Hepatoprotective effect of a fucoidan extract from Sargassum fluitans Borgesen against CCl4-induced toxicity in rats

Journal Pre-proof Hepatoprotective effect of a fucoidan extract from Sargassum fluitans Borgesen against CCl4-induced toxicity in rats

Juan Chale-Dzul, Rebeca Pérez-Cabeza de Vaca, Carlos QuintalNovelo, Leticia Olivera-Castillo, Rosa Moo-Puc PII:

S0141-8130(19)33830-9

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.183

Reference:

BIOMAC 14211

To appear in:

International Journal of Biological Macromolecules

Received date:

22 May 2019

Revised date:

29 November 2019

Accepted date:

20 December 2019

Please cite this article as: J. Chale-Dzul, R. Pérez-Cabeza de Vaca, C. Quintal-Novelo, et al., Hepatoprotective effect of a fucoidan extract from Sargassum fluitans Borgesen against CCl4-induced toxicity in rats, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/j.ijbiomac.2019.12.183

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© 2019 Published by Elsevier.

Journal Pre-proof

Hepatoprotective effect of a fucoidan extract from Sargassum fluitans Borgesen against CCl4-induced toxicity in rats 1

Juan Chale-Dzul,2Rebeca Pérez-Cabeza de Vaca, 4 Leticia Olivera-Castillo, 5Rosa Moo-Puc.

3

Carlos Quintal-Novelo,

1

Laboratorio de Apoyo a la Vigilancia Epidemiológica, Hospital de Especialidades 1, Centro Médico Nacional Ignacio García Téllez, Instituto Mexicano del Seguro Social, C 41 No. 439 x 32 y 34, Col. Industrial, 97150 Merida, Yucatan, Mexico. 2

Research Coordination, Centro Médico Nacional “20 de noviembre” ISSSTE, Mexico City, Mexico. 3

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Unidad Médica de Alta Especialidad, Centro Médico Ignacio García Téllez, Instituto Mexicano del Seguro Social, C. 41, No. 439, Col. Industrial, 97150 Mérida, Yucatan, Mexico. 4

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Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Unidad Mérida, Carretera Antigua Progreso Km. 6, 97310 Mérida, Yucatán, Mexico. 5

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Unidad de Investigación Médica Yucatán, Unidad Médica de Alta Especialidad, Centro Médico Nacional Ignacio García Téllez, Instituto Mexicano del Seguro Social, C 41 No. 439 x 32 y 34, Col. Industrial, 97150 Mérida, Yucatan, Mexico.

Journal Pre-proof Abstract The in vivo antifibrotic effect of a fucoidan extract (FE) from Sargassum fluitans Borgesen was evaluated in a carbon tetrachloride-induced liver damage model in rats over twelve weeks. Chemical analysis showed the FE to contain carbohydrates, sulfates, uronic acids, protein, phenols, and to have a molecular weight of ~60 kDa. Physiological, biochemical, histological and genetic assays were done. Daily oral administration of FE (50 mg/kg) reduced liver enzymatic activity, liver infiltration of inflammatory cells, collagen fiber deposition and gene

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expression cytokines such as interleukin beta 1 (IL-β1), tumor necrosis factor alpha

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(TNF-α), transforming growth factor beta 1 (TGF-β1), Smad-3, Smad-2, collagen 1 alpha 1 (col1α1) and tissue inhibitor of metalloproteinase 1 (TIMP-1). It also

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increased RNA expression of Smad-7 and metalloproteinase 2 and 9 (MMP2 and

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MMP9). The fucoidan extract exhibited an antifibrotic effect mediated by the

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inhibiting TGF-β1/Smad pathway, as well as anti-inflammatory effects.

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Key words: Sargassum fluitans; Fucoidan; Hepatoprotective

Journal Pre-proof 1. Introduction Liver fibrosis is a pathologic process characterized by excessive production of extracellular matrix proteins (EMP) which can lead to development of cirrhosis, liver failure and mortality. Activated hepatic stellate cells (HSC) are believed to be the principal cells involved in liver fibrosis [1]. These cells are located in the space between the parenchymal cells and sinusoidal space in the hepatic lobule, the main function of which is to store vitamin A and regulate homeostasis of retinoic acid [2]. During liver damage, however, Kupffer cells and hepatocytes release

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various stimulants, such as reactive oxygen species (ROS), lipid peroxides and

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transforming growth factor beta 1 (TFG-β1), which induce activation of quiescent HSC into a myofibroblastic phenotype [2]. Transforming growth factor beta 1has

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been described as the main cytokine involved in HSC activation and transformation. Binding of TGF-β1 to type II receptors in HSC induces

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phosphorylation of type I receptors. This in turn phosphorylates Smad2/3 which

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forms a heteromeric complex with Smad-4 and migrates to the nucleus. Here it induces increased expression of genes related to EMP synthesis such as α-smooth

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muscle actin (α-SMA); procollagen type 1; collagen one alpha 1 (Col1α); and tissue inhibitor of metalloproteinase (TIMP1). It also decreases expression of matrix

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metalloproteinases (MMPs), responsible for EMP remodeling and degradation. This causes an imbalance between EMP remodeling and production [2,3].

