Protective effect against gastric mucosa injury of a sulfated agaran from Acanthophora spicifera

In this study, a polysaccharide from marine alga Acanthophora spicifera (PAs) was isolated and structurally characterized. Its protective potential against chemically-induced gastric mucosa injury was evaluated.

Carbohydrate Polymers 261 (2021) 117829 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Protective effect against gastric mucosa injury of a sulfated agaran from Acanthophora spicifera Lindauro C Pereira Júnior a, 1, Fernando G Nascimento b, 1, Samara R.B.D Oliveira c, Glauber C Lima a, d, Francisco Diego S Chagas a, Venicios G Sombra e, Judith P.A Feitosa e, Eliane M Soriano f, Marcellus H.L.P Souza c, Guilherme J Zocolo g, Lorena M.A Silva g, Regina C.M de Paula e, Renan O.S Damasceno h, *, Ana Lúcia P Freitas a a Departamento de Bioquímica e Biologia Molecular, Universidade Federal Cear´ a, 60455-760, Fortaleza, CE, Brazil Centro Universit´ ario INTA (UNINTA), 62050-100, Sobral, CE, Brazil c Departamento de Fisiologia e Farmacologia, Universidade Federal Cear´ a, 60430-270, Fortaleza, CE, Brazil d Centro Universit´ ario INTA (UNINTA), 62500-000, Itapipoca, CE, Brazil e Departamento de Química Orgˆ anica e Inorgˆ anica, Universidade Federal Cear´ a, 60455-760, Fortaleza, CE, Brazil f Departamento de Oceanografia e Limnologia, Universidade Federal Rio Grande Norte, 59072-970, Natal, RN, Brazil g Embrapa Agroindústria Tropical, 60511-110, Fortaleza, CE, Brazil h Departamento de Fisiologia e Farmacologia, Universidade Federal de Pernambuco, 50670-420, Recife, PE, Brazil b A R T I C L E I N F O A B S T R A C T Keywords: A spicifera Polysaccharide Gastric injury Oxidative stress In this study, a polysaccharide from marine alga Acanthophora spicifera (PAs) was isolated and structurally characterized Its protective potential against chemically-induced gastric mucosa injury was evaluated The gel permeation chromatography experiments and spectroscopy spectrum showed that PAs is a sulfated poly­ saccharide with a high molecular mass (6.98 × 105g/mol) and degree of sulfation of 1.23, exhibiting structural characteristic typical of an agar-type polysaccharide Experimental results demonstrated that PAs reduced the hemorrhagic gastric injury, in a dose-dependent manner Additionally, PAs reduced the intense gastric oxidative stress, measured by glutathione (GSH) and malondialdehyde (MDA) levels PAs also prevented the reduction of mucus levels adhered to the gastric mucosa, promoted by the aggressive effect of ethanol In summary, the sulfated polysaccharide from A spicifera protected the gastric mucosa through the prevention of lipid peroxi­ dation and enhanced the defense mechanisms of the gastric mucosa, suggesting as a promising functional food as gastroprotective agent Introduction Recent studies have demonstrated pharmaceutical and biotechno­ logical interest in marine algae, since they are important sources of bioactive molecules such as polyphenols, lectins and polysaccharides (Costa et al., 2010; Dan, Liu, & Ng, 2016) Polysaccharides are macro­ molecules formed by the polymerization of monosaccharide units linked by glycosidic bond sand which may contain substituted hydroxyl groups along the polymer chain (Xie et al., 2016) The galactans are known as polysaccharides of red marine algae, composed of galactopyranose disaccharide repeated units The first unit is always a β-D-galactopyranose (Unit A) while the second is a α-gal­ actopyranose (unit B) that can have D or L configuration (Florez et al., 2017; Wijesekara, Pangestuti, & Kim, 2011) When unit B possesses D configuration, galactan is considered as a carrageenan; when they are L, they are called agarans (Campo, Kawano, Silva, & Carvalho, 2009) In typical agarose polysaccharide, unit B is in the form of 3,6-anhy­ dro-α-L-galactose Agarans are sulfated biopolymers that can be defined as hydrocolloids that have gelling activities, found in a series of red algae genera, mainly in Gelidium, Pterocladiella, Gelidiella, Gracilaria Abbreviations: FTIR, Fourier transform infrared; GPC, gel permeation chromatography; GPx, glutathione peroxidase; GSH, glutathione; Hb, hemoglobin; MDA, malondialdehyde; NMR, Nuclear magnetic resonance; ROS, reactive species derived from oxygen * Corresponding author E-mail address: renan.oliveirasilva@ufpe.br (R.O.S Damasceno) Both authors contributed equally to the development of this work https://doi.org/10.1016/j.carbpol.2021.117829 Received 12 August 2020; Received in revised form February 2021; Accepted 12 February 2021 Available online 17 February 2021 0144-8617/© 2021 Elsevier Ltd This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/) L.C Pereira Júnior et al Carbohydrate Polymers 261 (2021) 117829 (Pereira, Gheda, & Ribeiro-Claro, 2013) and Acanthophora (Duarte