Protective effect of crude sulphated polysaccharides from Sargassum Swartzii (Turn.) C.Ag. against acetaminophen induced liver toxicity in rats
Polysaccharides from seaweeds have been reported to possess biological activities with potential medicinal value. Present study was aimed to investigate hepatoprotective effect of crude sulphated polysaccharides extracted from Sargassum swartzii against acetaminophen-induced liver injury.
The polysaccharides from S. swartzii was extracted at room temperature and at 70 °C and named as EW1 and EW2. These fraction was given orally to rats at 200 mg/kg body weight. Liver injury was induced by single intraperitoneal injection of acetaminophen. Hepatic marker enzymes; alanine aminotransferases (ALT), aspartate aminotransferases (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and other biochemical parameters; glucose, triglycerides, cholesterol, urea and creatinine were estimated in serum, while hepatic glutathione (GSH) and lipid peroxidation were measured in liver tissue. Histopathology of liver tissues was also carried out.
Treatment with polysaccharides EW1 & EW2 fractions significantly (p < 0.05) reduced the hepatic marker enzymes and other biochemical parameters along with increased GSH and reduced lipid peroxidation. The EW1 fraction of crude sulphated polysaccharides produced hepatoprotection more or less equivalent to silymarin (35 mg/kg), a commercial herbal drug, while some parameters showed better results than silymarin. These results were further confirmed with histology of liver.
This study suggests that crude polysaccharides of S. swartzii has ability to protect against liver toxicity similar and/or better than silymarin (a standard drug) based on biochemical and histological findings. However toxicological studies would be recommended to evaluate any toxic effect of Sargassum swartzii.
KeywordsSargassum swartzii Sulphated polysaccharides Hepatoprotective Acetaminophen Liver injury Silymarin
Polysaccharides extracted at room temperature
Polysaccharides extracted at 70 °C
- H & E
Hematoxylin and eosin
Wet liver tissue
Liver is the vital organ and major metabolic hub of the body. An inflammation of liver cells is called hepatitis, if not controlled effectively then may leads to chronic liver diseases [1, 2]. A number of drugs have been used to treat liver diseases but their successful use in treating liver diseases is very small, because the drugs used for treating the liver diseases may have side effects . The therapies based on natural substances are believed to be safer and better than drugs used in medical practices .
The marine environment is believed to be an incomparable reservoir of bioactive natural products, many of which exhibited structural features that have not been found in terrestrial plants . In marine environment algae are of two types, microalgae and macroalgae . Records showed that macroalgae (seaweeds) have been consumed in human diets particularly as a vegetable in Asia, since 600 BC . The Chinese used algae and seaweeds for medicinal purposes as early as 300 BC . The presence of reactive antioxidants (ascorbate and glutathione) along with carotenoides (α- and β-carotene, fucoxanthin, astaxanthin), mycosporine-like amino acids (mycosporine-glycine) and catechins (e.g., catechin, epigallocatechin), gallate, phlorotannins (e.g., phloroglucinol), eckol and tocopherols was reported in seaweeds .
Phytochemicals from marine macroalgae are found to be effective non-toxic substances with wide variety of pharmacological activities [10, 11]. Seaweeds possess biological activities, such as cholesterol lowering, immunomodulatory, anti-hepatotoxic . The polysaccharide from seaweeds specially agaro-oligosaccharides not only prevents the oxidative injury but also boosts host antioxidant enzyme system . Sulphated polysaccharides from Laminaria japonica are able to repair damaged or hypofunctional organs and tissues by enhancing the production of immunoregulatory and proinflammatory cytokines . Among different polysaccharides derived from brown and red seaweeds, sulphated polysaccharides (fucoidan) and lambda carrageenan exhibit hepatoprotective, antioxidant and free radical scavenging activity that might be due to their sulfur content  and molecular weight [16, 17]. Seaweed, polysaccharide can be used for the development of new drugs due to its many biological activities . The present study, further investigates the hepatoprotective effect of sulphated polysaccharides from Sargassum swartzii against acetaminophen (AAP) induced liver damage and associated complication.
