides and Laurencia majuscula, treated in distilled water at 80–85 °C were about 18.3%, 20.2%, 26.9% and 13%, respectively [20]. Thus, it appears that the yields of sulfated polysaccharides depend on species, environmental factors and extraction processes [43]. The results of different colorimetric assays demonstrated that the analyzed polysaccharide contained high amount of sugars (61 ± 3%) and low levels of proteins (2.3 ± 0.6%). It was also found to have high

Fig. 3. (A): NMR spectra of SP recorded in the D2O at 80 °C. (A): 1H NMR (500 MHz); (B): 13C NMR (125 MHz) recorded with the J-modulated spin-echo pulse sequence (JMOD).


Fig. 4. Refractive Index (plain line) and Light Scattering (dashed line) chromatograms of SP sample eluted in 0.1 mol L−1 NaNO3 at 0.5 mL·min−1 with TSK-GEL GMPWXL (tosohaas) column.

sulfate content (28.2 ± 2.4%). These results are in agreement with values reported in literature [20]. The amount of uronic acid was about 9.2 ± 1.3% which is very close with earlier reports on other Laurencia species (Laurencia papillosa and Laurencia pinnatifida) [44,45]. Moreover, infrared absorption spectra of SP extracted from the red alga Laurencia obtusa is basically in accordance with reported spectra

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[46] (Fig. 2). It showed a general absorption band at 3440 cm−1 corresponding to the stretching vibration of hydroxyl group (OH), a band at 2927 cm−1 due to the stretching vibration of C\\H bonds and a band at 1620 cm−1 attributed to the absorbance of the deprotonated carboxylic group (COO\\). Additional band of low intensity between 1447 cm−1 was associated to the C\\C\\H vibrations. Besides, a peak at 1235 cm−1 was assigned to the presence of sulfate ester groups (S_O) which is a characteristic component in sulfated polysaccharides; the region around 1075 cm−1 is equivalent to the skeleton of galactans [47]. Furthermore, the typical signals of sulfated gatactans with a broad band at 823 cm−1 with a small shoulder at 830 cm−1 were assigned to stretching vibration of CO\\SO4− bonds and corresponded to α-D-galactose-6-sulfate and galactose-2-sulfate, respectively [48,49]. The absence of corresponding bands of carrageenan (κ-carrageenan: 850 cm−1 and ι-carrageenan: 805 cm−1). The 1D (1H and 13C NMR) of SP from Laurencia obtusa were displayed in Fig. 3. Based on chemical shifts reported in the literature, the signals of SP in 1H NMR spectrum were assigned (Fig. 3A). Briefly, the anomeric protons signals observed at δ 4.5–4.6 and δ 5.00–5.30 ppm are assigned to the β-D-galactose and the α-L-galactose, respectively [50]. The proton signal at δ 5.28 ppm is attributed to the anomeric proton of the agarose precursor 4-O-linked α-L-galactose-6sulfate units [51]. Besides, chemical shifts from δ 3.6 to δ 4.0 ppm are assigned to the ring osidic protons (H2-H5) [52]. In addition, the resonances at δ3.4 ppm correspond to the methyl group of 6-O-methyl-Dgalactose [53]. 1H NMR spectra shows also signal at δ 2.2 ppm attributed to CH3 protons of the O-acetyl groups [51,54].

Fig. 5. Flow Cytometric analysis of apoptosis/necrotic cell death using Annexin-V-FITC/PI staining of THP-1 cells treated with DMSO (negatif control), Celastrol (50 μM) (positif control) and SP (10–200 μg·mL−1). Lower left quadrant (Annexin V (−)/PI (−)) represents live cells, lower right quadrant (Annexin V (+)/PI (−)) represents early apoptotic cells, upper right quadrant (Annexin V (+)/PI (+)) represents late apoptotic cells, upper left quasrant (Annexin V (−)/PI (+)) represents cells in late necrosis or cellular debris.