et al., 2004) Currently, polysaccharides from marine algae are used in the food and pharmaceutical industry, resulting in their usefulness and applica­ tion as hydrocolloids with the ability to form gels with antioxidant po­ tential, besides interacting with various systems promoting biologically ´rez-Ferna ´ndez, & Domínguez, 2019) in vivo active effects (Torres, Flo experimental studies showed that polysaccharides are able to reduce free radicals, suggesting their use in conditions with change in homeo­ stasis mechanisms and intense oxidative stress (Costa et al., 2010) Acanthophora spicifera is a red marine alga found in tropical and subtropical regions, native of the Caribbean and Florida, and widely ˜o to Rio Grande Sul distributed in the Brazilian coast from Maranha (Horn, 2012) Biologically active substances have been extracted from A spicifera, including flavonoids with antibacterial, antitumoral, pro­ coagulant and antioxidant effects, antiproliferative phenolic compounds and polysaccharides with great functional activity (Anand et al., 2018; Murugan & Iyer, 2014) The main components of sulfated poly­ saccharide from A spicifera are galactose and 3,6-anhydrogalactose (80–85 %) (Duarte et al., 2004; Ganesan, Shanmugamb, Palaniappanc, & Rajauria, 2018; Anand et al., 2018) but small amounts of xylose, mannose and arabinose, as well as, pyruvic acid has been also detected (Duarte et al., 2004; Ganesan et al., 2018; Anand et al., 2018) Fig shows a structural representation of A spicifera polysaccharide based on previous reports (Duarte et al., 2004; Ganesan et al., 2018; Anand et al., 2018) Fractions of A spicifera polysaccharide has been reported as potential anticancer activity against A549 cell lines (Anand et al., 2018) and antiviral activity against HSV-1 and HSV-2 (Duarte et al., 2004) However, studies on the effect of these polysaccharides under conditions involving intense oxidative stress such as gastric damage, have not been conducted The excessive consumption of alcohol is responsible for intense pathological aggression in the gastrointestinal tract, characterized by hemorrhage followed by exfoliation of gastric mucosa cells, decreased levels of mucus and a consequent lipid peroxidation and depletion of antioxidant defense (Bujanda, 2000) Due to the imbalance between gastric aggressive and protective factors, it is important to develop studies aimed at the search for effective molecules to prevent patho­ logical events caused by alcohol Thus, the present study aimed to isolate and elucidate the structure of a polysaccharide from A spicifera and to investigate its gastro­ protective potential to prevent the gastric mucosa injury in mice Materials and methods 2.1 Marine alga Specimens of marine alga A spicifera were collected in August 2016 at Búzios Beach, Nísia Floresta, Rio Grande Norte-RN, Brazil (06◦ 00′ 43.3′′ S and 35◦ 06′ 27.2′′ O) After collection, the material was cleaned of epiphytes, washed with distilled water and stored at 20 ◦ C A voucher specimen (no 62375) was deposited in the Prisco Bezerra ´, Brazil Herbarium, Federal University of Ceara 2.2 Extraction The polysaccharide from A spicifera (referred to as “PAs”) was iso­ ˜o, lated as described previously (Farias, Valente, Pereira, & Moura 2000), with modifications The dried tissue (5 g) was macerated in liquid nitrogen and suspended in a sodium acetate buffer (0.1 M, pH 5.0) containing mM EDTA, mM cysteine and papain, and incubated for h at 60 ◦ C Then, the material was filtered and centrifuged at 8000×g for 20 at 25 ◦ C, and precipitated with 10 % cetylpyridinium chlo­ ride The precipitate was dissolved in NaCl:ethanol (2 M, 100:15, v/v) and precipitated by the addition of ethanol for 24 h at ◦ C Finally, the precipitate was washed with acetone and dried with hot air flow 2.2.1 Chemical composition Carbon, nitrogen and sulfur contents were determined using a Per­ kinElmer 2400 Series II CHNS analyzer (PerkinElmer, Waltham, MA, USA) (Maciel et al., 2008) The degree of substitution for sulfate groups (DS sulfate) was calculated by the percentage of sulfur and carbon atoms (Melo, Feitosa, Freitas, & de Paula, 2002), as described by the Eq (1): DS = %S/(atomic mass of S) %S = 4.5 x %C/ %C (atomic mass of C × 12) (1) NaSO3 content was calculated as described in Eq (2): %NaSO3 = 103 × %S 32 (2) Where 103 and 32 are NaSO3 and S molar masses, respectively (in g/ mol) The determination of the carbohydrate content was carried out in accordance with the sulfuric acid-UV method (Albalasmeh, Berhe, & Ghezzehei, 2013), using D-galactose as a standard The protein content was measured using the Bradford method (Bradford, 1976) 2.2.2 Xylose determination The polysaccharide hydrolysis was carried out for 3, 4, 5, and 12 h, at 100 ◦ C, with M trifluoroacetic acid The hydrolyzate was analyzed