Materials and methods
Collection of seaweeds
Seaweeds were collected at low tide from Buleji beach, Karachi coast. These seaweed species were washed thoroughly in order to remove salts, debris and epiphytes. Voucher specimens and herbarium sheets of the seaweeds were prepared and record (KUH-SW) was kept in the Seaweed Herbarium, MAH Qadri Biological Research Center, University of Karachi. Professor Dr. Mustafa Shameel (Department of Botany, University of Karachi) identified the seaweeds. Seaweeds were dried under shade, grinded and fine powder was used for experimental purpose.
Extraction of polysaccharides
The extraction of polysaccharides from seaweeds was carried out by slightly modified method of Ponce et al. . The coarse powder of dry seaweeds (100 g) was extracted with 500 ml of 80% ethanol first at room temperature and then at 70 °C with constant stirring for 24 h in order to remove proteins, peptides, amino acids, vitamins, polyphenols, mannitols and other monosaccharides. The liquid portion was centrifuged off and residual seaweed was extracted with 250 ml of water at room temperature and at 70 °C for 7 h with constant stirring and labeled as EW1 and EW2 fractions of crude sulphated polysaccharides respectively. The EW1 and EW2 fractions of crude sulphated polysaccharides were dried on Eyela FD-1, lyophilizer and stored at − 20 °C.
Healthy male albino rats of Wistar strain (140–170 g) were purchased from Dow University of Health Sciences, Karachi. All the animals were housed in prebedded polyethylene cages (3 rats/cage and 6 rats were used per group) with standard laboratory conditions (temperature 25 ± 2 °C and 12 h light/dark cycle), fed with standard pellet diet and water ad libitum. The animals were kept in the laboratory for 1 week before starting the experiment to acclimatize animals with laboratory conditions.
Induction of hepatotoxicity
Acetaminophen (Sigma Aldrich, U.S.A.) was administered intraperitonealy @ 1 g/kg, body weight and the dose was prepared in 40% polyethylene glycol (Sigma Aldrich, U.S.A.) with constant stirring and mild heating.
Group 1 received the distilled water daily for 14 days and served as normal control. Group 2 rats received distilled water daily for 14 days but injected with acetaminophen, 24 h prior to decapitation and served as AAP control. Group 3 orally administered with reference drug, sliymarin (Sigma Aldrich, U.S.A.) @ 35 mg/kg b.w. daily for 14 days. Group 4 and 5 received the EW1 and EW2 polysaccharide fractions of S. swartzii respectively (200 mg/kg b.w.) daily for 14 days. On last day, all the rats from group 2, 3, 4 and 5 received single intraperitoneal injection of acetaminophen. All the animals were fasted overnight and decapitated after 24 h of AAP injection. Normal control rats were injected with vehicle, 1 day before to decapitation.
Assessment of hepatic damage
Serum was separated from blood by centrifugation and used for the determination of alanine aminotransferases (ALT), aspartate aminotransferases (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), urea, creatinine, bilirubin, triglycerides, cholesterol and glucose using kits from Merck (France) and Ecoline (Germany). The liver tissue was excised immediately after decapitation and washed with ice cold normal saline. Homogenate (10%) was prepared by homogenizing liver tissue with Tris HCl buffer (pH 7.4). Freshly prepared homogenate was used for the estimation of glutathione and lipid peroxidation.