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Activation of HSC via the TGF-β1 pathway and fibrogenic gene expression are therefore considered hallmarks of liver fibrosis; regulation of HSC activation has been proposed as a therapeutic target in fibrosis treatment [4,5]. No currently available treatment can reverse liver fibrosis. Research into new therapeutic alternatives is therefore important, and natural source derivatives are a promising option. Compounds or extracts with an antioxidant effect can contribute to preventing chronic diseases related to oxidative stress, such as cancer, neurodegenerative conditions, cardiovascular conditions and liver disease (fibrosis) [6,7]. For example, an extract of Citrus limon fruit was reported to induce a hepatoprotective effect in Wistar rats attributable to the presence of different antioxidants which may neutralize ROS generated by carbofuran, a hepatotoxic

Journal Pre-proof agent [8]. An aqueous extract of Aloe vera leaf was found to protect the liver of rats against damage inducedby the hepatotoxic agents cartap and malathion, an effect linked to the extract’s antioxidant properties [9]. Silymarin and curcumin are reported to exercise an antifibrotic effect by decreasing HSC activation and reducing collagen synthesis activities associated with their antioxidant potential [10,11]. Recent research has focused on a group of macromolecules obtained from marine sources, known as fucoidans, which are sulfated polysaccharides derived from the cell wall ofbrown seaweeds. Composed principally of L-fucose units and

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sulfate groups,it also contains small quantities of other sugars such as of Dgalactose, D-mannose and D-xylose, as well as uronic acids and protein [12].

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Fucoidans have pharmacological properties such as anticoagulant, antitumoral,

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antiviral, anti-inflammatory, antibacterial and immunomodulatory, as well as strong antioxidant and antifibrotic effects. They have understandably been proposed as a

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natural ingredient in functional foods [13,14].

Fucoidans have a notable antifibrotic effect [15], which is mediated by regulation of

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inflammation and the TGF-β1 pathway [16-18]. Our research group has been studying the antioxidant and cytoprotective effect of a fucoidan extract from

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Sargassum fluitans, a tropical brown seaweed that grows in the Caribbean Sea off the eastern Yucatan Peninsula. We have found that a fucoidan extract is capable

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of scavenging free radicals, inhibiting intracellular formation of ROS, increasing

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glutathione levels and restoring catalase activity in a human hepatic cell line [19]. Based on previous in vitro evidence,the present study objective was to evaluate the antifibrotic effect of a fucoidan extract from S. fluitans in an in vivo model of liver fibrosis induced by carbon tetrachloride (CCl4). 2. Material and Methods 2. 1. Chemical Reagents Carbon tetrachloride (CCl4), ethanol (EtOH), calcium chloride (CaCl2), sodium carbonate (Na2CO3), sodium chloride (NaCl), sulphuric acid (H2SO4), Folin– Ciocalteau, silymarin, phloroglucinol, fucoidan standard (from Fuscus vesiculosus), L-cysteine hydrochloride, Bradford reagent, pullulan and albumin were purchased

Journal Pre-proof from Sigma-Aldrich®(St. Louis, MO, USA). TaqMan Gene Expression Assays for the selected genes (Table S1) and Verso 1-Step qRT-PCR Kit Plus RoxVial were purchased from Thermo Fisher Scientific Co. (USA), and a RNAeasy Mini Kit from QIAGEN®.

2.2. Algal material Sargassum fluitans Borgesen was collected off the coast of Puerto Morelos, in the state of Quintana Roo, Mexico (20°50’51.9” N; 86°52’30.3” W) in February 2016.

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The collected biomass was centrifuged on-site to remove excess seawater using a commercial portable centrifuge, stored in plastic bags and kept on ice for transport

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to the laboratory. In the laboratory the biomass was washed again with freshwater

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to remove salts, sand, and epiphytes, and stored at -20°C. Voucher specimens were identified in the Alfredo Barrera Marin Herbarium of the Biological Sciences

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Campus, Faculty of Biology of the Autonomous University of Yucatan (UADY),

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Mexico.

2.3. Fucoidan extract from Sargassum fluitans Borgesen

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Fucoidan extract (FE) was obtained following Foley et al. [20] with modifications [21]. Briefly, seaweed previously stored at -20°C was thawed, washed with

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freshwater, and 100 g milled in a commercial blender to produce a homogeneous

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paste. This was pretreated with 500 mL EtOH (80% v/v) at room temperature for 12 h. The mixture was filtered and the solid fraction extracted with EtOH (80% v/v) at 70°C for 12 h. Successive extraction of the solid residue with Milli-Q H2O was done first at room temperature for 7 h, and then at 70°C for 7 h and 70°C for 4 h. The resulting aqueous extracts were treated with 2 M of a CaCl2 solution at room temperature to precipitate alginates. These were removed by centrifuging at 15652xg´ for 30 min, producing a fucoidan-rich extract. Salinity was further lowered by dialysis (using a SpectrumTM Labs Spectra/PorTM6-8 kDa) for 48 h with a Milli-Q H2O, with changes every 12 h. The fully processed FE was freeze-dried and stored until use.

Journal Pre-proof 2.4. Chemical analysis Total carbohydrates and sulfate contents of the FE were quantified using the phenol sulfuric acid method [22], and the turbidimetric method [23]. Uronic acid content was measured with a modification of the carbazole method employing a combination of sulfamate to suppress browning and carbazole to color the uronic acids, using D-glucuronic acid as a standard [24,25]. Total phenol content (TPC) was determined by spectrophotometry using Folin-Ciocalteau reagent according to Attard [26], with modifications. Briefly, 10 mg FE was dissolved in 1 mL deionized

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water, 10 µL of this solution mixed with 90 µL Folin-Ciocalteau reagent (1:10) in a 96-well microplate, and 80 µL 1M sodium carbonate added (Sigma-Aldrich, St.