on HPLC Shimadzu LC-20AD with refractive index detector (RID-10A) The separation was performed using a Rezex RCM-Monosaccharide Ca2+ (300 × 7.8 mm) 2.2.3 Gel Permeation Chromatography (GPC) The peak molar mass was performed by GPC with concentration of 0.5 % PAs and 0.1 M NaNO3 as solvent Chromatography was performed on a Shimadzu equipment at room temperature using a PolySep Linear (7.8 × 300 mm) column, flowing at 1.0 mL/min A differential refrac­ tometer and an ultraviolet photometer (at 280 nm) were used as de­ tectors and the elution volume was corrected for the internal marker of ethylene glycol at 11.25 mL Samples of pullulans (P-82, Shodex Denko), linear homopolysaccharides isolated from the fungus Aureobasidium pullulans (Leathers, 2003) of different molar masses were used, ranging from 103 to 106 g/mol (Mw of 5.9 × 103, 1.18 × 104, 4.73 × 104, 2.12 × 105 and 7.88 × 105 g/mol) The equation obtained from the calibration curve was log Mw = +13.94− 1.007.VEL, where VEL is the elution volume in mL The linear coefficient obtained for this equation Fig Chemical structure of the repeating disaccharide unit of A spicifera with the different types of sugar units and substituent L.C Pereira Júnior et al Carbohydrate Polymers 261 (2021) 117829 was 0.992 0.01 M DTNB Subsequently, the samples were shaken for and absorbance was measured at 412 nm using a spectrophotometer The results are expressed as microgram of GSH per gram of tissue (μg of GSH/g tissue) (Sedlak & Lindsay, 1968) 2.2.4 Fourier Transform Infrared (FTIR) spectroscopy The spectrum in the infrared region were obtained with a Shimadzu Fourier transform infrared spectrometer, model IRTracer-100, with a spectral region of 1400 to 700 cm− Potassium Bromide tablets (KBr) were used for the sample analysis 2.3.5 Malondialdehyde (MDA) concentration Stomach samples were homogenized in 1.15 % KCl Then, the ho­ mogenate was added with 1% H3PO4 and 0.6 % tert-butyl alcohol Then, this mixture was stirred and heated in a boiling water bath for 45 The preparation was cooled immediately in an ice water bath, followed by the addition of n-butanol The mixture was stirred and the butanol was removed via centrifugation at 1200 rpm for 10 min, and absorbance was measured at 520 and 535 nm using a spectrophotometer Results were expressed as nanomoles of MDA per gram of tissue (nmol of MDA/g tissue) (Uchiyama & Mihara, 1978) 2.2.5 Nuclear Magnetic Resonance (NMR) spectroscopy NMR spectroscopy was performed for the chemical characterization of the polysaccharide The spectrum were obtained on an Agilent 600MHz spectrometer equipped with a 5-mm (H-F/15N-31 P) inverse detection One Probe™ with actively shielded z-gradient The 1H NMR spectrum were acquired using 16 scans in a 16-ppm window and a relaxation delay of s The carbon nuclear magnetic resonance (13C NMR) spectrum was recorded with 10k of scans into a 220 ppm of spectral width and a relaxation delay of s The 2D hydrogen and car­ bon heteronuclear single quantum correlation spectrum(1H-13C HSQC) were also performed, carried out with 72 transients, delay time of s, spectral window of 200 ppm and 16 ppm for F1 and F2 respectively, and 192 and 1442 of points for F1 and F2, respectively The temperature was controlled at 333.1 K The polysaccharide from A spicifera was prepared by dissolution of 2.5 % m/v in deuterated water D2O containing 1% of the trimethylsilyl propanoic acid, (TSP, v/m) (Moraes et al., 2019) 2.3.6 Hemoglobin (Hb) concentration The Hb concentration was measured using a colorimetric test (LABTEST, Diagnostic SA, Minas Gerais, Brazil) Stomach samples were homogenized in a color reagent and centrifuged at 10,000 rpm for 10 Then, the supernatant was removed, filtered using a 0.22-mm filter and centrifuged at 10,000 rpm for 10 The absorbance was measured using a spectrophotometer at 540 nm and results were expressed in milligrams of Hb per gram of tissue (mg of Hb/g tissue (Medeiros et al., 2008) 2.3 Biological effects of PAs against gastric mucosa damage 2.3.7 Adhered mucus content Stomach samples were incubated in 0.1 % alcian blue solution pre­ pared in a solution of 0.16 M sucrose and sodium acetate (pH 5) The excess of alcian blue was removed by washes in 0.25 M sucrose Then, the dye content in gastric tissue was extracted with MgCl for h The extracted material was mixed with equal volume of diethyl ether and centrifuged at 3600 rpm for 10 and absorbance was measured using a spectrophotometer at 598 nm The adhered mucus content was calculated using a standard curve of alcian blue and the results were expressed in micrograms of alcian blue per gram of tissue (μg of alcian blue/g tissue) (Corne, Morrissey, & Woods, 1974) 2.3.1 Mice Male Swiss mice (25− 30 g, n = 6–7) obtained from the Central Vi­ varium at the Federal University of Cear´ a were maintained