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SP was also investigated by 13C NMR spectroscopy employing the Jmodulated spin-echo pulse sequence (JIMOD). The presence of a signal at δ 59.1 ppm indicated the presence of methylated units [55,56]. Typical signals at δ 98.7 (C-1), δ 78.5 (C-2), δ 78.3 (C-3), δ 77.2 (C-4) and δ 59.1 ppm (O-Me) indicated the presence of 2-O-methyl-3,6anhydro-L-galactose, the methylated unit which is commonly present in Laurencia polysaccharides [20,45,57]. Signals at δ 73 and δ 65.1 ppm may be attributed to the C-5 and C-6 of D-galactose-6-sulfate unit, respectively. Besides, 13C NMR spectra showed signal at δ 103.3 ppm corresponding to C-1 of D-galactose unit of agaran-6′-sulfate [20,51,58]. Finally, the absence of signal at δ 95 ppm, characteristic of C1 of the D-anhydrogalactose unit of carrageenan, confirm that this polysaccharide is not of carrageenan type [59,60]. It can be concluded from the above analysis that the polysaccharide from Laurencia obtusa is complex in nature made of sulfated and methylated units with agarose backbone. SP sample was analyzed by size exclusion chromatography with the two detectors MALS and DRI on-line. Experiments were carried out in 0.1 M NaNO3 to determine molecular weights and size information. The elution profile of SP is presented in Fig. 4. Light scattering chromatogram showed one single peak eluted between 5 and 6.5 mL thereby confirming the purity and homogeneity of the extracted polysaccharide. The integration of this peak gives an average molecular weight of 336,900 g·mol−1 with a polydispersity of 1.18. The radius of gyration of the extracted carbohydrate is about 53.1 nm showing that it's a polymer with flexible pelote. These results are in accordance with other studies in which high molecular mass have been described for other sulfated polysaccharides from red algae with values higher than 300,000 g·mol−1 and with polydispersity index b2.0 [61].

3.2. Apoptosis assay THP-1 cells were seeded in 96-well plates and were incubated, for 24 h, with increasing concentrations of tested compounds (10–200 μg·mL−1). Experiment was performed in triplicates and repeated three times. Positive control showed almost total inductions of cell death since 99% of cells were dead by apoptosis, whereas negative control showed only 1% of dead cells. SP exhibited high apoptotic activity at the dose of 200 μg·mL−1, it showed a high induction of cell death, 98.1% cells were dead by apoptosis (Fig. 5). The EC50 was 53 ± 3 μg·mL−1, curve is represented in Fig. 6. Several studies have reported that sulfated

polysaccharides have antiproliferative activity in cancer cell lines in vitro, as well as inhibitory activity of tumor growth in mice [5]. Recently Murad et al., [62] showed that the sulfated polysaccharide, extracted from the red alga Laurencia papillosa induces apoptosis and G1-phase arrest in MDA-MB-231 breast cancer cell line by activating caspase-3, increasing Bax/Bcl-2 ratio, and inducing ROS production. The basic chemical structure, or the sulfate amount, of the polysaccharide or, even, both could be at the origin of the antitumoral effects. Indeed, sulfated polysaccharides such as carrageenans interact with a variety of sulfated polysaccharides-binding proteins (including the family of toll-like receptors (TLRs)), activate immunostimulating cells: Nk cells, macrophages, T and B lymphocytes and increase proinflammatory cytokines production [63]. So, the tumor-inhibition by SP may be related to an immunostimulation activity. Thus, we conclude that SP is a potent antitumor agent which might be further used in cancer treatment to enhance tumor immune response. 3.3. Acute toxicity Results revealed that rats administered with SP at oral doses of 50, 100, 200, 400 and 500 mg·kg−1 showed no clinical signs of toxicity. No mortality was observed with the tested doses during all the treatment period. These observations showed that the SP extracted from the red alga Lauencia obtusa is not toxic up to the dose of 500 mg·kg−1 and hence LD50 could not be established. Other studies in literature demonstrated that several sulfated polysaccharides from algal sources are safely exhibiting no toxic effect even at high concentration in rats [64]. 3.4. Gastroprotective and antioxidant activities EtOH/HCl-induced gastric ulcer is regarded as a suitable model to study the cytoprotective activity of compounds [65]. The results of gastroprotective effect on gastric ulcer induced by EtOH/HCl solution are shown in Table 1. Oral administration of the damaging agent to the control group clearly produced a mucosal damage characterized by multiple hemorrhage red bands of different sizes along the long axis of the glandular stomach as described in other studies; the lesion index was 129 mm [66]. Upon its rapid penetration into the gastric mucosa, ethanol can either cause lipid peroxidation or metabolize to form superoxide anion and hydroxyl radicals in the gastric mucosa that can react with most of the cell components or be involved in other processes that ultimately result in oxidative damage, leading to a loss of epithelial gastric structure characterized by mucosal edema, and inflammatory infiltrate [67]. Pretreatment with SP at the doses 25, 50 and 100 mg·kg−1 produced a significant decrease in gastric hemorrhage (p b 0.01) and the lesion index was inhibited by 63.44, 78.42 and 82.15%, respectively. These inhibition percentages were comparables of those of the three classical ulcer drugs sucralfate (500 mg·kg−1), Table 1 Effects of SP from the red alga Laurencia obtusa, on gastric ulcer induced by EtOH/HCl in rats. Samples