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Louis, MO, USA). The mixture was incubated at room temperature for 20 min

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under darkness and absorbance recorded at 620 nmusing a MultiskanG0 (Thermo Fisher Scientific, Co, USA). The TPC (expressed as % dry weight) was calculated

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using a standard curve for phloroglucinol.

Soluble protein content was measured as described by Bradford [27], with

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modifications. Briefly, 10 mg FE was dissolved in 1 mL deionized water, 10 μL of this solution placed in a 96-well microplate and mixed with 90 μL Bradford reagent

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(Sigma-Aldrich, St. Louis, MO, USA).The plate was incubated at room temperature for 10 min, and absorbance measured at 595 nm in a Multiskan G0 (Thermo Fisher

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Scientific, Co, USA).

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Fucoidan content were analyzed by the cysteine-sulphuric acid colorimetric method as described by Dische et al.[28]. Briefly, 10 mg FE was dissolved in 10 mL Milli-Q H2O and 1 mL of this solution pipetted into test tubes and placed on ice before sulphuric acid (4.5 mL; 85%; 1:6 H2O:H2SO4) was added to each test tube, homogenized and incubated for 1 min. The tubes were closed to prevent evaporation and placed in a boiling water bath (100 °C) for 10 min. After cooling to room temperature, 0.1 mL 3% L-cysteine hydrochloride solution was added to each tube, mixed and allowed to stand for 30 min. Absorbance was measured at 396 nm and 427 nm using a Multiskan G0 (Thermo Fisher Scientific, Co., US). Fucoidan content (expressed as % dry weight) was calculated using a standard curve for the commercial fucoidan Fucus vesiculosus (Sigma-Aldrich, St. Louis, MO, USA).

Journal Pre-proof Absorbance values were calculated with the equation: Absorbance=(A396 nm A427 nm); this corrects for the presence of hexoses [28,29].

2.5. Fourier-transform infrared spectroscopy Fourier-transform infrared spectroscopy (FTIR) of the FE (15 mg) was done in KBr pellets. Spectra were recorded using the Thermo NicoletNexus670 FTIR spectrometer with a DTGS-KBr detector over a 750 to 3533 cm-1range.

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2.6. Molecular Weights

High performance liquid chromatography (HPLC) was run on a Dionex Ultimate

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3000 coupled with a refraction index (Thermos Fisher Scientific, Co, USA),

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employing a BioBasic SEC 1000 column (Thermo Fisher Scientific, Co, USA) operated at a 37°C column temperature. The mobile phase consisted of 0.2 M

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NaClat a 1.0 mL/min flow rate for 15 min. Sample injection volume was 25 μL.

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Briefly, FE was dissolved in 0.2 M NaCl solution to a 1.0 mg/mL concentration, filtered (0.45 µm) to eliminate dust particles and the sample injected under the above conditions. Estimation of fucoidan molecular weight was done by HPLC with

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high weight standards for column calibration, pullulans (MW = 800, 400, 200, 110, 50 and 22 kDa) and albumin (MW 66.3 kDa) (Sigma-Aldrich, St. Louis, MO, USA);

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conditions were those used above. The fucoidan, albumin (66.3 kDa), and pullulan

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(50 kDa) were dissolved in 0.2 M NaCl at 1 mg/mL, filtered, and injected. The resulting chromatograms were compared.

2.7. Model of induced liver fibrosis in rats 2.7.1. Animals Experimental animals were adult male albino Wistar rats (220-300 g/weight) purchased from the Animal Care Center of the Autonomous University of Yucatan (UADY), Mexico, and fed a regular lab rat diet (Harlan Teklad, Madison, WI). The rats were kept under a 12 h dark: 12 h light cycle, at constant temperature (23 ± 1 °C) and humidity (60%), and with free access to food and water. Housing conditions met U.S. National Institutes of Health (NIH) guidelines and was

Journal Pre-proof approved by the Research Ethics Committee of the National Medical Center of the Mexican Institute of Social Security (Instituto Mexicano del Seguro Social - IMSS).

2.7.2. Animal treatment The rats were randomly divided into four groups, six per group: a negative control (administered 0.60 mL/kg body weight of corn oil); a positive control (0.60 mL/kg body weight of CCl4); a protection control (0.60 mL/kg body weight of CCl4+ 100 mg/kg silymarin); and the experimental treatment (0.60 mL/kg body weight of

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CCl4+ 50 mg/kg FE). The CCl4 was prepared in corn oil and injected subcutaneously into the rats twice a week for twelve weeks. The FE treatment was

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administered once a day by oral gavage, one week prior to CCl4 administration and

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for twelve weeks thereafter. One week after final treatment application all animals were evaluated with the sleeping time assay. Twenty-four hours later they were

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killed by narcosis, blood samples collected individually by heart puncture and the

2.7.3. Sleeping time

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histological and RNA isolation.

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liver lobules immediately excised, photographed, weighed, and preserved for

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One week after final treatment administration the rats were made to sleep with pentobarbital (30 mg/kg, i.p). Time elapsed was recorded from injection to when

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the animal could no longer accomplish the righting reflex. The rats were then placed on their back in a standard cage, and time elapsed again recorded from this moment to when the righting response was regained, that is, when the rat was able right itself. Sleep latency was the time elapsed between injection and loss of righting reflex, and sleep time that elapsed between loss and return of the righting reflex [30].