in cages under a 12 h light/12 h dark cycle, controlled temperature (22 ± ◦ C) and with food and water ad libitum However, the animals were fasted for 14− 18 h prior to the experiments, with free access to water All animal treatments and procedures were performed according to the guidelines of the National Council for Control of Animal Experimenta­ tion/CONCEA and approved by the Ethics Committee in Research (Protocol nº 068/14) 2.3.8 Statistical analysis Data are described as means ± SEM or median with minimum and maximal, when appropriate One-way analysis of variance (One-way ANOVA) followed by Student-Newman-Keuls test was used to determine the statistical significance of the differences between the groups For histological assessment, the Kruskal-Wallis nonparametric test was used, followed by Dunn’s test for multiple comparisons P < 0.05 was defined as statistically significant 2.3.2 Effect of PAs on ethanol-induced gastric damage Mice were treated with PAs (0.3, 1, and 10 mg/kg, p.o) After h, the gastric damage was induced by the administration of ethanol (100 %, 0.5 mL/25 g) The control group received only saline (0.9 % NaCl) or ethanol One hour later, the animals were sacrificed and their stomachs removed, opened, washed with saline and photographed Gastric dam­ age was measured using a computer planimetry program (Image J®) (Medeiros et al., 2008) One sample was removed and fixed in 10 % formalin for histopathological analysis Other samples were collected for determination of hemoglobin (Hb), glutathione (GSH), malondialde­ hyde (MDA) and mucus levels Results and discussion 3.1 Isolation and structural elucidation 2.3.3 Histopathological analysis Four-micrometer-thick sections were deparaffinized, stained with hematoxylin and eosin, and then examined under a light microscope by a pathologist without the knowledge of the experimental groups The specimens were assessed according to criteria as described previously (Laine & Weinstein, 1988) In brief, the sections were assessed for epithelial cell loss (a score of 0− 3), edema in the upper mucosa (a score of 0− 4), hemorrhagic damage (a score of 0− 4) and the presence of in­ flammatory cells (a score of 0− 3) The sections were assessed by an experienced pathologist without knowledge of the experimental groups 3.1.1 Yield and chemical analysis of PAs From 10 g of dry mass of A spicifera, 2.40 g of total polysaccharide was obtained, corresponding to a yield of 24 % The yield of total polysaccharide from marine algae can be influenced by several factors, such as type species, extraction methods, alga life stage, seasonal vari­ ations and natural habitat The yield obtained in this study is in accor­ dance with other species of red marine algae of the Brazilian coast, which presented yield ranging from 2.4–46% (Marinho-Soriano & Bourret, 2003) The method used in the extraction of polysaccharides can explain the differences in yield found in this study (24 %), compared to the values found for other published data for the same algae (3.6–56.6%) (Duarte et al., 2004; Ganesan et al., 2018; Anand et al., 2018) and also the value found for sulfated polysaccharide from Acan­ thophora muscoides (11.6 %) obtained by aqueous extraction at 100 ◦ C (Quinder´ e et al., 2013) In general, higher yields of total polysaccharides 2.3.4 Glutathione (GSH) levels Stomach samples were homogenized in 0.02 M EDTA Then, the homogenate was mixed with distilled water and trichloroacetic acid (50 %, w/v) and centrifuged at 3000 rpm for 15 at ◦ C Next, the su­ pernatant was mixed with tris buffer (0.4 M, pH 8.9) and added with L.C Pereira Júnior et al Carbohydrate Polymers 261 (2021) 117829 are expected in aqueous extraction, since it involves a much smaller number of steps during the process The results of the present study demonstrated that A spicifera presents high yield in the enzymatic extraction of polysaccharides, even when compared to other marine algae with high yield using the same extraction method, such as Solieria filiformis, which presented a recovery of 19.14 % (Araújo et al., 2011) The carbohydrate content (galactose) of A spicifera was 83 % The PAs also presented 6.48 % of sulfur, 4.95 % of hydrogen and 23.76 % of carbon The degree of sulfatation was calculated based on elemental analysis The DS express the amount of DS sulfate is defined as the number of OSO–3 or sulfur atoms, per disaccharide repeat unit (gal­ actose + anhydrogalactose) DS was calculate taking into account the % of S and C (Eq 1) and the value obtained shows that PAs has a degree of sulfatation of 1.23 The % of NaSO3 is 20.86 % for A spicifera poly­ saccharide, this value was in the average of the observed for previously reported data for the same polysaccharide (9.8–26.6 %) (Duarte et al., 2004; Ganesan et al., 2018; Anand et al., 2018) The