Estimation of hepatic glutathione
Reduced glutathione was estimated according to the method of Samarth et al.  with slight modifications. Homogenate (0.1 ml) was mixed with 100 μl of 25% trichloroacetic acid (TCA) and kept for 5 min at room temperature in order to precipitate protein. The mixture was centrifuged at 3000 rpm for 10 min. 0.2 ml of the supernatant was mixed with 1.8 ml of 0.1 mM DTNB (5,5′-Dithiobis-2-nitrobenzoic acid; in 0.3 M sodium phosphate buffer pH 8). The tubes were incubated for 10 min and absorbance was recorded against reagent blank at 412 nm using UV-visible spectrophotometer (HeλIOS α, Thermo Spectronic, UK). Mixture of all reagents except sample served as reagent blank.
Estimation of lipid peroxidation
The extent of lipid peroxidation was determined by quantifying the concentration of malondialdehyde (MDA) as described by Ohkawa et al. . The tissue homogenate (0.1 ml) was mixed with 0.1 ml of 8.1% SDS and incubated for 10 min at room temperature. 750 μl of 20% acetic acid (pH 3.5) and 750 μl of 0.8% thiobarbituric acid (TBA) were added and volume was adjusted to 2 ml with water. The tubes were placed in water bath at 95 °C for 60 min and cooled under tap water. The final volume was adjusted to 2.5 ml with distilled water. The same volume of butanol and pyridine (1:1) mixture was added in each tube, vortex and centrifuged at 4000 rpm for 10 min. The upper organic layer was removed and absorbance was recorded against reagent blank at 532 nm using UV-visible spectrophotometer.
A small portion of liver tissue was fixed in formalin (5%) and kept in laboratory for 48 h. Tissue was embedded in paraffine in order to cut sections of 3 to 4 μm thick and stained with hematoxylin and eosin (H & E) staining solutions. The extent of damage was assessed by using microscope (Nikon FX-35A, Japan) at 100X and 200X.
Data was analyzed and subjected to Analysis of Variance (ANOVA) and means were separated using Duncan’s multiple range test (DMRT) according to Armitage & Berry . P-value < 0.05 was considered as a level of significance.
Effect of EW1 & EW2 fraction of S. swartzii on serum markers in acetaminophen intoxicated rats
Effect of Crude Sulphated Polysaccharides of Sargassum swartzii on Liver Enzymes and Bilirubin in Acetaminophen-Intoxicated Rats
27d ± 1.7
88d ± 8
45c ± 6.2
238.6d ± 10.2
0.56c ± 0.051
Acetaminophen (AAP) control
64a ± 7.8
187.6a ± 9.29
124a ± 4.3
542.6a ± 17.6
0.93a ± 0.054
Silymarin (35 mg/kg) + AAP
37.3bc ± 4.5
139bc ± 8.88
68.3b ± 7.5
375b ± 17.5
0.76b ± 0.056
S. swartzii EW1 + AAP
33.3cd ± 2.5
121c ± 12.5
47.6c ± 1.5
318c ± 10.5
0.76b ± 0.06
S. swartzii EW2 + AAP
45b ± 4
154b ± 14
60.3b ± 0.57
375b ± 17.5
0.73b ± 0.052
Effect of Crude Sulphated Polysaccharides of Sargassum swartzii on Glucose, Urea, Creatinine, Triglycerides and Cholesterol in Acetaminophen-Intoxicated Rats
108.6c ± 3.5
84b ± 5.2
84.6a ± 2.3
30d ± 1
0.9c ± 0.1
Acetaminophen (AAP) control
162a ± 6.5
167.3a ± 10.06
64.6bc ± 5.8
64a ± 6.2
1.23a ± 0.11
Silymarin (35 mg/kg) + AAP
118.3bc ± 8.6
92b ± 23.06
68.6b ± 4.04
35cd ± 2.6
1.03bc ± 0.057
S. swartzii EW1 + AAP
127b ± 7.5
108b ± 17.08
65ac ± 3.6
38.6bc ± 2.5
1bc ± 0.1
S. swartzii EW2 + AAP
169a ± 8
106b ± 13.11
58.3c ± 3.7
43.6b ± 2.08
1.1ab ± 0
Effect of EW1 & EW2 fraction on glutathione (GSH) and lipid peroxidation
Administration of AAP induced significant increase in lipid peroxidation as observed in high concentration of TBARS as compared with normal control rats. However significant decline in TBARS level was found in rats pretreated with EW1 and EW2 fraction of S. swartzii following the acetaminophen intoxication. But good protective effects were observed in EW1 fraction of S. swartzii followed by EW2 fraction of S. swartzii (Fig. 1). The crude sulphated polysaccharides (EW1) fraction of S. swartzii restored hepatic glutathione level and reduced hepatic lipid peroxidation almost near to the standard drug (silymarin).