2.7.4. Determination of biochemical markers of hepatic injury Serum levels were quantified of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), total protein (TP) and albumin (ALB). These were quantified by an automatic analyzer (VITROS System

Journal Pre-proof Integrated 5600; Ortho Clinic Diagnostic) at the Ignacio García Téllez National Medical Center, Clinical Laboratory of the IMSS.

2.7.5. Liver histology Liver lobes were excised and washed in phosphate buffered saline (PBS), fixed in 10% paraformaldehyde in 0.1% PBS, embedded in paraffin and stored until use. A microtome was used to section (4-5 µm) the paraffin-embedded tissue and this stained with hematoxylin-eosin (H&E) or Masson trichrome (MT) stains.

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Measurement of hepatocyte degeneration and fibrosis was done by histological

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analysis of the H&E- and MT-stained sections. Quantitative fibrosis analysis was done with the NIH ImageJ program according to Chen et al.[31]. Fibrosis was

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calculated as a percentage of total hepatic area and expressed as the average of five randomly selected tissue sections from each liver, normalized versus a

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negative control. All images were taken using a high-resolution video camera

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(Canon PC1089) coupled to an Axiostar Plus Carl Zeiss microscope at 20X magnification. These were taken by the same operator following a blinded

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procedure.

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2.7.6 Immunohistochemistry

Formalin-fixed, paraffin-embedded (FFPE) liver tissue samples were sectioned (4-

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5 μm) with a microtome. Sections were mounted on glass slides, coated with polyL-lysine solution, and microwaved in preparation for deparaffinization. Xyleneethanol deparaffinization and rehydration were followed by citrate-buffer antigen retrieval. Immune staining antibodies were diluted at a1:50 TGF-β1 antibody (TB21): sc-52893 ratio and viewed in a Dako® HRP-DAB system. Images were taken with a high-resolution video camera (Canon® PC1089) connected to a light microscope (Axiostar Plus Carl Zeiss®) at 40X magnification. These were taken by the same operator in a blinded manner. Quantitative measurements were analyzed using the NIH ImageJ Analysis Program.

Journal Pre-proof 2.7.7. Real-time reverse transcription polymerase-chain reaction (qRT-PCR) Total RNA from liver tissue was isolated using the RNeasy Mini Kit (Qiagen®) following

manufacturer

instructions,

and

the

isolated

RNA

quantifiedby

spectrophotometry at 260 nm using a Nanodrop (Thermo Fisher Scientific, Co, USA). The quantified RNA was amplified using the TaqMan® gene expression assay and Verso 1-step qRT-PCR kit plus ROX vial (Thermos Fisher Scientific, Co, USA) in a Rotor-Gene (Qiagen®). Each 25 µL of reaction volume contained 400 nM forward and reverse primer, 250 nM probe and 5 ng RNA. Reaction conditions

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were as follows: cDNA synthesis 1 cycle for 15 min at 50 °C; thermo-star activation 1 cycle for 15 min at 95 °C; denaturation 40 cycles for 15 s at 95 °C; and

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annealing/extension 40 cycles for 60 s at 60 °C.In each run, the glyceraldehyde-3-

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phosphate dehydrogenase (GAPDH) reference gene was used as an internal control, and a negative template control (NTC) was used to prevent false positives.

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Expression levels relative to the control were calculated using the formula ΔΔCt

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2.7.8. Statistical analysis

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(ΔCt sample – ΔCt control) with the 2ΔΔCt method [32].

Results were expressed as the mean ± standard deviation (SD). A one-way analysis of variance (ANOVA) was applied to assess significant differences

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between the groups of animals, followed by a Tukey post hoc test. Statistical

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significance (p<0.05) was determined with the GraphPad Prisma ver. 4.0 software (San Diego, CA, USA).

3. Results and Discussion 3.1.Fucoidan extraction and chemical analysis Under the present extraction conditions 14.70 g FE were obtained from S.fluitans Borgesen,

equal

to

0.84%

(dry

weight)

yield.

This

contained

25.80%

carbohydrates, 7.56% sulfates and 0.32% uronic acid (Table 1). Carbohydrate, uronic acid and sulfate ester group contents can vary slightly due to extraction techniques, harvest location, harvest season and alga species [33,34]. Variability in

Journal Pre-proof components has been documented for other seaweeds such as S. polycystum, S. graminifolium and S. wightii [35-37]. Carbohydrates content in the FE was lower than in a previous report [19]. Fucoidan has been shown to bind to a large number of compounds, including proteins and sulfate groups. Binding affinity appears to be mainly determined by the polymer’s negative charge, molecular weight and degree of sulfation, rather than by any specific structure of the carbohydrate [38-40]. In the present results the FE contained 1.16% soluble protein, considered to be the principal impurity in fucoidan extracts [41,42]. This amount of protein is below that

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reported for fucoidans isolated from S. filipendula [43], S. polycystum [35] and S. muticum [44]. As mentioned previously, this variability may be due to harvest

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season, collection site and algae species [45]. Total polyphenol content (TPC) of

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the FE was1.99%, similar to values reportedfor fucoidans fromS. Glaucescens [46] and S. fluitans [19].