isolation process was very efficient in remove proteins as no nitrogen was detected by elemental analysis et al., 2004; Ganesan et al., 2018; Anand et al., 2018) but it is within an expected range, since biological macromolecules are already known to have high molar mass (104-1010 g/mol) (Ho, Chiang, Lin, & Chen, 2011) The value found was higher than that of sulfated polysaccharide from marine alga Gracilaria caudata, which had its molar mass estimated at 2.5 × 105 g/mol (Barros et al., 2013) and Gracilaria birdiae estimated at 3.7 × 105 g/mol (Souza et al., 2012) Both sulfated polysaccharides were obtained by aqueous extraction 3.1.3 Chemical characterization by FTIR spectroscopy FTIR spectroscopy is a very useful technique for the elucidation of structures of chemical compounds, especially in polysaccharides ´mez-Ordo ´n ˜ ez, Jim´enez-Escrig, & Rup´erez, 2014) The infrared (Go spectrum of a compound is quite characteristic for the molecule in question and can be used as its chemical signature Therefore, infrared spectroscopy has extensive application in the identification of molecules In sulfated polysaccharides from marine algae, the FTIR spectrum bands can be used to distinguish algae producing carrageenans and ´mez-Ordo ´n ˜ ez & Rup´ agarans (Go erez, 2011) The Fig 1B shows the infrared spectrum of PAs expanded in the band region between 1400 and 700 cm− to better identify the sulfated groups present The FTIR (Fig 2B) showed band profiles similar to those related for algae poly­ saccharides (Alencar et al., 2019; Chagas et al., 2020) Moreover, one can assume that the polysaccharide is an agar, due to the presence of the band found at 893 cm− 1, corresponding to the agar group (Mollet, Rahaoui, & Lemoine, 1998), besides evidencing the presence of sulfated – O) groups in the molecule perceived by the band at 1249 cm− (S– (Chopin & Whalen, 1993; Lloyd, Dodgson, Price, & Rose, 1961) The region between 800 and 850 cm− is used to infer the position of the sulfate groups on agar-type polysaccharide The 845 and 830 cm− bands can be assigned, respectively, to 4-O-sulfate and 2-O-sulfate of Dgalactose residues, while bands at 820 and 805 cm− can respectively be assigned, C6 of L-galactose and C2 of the 3,6-anhydro-L-galactose (Chopin & Whalen, 1993; Lloyd et al., 1961) The PAs analysis by FTIR spectroscopy shows two bands in this region one in 820 cm− due to C-2 of the 3,6-anhydro-L-galactose and 830 cm− due to 2-sulfate-D-ga­ lactose The absorption spectrum showed bands characteristic of algae polysaccharides, such as 1249, 1157, 1072, 931 and 893 cm− The band with a wave number of 1072 cm− is attributed to the carbonic skeleton of galactan-type polysaccharide, whereas the band observed at 931 cm− is characteristic of type 3,6 sugar, which is based on the re­ sults obtained in the literature (Chopin & Whalen, 1993; Rochas, Lahaye, & Yaphe, 1986) 3.1.2 Determination of the molar mass by GPC Polysaccharides, being natural polymers, are polydispersed, which means that they not exhibit precisely defined molecular weights, but rather a mean of the molar mass representing a distribution of almost identical molecular species in structure but varying in chain length (Stephen, Philips, & Williams, 2016) The chromatogram (Fig 2A) shows a single peak with an elution volume of 8.032 mL at its apex, behaving as a homogeneous system The peak molar mass was estimated to be 6.98 × 105 g/mol This molar mass was higher than the values obtained from previous works (1.44 × 105 to 4.7 × 105 g/mol) (Duarte 3.1.4 Chemical characterization by NMR spectroscopy NMR spectroscopy is a powerful technique for the elucidation of polysaccharide structures Therefore, the characterization of PAs was performed by employing one- and two-dimensional NMR spectroscopy The 1H, 13C, DEPT 135 and HSQC spectra are shown in Fig H and 13C NMR spectra of the sulfated polysaccharide from A spicifera in the anomeric region is similar to the observed by Duarte at al (2004) where three main anomeric signals, although they are over­ lapped in some degree, are observed Signals in the region of δ101.8 is due to α-L-Galp-6-sulfate (L6S), and δ100.8 corresponding to C-1 of β-Dgalap-2-sulfate (G2S) (δ100.8), while the signal at δ98.5 is due to 3,6anhydro-α-L-galp anomeric carbon (LA) HSQC spectrum shows that anomeric carbons correlates with three protons (13C/1H): δ101.8/5.22 (L6S), δ100.8/4.67 (G2S) and δ98.5/5.12 (LA) It is also observed two methyl groups in the HSQC spectrum, which was confirmed by the DEPT experiment (Fig 2) one at proton signal at δ 1.50 correlating with a carbon at δ 25.3, assigned as 4,6-O- (1’-O-carboxyethylidene) pyruvate group (Duarte et al., 2004) and also a methoxylated form of this poly­ saccharide since one can note the signal at δ 3.51 with carbon at δ 57.7, attributed to methyl group