Dose (mg·kg−1)

Ulcer index (mm) (m ± s.e.m) (n = 6)

Ulcer inhibition (%)

Control (0.9% NaCl) SP

– 25 50 100 60 30 500

129 ± 11.62 45.33 ± 4.22⁎ 34.66 ± 5.37⁎ 18.50 ± 2.73⁎ 43.38 ± 4.35⁎ 17.50 ± 1.38⁎ 16.23 ± 1.68⁎

– 64.86 73.13 85.65 66.37 86.43 87.41

Ranitidine Omeprazole Sucralfate Fig. 6. Dose response curve and EC50 value for apoptotic activity of SP after 24 h incubation in THP-1 cells (n = 3 independent experiments in triplicates, 2000 events per sample were analyzed).

Values are expressed as mean ± s.e.m. n = 6 animals. Bold values indicates significance at p b 0.01. ⁎ p b 0.01.


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Fig. 7. The dose response curves for percentage scavenging activity of DPPH by SP in comparison with ascorbic acid and quercetine. The results are presented as mean ± s.e.m. for SP (n = 3).

ranitidine (60 mg·kg−1) and omeprazole (30 mg·kg−1) which showed an inhibition percentage of gastric lesions of 87.41%, 66.96 and 86.67%, respectively. Many study demonstrated that sucralfate present cytoprotective properties. It releases prostaglandins [68], stimulates mucus production [69] and bicarbonate secretion [70] and promotes epithelial cell renewal from the mucosa of rats [71]. The ability of SP to exhibit antiulcer activity may be attributed to the same gastroprotective effect mechanisms of sucralfate. In fact, many studies demonstrated that sulfated polysaccharides induced prostaglandins production by increasing the expression of COX-2 and iNOS [72]. In the other hand, prostaglandins were reported to have a cytoprotective effect by stimulating the production of mucus and bicarbonate that will protect the gastric mucosa from ulcer formation [73]. In the other hand, oxidative stress is believed to initiate and aggravate many digestive system diseases, including gastric ulcers [74]. Antioxidants play a major role in counteracting excessive free radical generation that may occur during ulcer formation by scavenging free radical formation [75]. In light of this, the antioxidant activity of SP was evaluated in vitro and in vivo. The DPPH assay is a preliminary in vitro test to investigate the antioxidant potential of extracts. Antioxidants, on interaction with DPPH, either transfer an electron or hydrogen atom to DPPH, thus