Effect of crude Sulphated polysaccharides fractions (EW1 & EW2) on hepatic histology
Presence of polysaccharides in seaweeds has been reported for various biological activities including cholesterol lowering, immunomodulatory, In vitro and In vivo anti-hepatotoxic activity [23, 24] and antiproliferative activity . Seaweeds contain commercially important polysaccharides, named as agar, fucoidan, alginate, carrageenan, laminaran and sulphated galactan . In the present study intoxication of acetaminophen (AAP) in experimental rats caused liver damage as evident from the increase in the level of hepatic enzymes; AST, ALT, ALP and LDH along with increase in serum bilirubin, glucose, triglycerides, urea and creatinine. It is well established that when the liver is injured as a result of introduction of infectious agents or chemicals, the serum levels of GPT (ALT) and GOT (AST) are raised significantly , along with leakage of ALP and LDH  reflecting the severity of liver injury . In present study the treatment with both fractions of crude sulphated polysaccharides, EW1 and EW2 fraction of Sargassum swartzii significantly (p < 0.05) reduced the elevated level of ALT, AST, ALP, and LDH and all other biochemical parameters. The highest activity was found in EW1 fraction of S. swartzii. All parameters including liver marker enzymes, bilirubin, triglycerides, cholesterol and urea were statistically equal in both silymarin and EW1 fraction of S. swartzii in acetaminophen-induced liver injury. Silymarin regenerates liver tissues, help in protein synthesis, reverses glutathione fall in tissue, enhanced glucuronidation and reduced inflammation, hence it acts as antioxidant, antifibrotic, anti-inflammatory, membrane stabilization and immunomodulatory agent . In current study the crude sulphated polysaccharides (EW1) extracted at room temperature showed its maximum protective effect might be due to its antioxidant properties and presence of higher concentration of sulphated polysaccharide as reported by Ponce et al.  in a brown seaweed Adenocystis utricularis. They have reported 30% sulphate in EW1 as compared to 8% in EW2. Vijayabaskar et al.  reported antioxidant activity of Sargassum swartzii due to presence of 10.43% crude sulfated polysaccharide in seaweed. The hepatoprotective effect of crude sulphated polysaccharides have also been reported by other researchers [31, 32, 33]. The sulphated polysaccharides from Sargassum polycystum showed protective effect against paracetamol induced toxic hepatitis in rats . Ross et al.  also reported that sulphated polysaccharides from Sargassum siliquosum produced hepatoprotective activity against acetaminophen induced liver damage. Sulphated polysaccharides have ability to inhibit viral replication against HIV, hepatitis and herpes viruses , to stimulate immune system  and to protect against hepatocellular carcinomas , may be due to the prevention of increased pro-inflammatory cytokines, IL-6 and IL-12 .