linked at C6 of the galactosepyranose residue (G6M-OMe) (Barros et al., 2013; Seedevi, Moovendhan, Viramani, & Fig GPC curve (A) and FT-IR spectrum (B) in KBr of sulfated polysaccharide from A spicifera L.C Pereira Júnior et al Carbohydrate Polymers 261 (2021) 117829 Fig NMR spectra of A spicifera (A) 1H spectrum; (B) DEPT 135 spectrum; (C) 13 C spectrum; (D) HSQC spectrum Shanmugam, 2017) The proportion of main units was calculated based on NMR integrated spectum, as well as, the proportion of pyruvic groups using the signal of methyl groups (Chagas et al., 2020) The proportion of LA:L6S:G2S was 0.75:0.99:1.00 which gives in percentage 27.4 % of LA, 36.1 % of L6S and 36.5 % of G2S The proportion of pyruvic groups was 34.7 % of the total monosaccharide units, which is present as G2S-P The proportion of galactose and anhydrogalactose observed in this work is in the average of the one observed in previous works (Duarte et al., 2004, Ganesan, Shanmugamb, Palaniappanc, & Rajauria, 2018) The main difference found in this work was the absence of xylose The HPLC analysis of polysaccharide in different hydrolyse times did not show the presence of xylose 3.2 Biological activity of PAs 3.2.1 PAs prevents the ethanol-induced gastric damage The ethanol promotes damage against gastric mucosa by rapidly penetrating the phospholipid membrane, providing the appearance of lesions characterized by alterations in the cellular membrane structure, intense hemorrhage, production of reactive species derived from oxygen (ROS), destruction of mucosa barrier mechanisms and increased vascular permeability (Nassini et al., 2010) In the present study, the administration of ethanol (50.0 ± 14.0 % of damaged area) promoted the formation of extensive hemorrhagic macroscopic lesions in the gastric mucosa, compared to the saline group In addition, treatment with polysaccharide from A spicifera (PAs) significantly (P < 0.05) prevented the ethanol-induced macroscopic gastric damage, in a dose-dependent manner, at all doses tested, with the maximum effect at 10 mg/kg (12.4 ± 8.2 % of damaged area) (Fig 4) Corroborant with macroscopic analysis, the results from the histo­ pathology showed that mice treated with PAs have prevention in the gastric microscopic damage (edema, hemorrhage and loss of epithelial cells) provided by the administration of ethanol, showing gastro­ protective effect The saline group did not show any alteration in the gastric mucosa (Table and Fig 5A–C) Several studies in the literature have demonstrated the protective potential of polysaccharides from marine algae against lesions in the gastrointestinal tract For example, polysaccharides from Hypnea mus­ ciformis (Damasceno et al., 2013) and G caudata (Silva et al., 2011) have Fig PAs prevents ethanol-induced macroscopic gastric damage Results are expressed as mean ± SEM (6-7 animals per group) #P < 0.05 vs saline group; *P < 0.05 vs ethanol group; ANOVA and Newman-Keuls test Table Polysaccharide from A spicifera reduces ethanol-induced microscopic gastric damage Experimental groups Hemorrhagic damage (score, 0− 4) Edema (score, 0− 4) Epithelial cell loss (score, 0− 3) Total (score, 0− 14) Saline Ethanol (0− 1) (3− 4)* (0− 0) (1− 4)* (0− 0) (3− 3)* PAs 10 mg/kg (1− 3)# (0− 0)# 1.5 (1− 3)# (1− 3) 10 (7− 11)* (2− 6)# Results are expressed as the means ± S.E.M of 6–7 mice per group * P < 0.05, compared to saline group # P < 0.05, compared to ethanol group gastroprotective activity in ethanol-induced gastric damage, whereas polysaccharide from G birdiae reduced the TNBS-induced colitis (Brito et al., 2014) and naproxen-induced gastrointestinal damage (Silva et al., 2012) These authors suggested that the mechanism of action is L.C Pereira Júnior et al Carbohydrate Polymers 261 (2021) 117829 Fig PAs reduces ethanol-induced microscopic gastric damage and hemorrhage Photomicrographs of gastric mucosa (100x) showing (A) saline, (B) 100 % ethanol and (C) PAs 10 mg/kg plus ethanol; (D) hemoglobin levels Results are expressed as mean ± SEM (6–7 animals per group) *P < 0.05 vs saline group; #P < 0.05 vs ethanol group; ANOVA and Newman-Keuls test dependent, at last in part, on the reduction of oxidative stress damage membrane and cause ulcers, increasing the level of MDA, a marker of lipid peroxidation (Medeiros et al., 2008) The data from this study showed that the PAs treatment (70.4 ± 12.2 nmol/g) significantly (P < 0.05) prevented the increase in MDA concentrations in the gastric tissue altered by the administration of ethanol (112.7 ± 12.3 nmol/g tissue) The saline group (59.3 ± 5.8 nmol/g tissue) showed the basal levels of MDA concentra­ tion (Fig 6B) Sulfated polysaccharides from marine algae are effective in the reduction of oxidative damage in the gastrointestinal tract, due to a mechanism related to the prevention of lipid