neutralizing its free radical character. In fact, free radical scavenging method (DPPH) shows the reduction of alcoholic DPPH solutions in the presence of hydrogen donating antioxidant [76]. The color changes from purple to yellow and its absorption at wavelength 517 nm decreases. Fig. 7 showed that the radical-scavenging activity SP, ascorbic acid and quercetine on DPPH radicals increased in dose-dependent manner. The IC50 values calculated from the graph show that SP exhibited significant antioxidant activity with IC50 value of 24 ± 5 μg·mL−1 which was found to be comparable with that of ascorbic acid (17 ± 3 μg·mL−1) and quercetine (18 ± 2 μg·mL−1). Ferric ion reducing power assay measures the electron donating capacity of an antioxidant. The presence of reducing agents causes the reduction of the Fe3+/ferricyanide complex to the ferrous form Fe2+. The absorbance measured at 700 nm of the resultant blue-green colored solution is proportional to the amount of Fe2+. Therefore an increased absorbance is indicative of higher reducing power. The reducing potential of SP, ascorbic acid and quercetine increased with concentration increase (Fig. 8). SP showed a high reducing power with IC50 value of 92 ± 2 μg·mL−1 but slightly lower than ascorbic acid (62 ± 8 μg·mL−1) and quercetine (83 ± 4 μg·mL−1). Among the reactive oxygen species, the hydroxyl radical is the most reactive and induces severe damage to adjacent biomolecules. The

Fig. 8. Dose response curves of the reductive power of SP in comparison with ascorbic acid and quercetine. The results are presented as mean ± s.e.m. for SP (n = 3).

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Fig. 9. Dose response curves of Hydroxyl radical scavenging activity of SP in comparison with ascorbic acid and quercetine. Results are presented as mean ± s.e.m. for SP (n = 3).

scavenging effects of SP, ascorbic acid and quercetine on hydroxyl radicals are shown in Fig. 9. The results obtained for the inhibition of hydroxyl (OH−) radical formation demonstrated that the scavenging activity of SP increased for higher polysaccharide concentrations. The IC50 value of SP eliminating OH− was about 113 ± 9 μg·mL−1, showing a scavenging effect lower than that of ascorbic acid (75 ± 7 μg·mL−1) and quecetine (83 ± 3 μg·mL−1). It has been demonstrated that the DPPH and hydroxyl radicals scavenging activities and reducing power of sulfated polysaccharides have significant correlations with sulfate and uronic acid contents [77,78]. Meanwhile, SP was determined to have a considerable amount of uronic acid and a high content of sulfate groups which might explain the antioxidant activity. Furthermore, our results showed that oral administration of EtOH/ HCl in rats was associated with a significant increase in MDA concentration (320.74 ± 16.54 nmol/g tissue, p b 0.05) and decrease in GSH levels (77.24 ± 6.32 μg/g tissue, p b 0.05) when compared to the saline group which presented normal concentrations of MDA and GSH (98.53 ± 5.23 nmol/g tissue and 295.12 ± 15.02 μg/g tissue, respectively). Oral pretreatment with SP at the doses of 25, 50 and 100 mg·kg−1 significantly decreased MDA levels (Fig. 10A) and increased GSH concentrations (Fig. 10B) in stomach tissues and this in a dose dependent

manner when compared to the control group. Indeed, oxidative stress is involved in the pathogenesis of EtOH-induced gastric damage through the formation of free radicals and lipid peroxidation, which is demonstrated by the decrease of GSH and increase of MDA levels in stomach tissue, two markers of the oxidative stress [79]. Our results are consistent with previous reports that demonstrate the involvement of decreased oxidative stress in the action gastroprotective of several SPs [80]. Despite its high molecular weight, the pretreatment with SP showed a significant prevention of EtOH/HCl-induced damage in the superficial layers of the gastric mucosa. Therefore, the combination effect of both the reinforcement of resistance of the mucosal barrier by a protective coating and oxygen radical scavenging activity might be involved in the gastroprotective effect. Several studies explain the ability of polysaccharide to bind to the gastric mucosa surface and function as a protective coating and to protect the mucosa by increasing mucus synthesis and scavenging radicals [81]. 4. Conclusion In conclusion, our results suggest that SP may have applications in the therapeutic field against acute myeloid leukaemia and a beneficial

Fig. 10. Effect of SP on malondialdehyde (MDA) (A) and glutathione (GSH) (B) levels in rats with EtOH/HCL-induced gastric damage. Animals were treated orally with either saline (0.9% NaCl, 2.5 mL·kg−1) or SP (25, 50 and 100 mg·kg−1). 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. The results are expressed as the mean ± s.e.m. (n = 6). * p b 0.05 versus normal group; ** p b 0.05 versus EtOH/HCl group.


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