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In the present results the S. fluitans FE contained 11.67% fucoidan (Table 1),

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which is higher than reported for other sargassum species such as S. bindery (6.16%)[29]. Fluctuations in fucoidan content can respond seasonality, as shown in a study of fucoidan content in S. vulgare during the winter (3.78%), spring (3.59%),

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summer (3.96%) and fall (5.32%) [47]. Variation in fucoidan content can also occur in response to temperature variations, such as the 16.32 to 32.50% range reported

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for Sargassum sp. due to temperature changes in the extraction method[48].

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Variations in fucoidan content have also been reported for S. myriocystum (12.79% to 15.32%), S. marginatum (13.15% to 14.66%), S. ilicifolium (13.24% to 13.88%) and S. wightii (17.33% to 23.25%) due to extraction method, and the algal specie [49].

3.2. Fourier-transform infrared spectroscopy (FTIR) The representative FTIR spectrum was employed to identifythe presence of characteristic functional groups in the FE. The FTIR spectrum (Figure1) exhibited typical polysaccharide absorption bands. An intense band was observed at 3407 cm-1, which corresponds to OH and H2O stretching sugar ring vibration, and another was present at 2921 cm-1, which corresponds to C-6 of the fucose

Journal Pre-proof pyranoid ring [43,50]. Two intense signals were also recorded between 1631 and 1425 cm-1, probably corresponding to uronic acid content [43,50], which would coincide with the uronic acid content recorded in the chemical analysis. A band characteristic of fucoidans (sulfate ester group) was observed at 1228 cm -1, and another occurred at 1027 cm-1, which is characteristic of the glycosidic bond. More interesting was a band at 823 cm-1 indicating the presence of S=O groups in the equatorial position of the C-2 and C-3 of the fucopyranose residue [43,50]. Sulfate groups content and position are reported to be important for their biological

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activities. For example, Chevolot et al. [51] suggested that anticoagulant activity is related to molecular weight, sulfate content, and, of particular interest, sulfation in

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the C-2 and C-3 of fucopyranose residues. Haroum-Bouhedja et al. [51], reported

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that a fucoidan containing >20% sulfate reduced proliferation of hamster kidney fibroblasts (CCL39) and increased anticoagulant activity, whereas decreased

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sulfate content caused the opposite effect.

3.3.Molecular Weights

The molecular weight of the S. fluitans fucoidan was determined from a HPLC

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profile done to compare standards of different molecular weights (see section 2.6). Three peaks with different retention times (Rt) appeared (Figure 2): 5.46 min

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(albumin); 5.72 min (pullulan); and 5.73 min (FE). The FE peak had an estimated

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molecular weight of ~60 kDa, similar to an albumin (66.3 kDa) and a pullulan (50 kDa) standard. These results partially approximate those for a native fucoidan from Ascophyllun nodosum which produced molecular size fractions of two sizes: a very large one (~420 kDa) and a smaller one (~47 kDa) similar to the FE peak identified here [20].However, the authors expressed caution that the higher molecular weight may be no more than an approximation since non-sulfate polysaccharide standards are not available for such high molecular weights. In addition, determining the molecular weights of fucoidans is especially difficult due to their heterogeneous composition [53].

3.4. Induced liver fibrosis in rats

Journal Pre-proof Antioxidant and cytoprotective effects were identified in a previous study of S. fluitans FE [19]. As a continuation of this study we analyzed the FE’s hepatoprotective potential in an in vivo model of chronic liver damage induced with CCl4, which is commonly used to evaluate hepatoprotective potential. During the experimental period body weight was recorded weekly and total weight gain (TWG) and relative liver weight (RLW) calculated (Table S2). As expected, animals in the positive control (CCl4) exhibited a 13.59% decrease in TWG versus the negative control. In contrast TWG increased (versus the positive control) by 7.69% in the

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protection control (CCl4 + silymarin) and 22.58% in the treatment (CCl4 + FE); these TWG values are comparable to that of the negative control. Feeding rats or

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mice fucoidans is known to induce an increase in total body weight. For example, a

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study evaluating the hepatoprotective potential of fucoidan from the brown seaweed Fucus vesiculosus (Sigma Aldrich) in mice intoxicated with ethanol found

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a 3% increase in total body weight versus the negative control (no fucoidan) at a

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dose of 30 mg/kg fucoidan,and a 3.4% increase at 60 mg/kg fucoidan [54]. It remained unclear, however, why fucoidan administration increased total body weight, although it may be related to nutritional support or cell protective effects

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observed in the mice. In another report by the same group administration of fucoidan from F. vesiculosusin mice raised total body weight compared to the

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negative control (no fucoidan) by 11% at 50 mg/kg and 15% at 100 mg/kg [55].

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This kind of weight gain in response to fucoidan intake suggested the possibility of obesity. In response,obesity was induced in mice with a high fat diet and fucoidan co-administered in two treatments. Compared to the obesity control, obesity was reduced by 6% at 50 mg/kg fucoidan and 4% at 100 mg/kg. It was concluded that fucoidan is capable of regulating mRNA gene expression of 3-hydroxy-3methylglutaryl-CoA reductase, an enzyme involved in lipid metabolism [55]. Further research is needed to determine why administration of the FE in the present study caused weight gain. Liver damage as represented by RLW increased 0.45% in the positive control versus the negative control, possibly due to excess deposition of collagen fiber. This increase was largely mitigated in the protection control (0.40% reduction) and

Journal Pre-proof the FE treatment (0.38% reduction), attaining values similar to that of the negative control. Similar results have been reported for fucoidans from F.vesiculosus in a study evaluating hepatoprotective effect in a dimethyl nitrosamine-induced liver damage model in rats [16].