peroxidation, increasing tissue GSH levels (Damasceno et al., 2013; Silva et al., 2011) Thus, based on the literature and results of the present study, polysaccharide from A spicifera reduced the hemorrhagic damage in the gastric mucosa promoted by the administration of ethanol, by mechanisms that involve, at least in part, increase in antioxidant defense and reduction of harmful effects mediated by free radicals increased by ethanol (Costa et al., 2010; Tariq et al., 2015) 3.2.2 PAs reduces the gastric Hb in the ethanol-induced damage Gastric alterations mediated by the administration of ethanol also indirectly involves change in the gastric blood flow, marked by mucosal and submucosal hemorrhage In the stomach, the blood flow contributes to the protection through the supply of oxygen, nutrients and bicar­ bonate, removal of hydrogen (H+) of epithelial cells and toxic substances that diffuse from the lumen to the gastric mucosa (Tarnawski, Ahlu­ walia, & Jones, 2012) Thus, in the present study, the hemoglobin (Hb) concentration in the gastric mucosa was used as an indirect marker of ethanol-induced hemorrhagic damage (Medeiros et al., 2008) The results showed that the treatment with PAs (0.410 ± 0.040 g/g tissue) significantly (P < 0.05) reduced the increase of Hb concentra­ tion, an indication of hemorrhagic gastric damage, in the gastric mucosa of animals submitted to ethanol damage (0.770 ± 0.050 g/g tissue) The saline group (0.240 ± 0.002 g/g tissue) showed the basal levels of Hb (Fig 5D) Thus, the gastric Hb concentration confirmed the data from macro­ scopic and microscopic analyses, and showed a significant protection of this macromolecule, suggesting that the cascade of ethanol-mediated harmful events to the gastric mucosa are negatively modulated Simi­ larly, a study using polysaccharide from marine alga S filiformis showed its capacity to prevent the increase of Hb concentration in the gastric mucosa of animals submitted to the ethanol damage protocol (Sousa et al., 2016) 3.2.4 PAs prevents the reduction of adhered gastric mucus after the administration of ethanol The organism is often exposed to harmful agents that can cause serious damage to the gastric mucosa, including H pylori, drugs (i.e NSAIDs and alendronate) and chemical substances (Bhattamisra et al., 2019; Medeiros et al., 2008; Silva et al., 2014) However, the gastric mucosa has defense mechanisms against these agents Mucus secretion on the surface of epithelial cells acts as a first line of defense of the gastric mucosa, delaying the diffusion of acid content from the gastric lumen to the gastric mucosa, providing lubrication for the passage of food, protecting the mucosa from mechanical and chemical tensions that assist in the process of regeneration of gastric lesions and mucosa maintenance (Dong & Kaunitz, 2006) In this context, the present study also showed that the administration of ethanol (684.1 ± 117.0 μg/g tissue) promoted a significant (P < 0.05) decrease in mucus adhered to gastric mucosa, compared to the saline group (1,196.0 ± 156.7 μg/g tissue), probably by mobilizing mucopolysaccharides from the mucosa to the lumen, decreasing the secretory capacity of mucus in the stomach, contributing to the mucosa damage (Silva et al., 2016) On the other hand, mucus loss was pre­ vented by the administration of polysaccharide from A spicifera (1, 005.0 ± 172.0 μg/g tissue) in ethanol-treated animals, confirming a gastroprotective effect of gastric mucosa (Fig 6C) Thus, we observed a straight correlation between the gastroprotective effect and the con­ centration of mucus adhered to the gastric mucosa, confirming a gas­ troprotective effect to the gastric mucosa against damage caused by the administration of ethanol 3.2.3 PAs prevents the consumption of gastric GSH and increase in MDA due to the administration of ethanol The next step of our work was to evaluate important parameters related to the redox balance, such as the GSH levels and MDA concen­ tration in gastric mucosa of animals submitted to ethanol-induced injury GSH is a tripeptide found in high concentrations in the gastric mu­ cosa cells, acting as a blocker of free radical production and as a sub­ strate for glutathione peroxidase (GPx), metabolizing hydrogen peroxide (H2O2) and other hydroperoxides (Song et al., 2014) Thus, we investigated the gastric GSH levels, as a relevant protective mechanism preventing and/or controlling free radical formation Similar to another study (Shine et al., 2009), our results showed that the administration of ethanol (107.4 ± 12.7 μg/g tissue) promoted a significant (P < 0.05) decrease in GSH levels, compared to the saline group (178.1 ± 10.7 μg/g tissue) However, the treatment with PAs (154.0 ± 13.5 μg/g tissue) prevented this GSH consumption (Fig 6A) Thus, the PAs may work by decreasing