Glutathione is most abundant tripeptide endogenous antioxidant present in liver. It protects liver by binding with reactive metabolites formed by AAP, thus preventing their covalent binding with liver proteins. Glutathione helps in scavenging free radical species and maintenance of membrane protein thiols . Taye & Abdel-Raheem,  reported significant reduction in GSH level of kidney and lungs tissues after CCl4 intoxication. Other researcher also proved the significant decrease in the GSH content in different organs of rats when injected with CCl4 . The rats pretreated with crude polysaccharides (EW1 and EW2 fraction) of S. swartzii significantly ameliorated the acetaminophen-induced reduction in glutathione level. The glutathione status of body was significantly improved and lipid peroxidation was reduced in rats pretreated with crude polysaccharides extracted at room temperature (EW1 fraction) when compared to silymarin (35 mg/kg) pretreated rats intoxicated with acetaminophen. The polysaccharides present in seaweeds prevent the elevation of pro-inflammatory cytokines IL-6 and IL-12  and showed hepatoprotective effect [17, 31]. The sulphated polysaccharides from S. wightii restored the disturbed level of hepatic enzymes, antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase), non-enzymatic antioxidants (glutathione, vitamin C and E), lipid peroxidation, protein carbonyls and ATPases . Kawano et al. [39, 40] reported that dietary fiber from Laminaria sp., Sargassum fulvellum and Eisenia bicyclis have ability to suppress D-galactosamine (D-GalN)-induced liver malfunction, might be due to the presence of fucoidan. The crude sulphated polysaccharides from Laminaria japonica and Ulva lactuca have ability to improve liver function by scavenging free radicals [41, 42, 43].
The intraperitoneal administration of acetaminophen caused marked hepatic necrosis with fatty changes, infiltration of polymorphonuclear cells, abnormal central vein and sinusoidal morphological pattern. Crude sulphated polysaccharides from S. swartzii (EW1 fraction) showed considerable reduction in pathological changes that produced after acetaminophen administration. The sulphated polysaccharides from S. swartzii extracted at room temperature (EW1 fraction) prevented hepatic necrosis similar to the rats pretreated with silymarin (35 mg/kg) in acetaminophen-intoxicated rats. Raghavendran & Srinivasan , reported that a number of necrotic cells were depleted in rats co-treated with sulphated polysaccharides and acetaminophen. This might be due to the presence of high sulfate content in seaweed polysaccharides, which inhibit oxidative stress . Ross et al.  reported that administration of sulphated polysaccharides from Sargassum siliquosum produced mild hepatocellular degeneration suggesting their protective role. The sulphated polysaccharides from Sargassum wightii showed considerable reduction in the pathological changes produced after cyclosporine A-induced liver injury animals . This protective action may possibly be due to its effect of preserving the cellular membrane of the hepatocytes from breakage by the reactive metabolites . The sulphated polysaccharides extracted at room temperature and at 70 °C had completely different chemical compositions .
Nature based treatment of liver diseases has gained tremendous attention worldwide in past few decades. Seaweeds have characteristic of having unique polysaccharides in their cell walls that are not found in land plants. Current study has focused on the extraction of polysaccharides and their hepatoprotective role as polysaccharides are assumed to be good antioxidants. Results of present study showed that polysaccharides extracted at room temperature (EW1 fraction) from Sargassum swartzii have reduced the acetaminophen attenuated disturbed liver marker enzymes and other biochemical parameters, supported by histopathological findings. The polysaccharides from Sargassum swartzii also increased the antioxidant status of body. This study suggests that crude polysaccharide of S. swartzii has ability to protect against liver toxicity similar and/or better than silymarin (a standard drug). However toxicological studies would be recommended to evaluate any toxic effect of Sargassum swartzii.
They are also thankful to the Dr. Aisha Begum, Assistant professor, Department of Botany for their kind help in identification of seaweed.
Authors are thankful to the Dean, Faculty of Science, University of Karachi for fincial assistance.
Availability of data and materials
Research data (Lab notebook) and materials can be provided on request.
KH & VS conceived and designed the study. KH performed experimental work. KH & SE analyzed and interpreted the results. NK supervised histo-pathological work. KH wrote the manuscript. KH, SE & JA improved the manuscript. All authors read and approved the final manuscript.
The experiment was conducted according to the rules of Institutional Animal Ethics Committee (IAEC), University of Karachi.
Consent for publication
The authors declare that they have no competing interests.
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