3.5. Sleeping time The liver is the main organ involved in detoxification of myriad drugs, including the common anesthetic sodium pentobarbital. Liver damage reduces cytochrome P450 activity

and

expression,

negatively

affecting

biotransformation

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(CYP)

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pentobarbital and prolonging its effect [56,57]. In the negative control sleep

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latency(the time between pentobarbital administration and loss of the righting

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reflex) was 451.10 sec, considered here to represent 100% sleep latency (Figure 3A). Liver damage induced by CCl4 (i.e. positive control) reduced latency by 33%

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over the negative control. In contrast, the protection control and experimental

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treatment essentially reversed liver damage-induced latency compared to the negative control, lengthening it by 80% in the protection control and nearly 100% in the experimental treatment. Normal (100%) sleep time was 7.64 min in the

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negative control, which increased 383% in the positive control (Figure 3B). This effect was reduced by 33% in the protection control and 60% in the experimental

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treatment. These results indicate that daily oral admiration of FE provided a

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measure of protection against CCl4-induced liver damage. 3.6. Biochemical markers Levels of the liver enzymes ALT, AST, AP and ALB, and TP were employed as biochemical markers ofCCl4-induced chronic liver damage and to corroborate any hepatoprotective effect from FE (Table 2). The positive control increased the activity of ALT 28-fold, AST 2.8-fold and AP 1.4-foldover levels in the negative control. These levels are indicative of liver damage and were considered here as 100% damage. Treatment with FE (experimental treatment) reduced levels of ALT by 58%, AST by 99% and AP by 80%. Administration of silymarin produced similar but slightly lower reductions: 52% for ALT; 89% for AST; and 80% for AP.

Journal Pre-proof The liver is the main source of protein synthesis in the organism and liver damage alters this activity. Total protein (TP) was unaffected in all the treatments (Table 2). Albumin concentration, however, was 12% lower in the positive control versus the negative control. Both the protection control and experimental treatment restored albumin concentrations to levels near that of the negative control. This constitutes yet another indication that relatively low doses of a FE from S. fluitans exercise a hepatoprotective effect against CCl4-induced damage. This coincides with a number of previous studies reporting a hepatoprotective effect

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from seaweed fucoidans. Oral administration of 200 mg/kg fucoidans isolated from S. siliquosum was found to significantly reduce paracetamol-induced increases of

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AST, ALT and AP in rats [58].In another study intake of 100 mg/kg fucoidans from

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F. vesiculosus was found to lower AST, ALT and ALB levels in a CCl4-induced acute liver damage model in rats [59]. Recently, administration of 500 mg/kg

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fucoidans from Kjellmaniella crassifolia was reported to mitigate increases in ALT

3.7. Liver histology

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3.7.1. H&E and MT staining.

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and AST caused by CCl4 [15].

When stained with H&E the negative control exhibited normal structure

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surrounding the central vein, whereas liver tissue from the positive control animals

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had hepatocytes with severe damage to the cytoplasm, increased cell inflammatory infiltration and steatosis (Figure 4). Treatment with silymarin or FE reduced this effect. Staining with MT identified a lack of collagen fiber in the negative control, but increased collagen deposition around the central vein (interstitial fibrosis) in the positive control (Figure 4). Excessive deposition of collagen fiber declined in response to silymarin or FE administration, which coincides with previous reports [16,18]. Quantitative analysis corroborated these results. Treatment with CCl4 increased relative fibrosis 2.65-fold (considered 100% damage) over the negative control, while silymarin reduced this fibrosis by 41% and FE did so by 61%, approaching levels near that of the negative control (Figure 5A).

Journal Pre-proof 3.7.2. Immunohistochemistry. Down regulation of TGF-β1 is indicative of a reduction in liver fibrosis since it is the main protein involved in activation of HSC to a myofibroblastic phenotype, and specializes in production of collagen fiber [16,18]. The immunohistochemistry analysis indicated that TGF-β1 expressed differently in tissues from different treatments (Figure 4). Immunostaining in the positive control was widespread but was significantly reduced by silymarin or FE. Quantitative analysis showed relative expression of TGF-β1 in the positive control to be 2.31 (considered 100%). In

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contrast, treatment with silymarin reduced TGF-β1 expression by 42%, and FE reduced it by 46% (Figure 5B). A similar pattern of reduction of TGF-β1 protein was

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found in an immunohistochemistry study using commercial fucoidans from F.

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vesiculosus (50 mg/kg) to evaluate a CCl4-induced liver damage model [18].

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3.7.3. Gene expression

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Gene expression was evaluated to identify the genes involved in the TGF-β1 signaling pathway, which plays an important role in hepatic fibrosis. In the positive control CCl4 increased mRNA expression of TGF-β1 and related genes involved in

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Smad-2 and Smad-3 pathway signaling. It also decreased liver tissue levels of Smad-7 (responsible for blocking phosphorylation of Smad-3 and regulating the

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TGF-β1 pathway). Daily oral administration of FE lowered mRNA expression of

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TGF-β1 0.48-fold to a value similar to the protection control; this decrease coincides with the immunostaining results. In the FE treatment Smad-2 expression was reduced 0.51-fold and Smad-3 expression 0.35-fold (a level lower than in the protection

control).