the redox state in gastric injury caused by ethanol; another possibility is that an increase in GSH levels may be secondary to a decrease in free radical production Another marker assessed was MDA, a lipid peroxidation product, which has been used as an important marker of oxidative damage Its quantification was used to show a possible antioxidant activity (Kanter, 2005) Superoxide produced by peroxidase in stomach tissues can Conclusion The sulfated polysaccharide from A spicifera classified as agar-type polysaccharide showing high molecular mass They exert L.C Pereira Júnior et al Carbohydrate Polymers 261 (2021) 117829 Validation, Formal analysis, Investigation Fernando G Nascimento: Conceptualization, Methodology, Validation, Formal analysis, Investi­ gation, Writing - original draft Samara R.B.D Oliveira: Methodology, Investigation, Writing - original draft Glauber C Lima: Conceptuali­ zation, Formal analysis, Investigation, Data curation, Writing - original draft Francisco Diego S Chagas: Methodology, Investigation, Data curation Venicios G Sombra: Conceptualization, Methodology, Vali­ dation, Data curation, Investigation Judith P.A Feitosa: Methodology, Validation, Formal analysis Eliane M Soriano: Formal analysis, Vali­ dation, Formal analysis Marcellus H.L.P Souza: Methodology, Formal analysis, Resources, Funding acquisition Guilherme J Zocolo: Meth­ odology, Investigation, Writing - original draft Lorena M.A Silva: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing Regina C M de Paula: Conceptualization, Methodology, Writing - review & editing Renan O.S Damasceno: Conceptualization, Methodology, Writing - review & editing, Supervision, Project administration, Re­ sources, Funding acquisition Ana Lúcia P Freitas: Conceptualization, Methodology, Writing - review & editing, Resources, Funding acquisition Acknowledgements The authors gratefully acknowledge the financial support from the National Council for Scientific and Technological Development/CNPq (Brazil) and Coordination for the Improvement of Higher Education Personnel/CAPES (Brazil) The authors also wish to acknowledge Embrapa (Brazilian Agricultural Research Corporation) in Fortaleza, Cear´ a, Brazil for recording the NMR spectrum, the Department of ´ Organic and Inorganic Chemistry from the Federal University of Ceara (UFC) for recording FTIR spectrum and Ethics Committee in Research from UFC for providing the animals We also thank teacher Abilio Borghi for the grammar review of the manuscript References Albalasmeh, A A., Berhe, A A., & Ghezzehei, T A (2013) A new method for rapid determination of carbohydrate and total carbon concentrations using UV spectrophotometry Carbohydrate Polymers, 97(2), 253–261 https://doi.org/ 10.1016/j.carbpol.2013.04.072 Alencar, P O C., Lima, G C., Barros, F C N., Costa, L E C., Ribeiro, C V P E., Sousa, W M., … Freitas, A L P (2019) A novel antioxidant sulfated polysaccharide from the algae Gracilaria caudata: In vitro and in vivo activities Food Hydrocolloids, 90, 28–34 https://doi.org/10.1016/j.foodhyd.2018.12.007 Anand, J., Sathuvan, M., 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https://doi.org/ 10.1016/S0002-9270(00)02140-7 Fig PAs decreases the oxidative stress and prevents the loss of adhered mucus content to gastric mucosa in mice submitted to ethanol-induced gastric damage (A) GSH levels, (B) MDA concentration and (C) Adhered mucus con­ tent Results are expressed as mean ± SEM (6-7 animals per group) #P < 0.05 vs saline group; *P < 0.05 vs ethanol group; ANOVA and Newman-Keuls test There was no statistical difference between PAs groups and saline groups in any experiment carried out gastroprotective effect on ethanol-induced gastric mucosa damage by a mechanism that involves the prevention of lipid peroxidation and en­ hances defense mechanisms These observations raise the possibility that PAs may be used as a promising source with great gastroprotective potential and possible application as a new natural tool to improve the resistance of the mucosa to lesion promoted by alcohol, being able to prevent future pathologies in the mucosa caused by factors 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The main components of sulfated poly­ saccharide from A spicifera are galactose and 3,6-anhydrogalactose (80–85 %) (Duarte et al., 2004; Ganesan, Shanmugamb, Palaniappanc, & Rajauria, 2018; Anand... https://doi.org/10.1016/j.foodhyd.2018.12.007 Anand, J., Sathuvan, M., Babu, G V., Sakthivel, M., Palani, P., & Nagaraj, S (2018) Bioactive potential and composition analysis of sulfated polysaccharide from Acanthophora spicifera (Vahl)... Yield and chemical analysis of PAs From 10 g of dry mass of A spicifera, 2.40 g of total polysaccharide was obtained, corresponding to a yield of 24 % The yield of total polysaccharide from marine