Smad-7

expression

increased

1.64-fold

(Figure

6);

overexpression of Smad-7 has been reported to prevent HSC cell activation and liver fibrosis in rats [60]. The FE clearly had the ability to regulate this pathway. Inhibition of the TGF-β1 pathway causes a reduction in expression of fibrogenic genes, so expression of genes related to fibrosis and EMP were measured to quantify inhibition. As expected, the positive control increased expression of the col1α1 (an important component of EMP, found in late pathological changes) and TIMP1 genes, and decreased that of MMP9 and MMP2. The protection control

Journal Pre-proof inhibited the reduction in MMP2 and MMP9 expression induced by CCl4 but had no significant effect on col1α1 and TIMP1. The FE treatment reduced the increases in expression of col1α1 and TIMP1 caused by CCl4 to levels lower than in the protection control. It also increased expression of MMP2 and MMP9, enzymes which promote EMP degradation. Indeed, relative MMP9-gene expression was higher in this treatment than in the protection control (Figure 7). Natural compounds or extracts from terrestrial plant sources have been reported to regulate liver fibrosis by blocking TGF-β1/Smad signaling [10,11,15,61]. An

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example of a natural marine product with antifibrosis activity is a commercial fucoidan from F. vesiculosus (Sigma-Aldrich, St. Louis, MO, USA), which, at 100

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mg/kg, suppresses liver fibrosis via the TGF-β1/Smad pathway and regulation of

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oxidative stress [16]. In a more recent evaluation of the same commercial fucoidan at doses of 10, 25 and 50 mg/kg in a bile duct ligation- and CCl4-induced damage

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model, the 50 mg/kg dose was found to inhibit HSC activation, EMP formation and

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autophagosome formation through down regulation of the TGF-β1/Smads pathway [18].

Analysis of the pro-inflammatory cytokines IL-β1 and TNF-α found that their mRNA

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expression increased in response to CCl4 administration, a response reported previously [62]. This increase is reported to contribute to recruitment and activation

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of the granulocytes found in hepatic inflammation [63], which agrees with the

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increased cell inflammatory infiltration observed in the H&E staining. Treatment with silymarin decreased the expression of these cytokines (Figure 8). Apparently, the FE treatment effectively modulated these cytokines since it reduced the CCl4induced rise in mRNA gene expression 0.84-fold for IL-β1 and 0.79-foldfor TNF-α. Indeed, relative TNF-α gene expression was lower in this treatment than in the protection control, which was reflected in a reduction in the inflammatory infiltration observed in the H&E staining. Similar results have been reported for a fucoidan isolate from F. vesiculosus evaluated in rats with a dimethyl nitrosamine-induced liver damage model [16], and an acetaminophen-induced damage model [17]. To our knowledge this is the first report of potential antifibrotic activity mediated by TGF-β1/Smad at a low dose (50 mg/kg) of a crude fucoidan isolate from the

Journal Pre-proof tropical brown seaweed S. fluitans. This dose is low compared to most studies, although the range of studied crude fucoidans doses is very broad: from 200 mg/kg of S. siliquosum [46] to 75 mg/kg of Turbinaria decurrens [64] and 100, 300 and 500 mg/kg of a fucoidan from Kjellmaniella crassifolia [15].

4. Conclusions The present results confirm the antifibrotic effect of a low dose of fucoidan extract from Sargassum fluitans Borgesen. It exercises this effect by restoring biochemical

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and tissue function, modulating mRNA expression of the TGF-β1/Smads pathway, and of the extracellular matrix proteins and inflammation genes involved in the

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pathology of CCl4-induced liver fibrosis. The studied S. fluitans fucoidan extract is a

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potential ingredient in functional foods aimed at prevention of liver fibrosis.

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Conflicts of Interest

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The authors declare no conflict of interest.

Acknowledgements

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The research reported here was financed by the CONACyT (PDCPN 2014248004) and the Red Temática de Farmoquímicos (294727) through research

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collaboration. Rosa Esther Moo-Puc received a grant from the Fundación IMSS.

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The authors wish to thank CINVESTAV, Unidad Mérida, and Facultad de Química from Universidad Autónoma de Yucatán for access to infrastructure. Particular thanks are due Dr. Yolanda Freile-Pelegrin, Crecencia Chavez-Quintal and Gonzalo Mena-Rejón for their assistance.

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Journal Pre-proof Figure captions Figure 1. Fourier transform infrared spectra (FTIR) of a fucoidan extract from S. fluitans Borgesen. Figure 2. HPLC chromatogram ofa fucoidan extract from S. fluitans Borgesen, compared to albumin and pullulan. Peaks are albumin (66.3 kDa, RT: 5.46 min), pullulan (50 kDa, RT: 5.72 min), and fucoidan fraction (RT: 5.73 min).

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Figure 3. Effect of a fucoidan extract fromS. Fluitans Borgesen on function of cytochrome p450 in the liver. (A) sleep latency and (B) sleep time induced with pentobarbital. All data expressed as the mean ± SD (n=6). One-way ANOVA with a Tukey post hoctest. p-Values:#<0.05 vs. CN;##<0.001vs. CN;*<0.05 vs. CCl4;**<0.001 vs. Silymarin. CCl4: carbon tetrachloride (positive control); Silymarin (protection control); FE (fucoidan extract).

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Figure 4. A fucoidan extract (FE) from S. fluitans Borgesen attenuates CCl4induced liver fibrosis in rats. Rats were treated with CCl4(0.60 mL/kg)+ FE (50 mg/kg)for twelve weeks, killed, and liver tissue samples taken. Liver sections from each rat were fixed in formalin, embedded in paraffin, cut with a microtome, stained with hematoxylin eosin (H&E) or Masson trichrome (MT) stain,or immunohistochemical stain (TGF-β1antibody). A representative view of each group (n=6) is shown. CV: central vein, long arrow indicates presence of inflammatory cells and short arrow indicates steatosis (H&E) and presence of TGF-β1. CCl4: carbon tetrachloride (positive control); Silymarin (protection control); FE (fucoidan extract)

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Figure 5. Effect of a fucoidan extract from S. fluitans Borgesen on reduction of liver fibrosis. A)Quantification of liver fibrosis versus collagen content (Masson trichrome stain); B) Quantification of relative TGF-β1 expression (immunohistochemistry stain). All experiments were run three times and dataare expressed as the means ± SD (n=6) normalized to the negative control. One-way ANOVA followed by the Tukey post-test.p-values: *<0.01 vs. CCl4; **<0.001vs. CCl4. Silymarin. CCl4: carbon tetrachloride (positive control); Silymarin (protection control); FE (fucoidan extract). Figure 6. Effect of a fucoidan extract (FE) from S. Fluitans Borgesen on relative RNA expression of profibrotic genes during liver fibrosis. Rats were treated with CCl4 (0.60 mL/kg) + FE (50 mg/kg) for twelve weeks, killed and liver tissue samples taken. Total mRNA was extracted and real-time RT-PCR with specific TaqMan probes used to measure TGF-β1, Smad-2, Smad-3 and Smad-7 gene expression. All experiments were run three times and data are expressed as the means ± SD (n=6) normalized to the negative control. One-way ANOVA followed by the Tukey post-test. p-values: *<0.05 vs. CCl4; **<0.01vs. CCl4;***<0.001 vs. CCl4;#<0.001 vs. Silymarin. CCl4: carbon tetrachloride (positive control); Silymarin (protection control); FE (fucoidan extract).

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Figure 7. Effect of a fucoidan extract (FE) from S. fluitans Borgesen on relative expression of fibrotic and EMP degradation modulator genes during liver fibrosis. Rats were treated with CCl4 (0.60 mL/kg) + FE (50 mg/kg)for twelve weeks, killed, and liver tissue samples taken. Total mRNA was extracted and real-time RT-PCR with specific TaqMan probes used to measure Col1α1, MMP2, MMP9 and TIMP1 gene expression. All experiments were run three times and data are expressed as the means ± SD (n=6) normalized to the negative control. One-way ANOVA followed by the Tukey post-test.p-values: *<0.05 vs. CCl4; **<0.001; #p<0.01 vs. Silymarin; ##p<0.001 vs. Silymarin: carbon tetrachloride (positive control); Silymarin (protection control); FE (fucoidan extract).

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Figure 8.Effect of a fucoidan extract (FE) from S. fluitans Borgesen on proinflammatory genes during liver fibrosis. Rats were treated with CCl4 (0.60 mL/kg) + FE (50 mg/kg) for twelve weeks, killed, and liver tissue samples taken. Total mRNA was extracted and real-time RT-PCR with specific TaqMan probes used tomeasureIL-β1 and TNF-α gene expression. All experiments were run three times and data are expressed as the means ± SD (n=6) normalized to the negative control. One-way ANOVA followed by the Tukey post-test. p-values: *<0.05 vs. CCl4; **<0.001 vs. CCl4;#p<0.01 vs. Silymarin. Silymarin: carbon tetrachloride (positive control); Silymarin (protection control); FE (fucoidan extract).

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Table 1. Yield and chemical composition (% dry weight ± standard deviation) of a fucoidan extract from S. fluitans Borgesen. Yield

Carbohydrates

Protein

Uronic acids

TPC

Fucoidan content

7.56 ± 1.54

0.32±0.02

1.99 ± 0.15

11.67 ± 0.64

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0.83 25.80±1.56 1.16± 0.008 TPC: Total phenolic content

Sulfate

Journal Pre-proof Table 2. Effect of a fucoidan extract (FE) from S. fluitans Borgesen on serum biochemical parameters in rats with CCl4-induced liver injury. Groups Negative control Positive control (CCl4) Protection control (CCl4+silymarin) Treatment (CCl4+FE)

ALT (U/L) 6.83 ± 13.47

AST (U/L) 93.83 ± 16.12

AP (U/L) 140.33 ± 29.43

TP (g/L) 5.67 ± 0.15

192.50 ± 12.08++

264.83 ± 56.38++

201.17 ± 28+

5.46 ± 0.39

95.83 ± 10.46**

112.23 ± 23.52**

152.67 ± 26.50*

5.50 ± 0.25

82.83 ± 12.25**

94.81 ± 12.92**

127.83 ± 26.51**

5.59 ± 0.27

+

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Results are expressed as mean ± SD (n=6). One-way analysis of variance (ANOVA) with a Tukey post hoc test. negative control and *p<0.05, **p<0.001 vs. positive control. AST: aspartate amine transferase, AST: alanine amine tra phosphatase, TP: total protein, ALB: albumin.

Journal Pre-proof Graphical abstract

Highlights 

Hepatoprotective activity of FE from S. fluitans Borgensen was evaluated.



The molecular weight of the fucoidan extract was approximately ~60 kDa

 Fucoidan extract protect against CCl4- induced liver damage.

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Fucoidan extract modulating mRNA expression of the TGF-β/Smad

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pathway.

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

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8