Hepatoprotective activity against acetaminophen-induced liver dysfunction and GC-MS profiling of a brown algae Sargassum ilicifolium

Drug-induced hepatotoxicity is one of the most important causes of liver dysfunction. Acetaminophen (paracetamol) an analgesic-antipyretic drug is generally considered safe but its overdose may cause liver toxicity. Marine macro-algae (seaweeds) especially brown seaweeds possess unique biological activities including hepatoprotective potential. The current study focused on the hepatoprotective effect of different solvent fractions of Sargassum ilicifolium and characterization of its n-hexane soluble fraction. The ethanol extract (20 g) of S. ilicifolium was mixed with solvents of increasing polarity, starting with n-hexane followed by chloroform and methanol. All three (n-hexane, chloroform and methanol) soluble fractions were administered to the rats at dose of 150 mg/kg, b.w. Intraperitoneal administration of acetaminophen (600 mg/kg b.w.) to rats was used to cause liver injury. The hepatic damage was evaluated by liver markers enzymes; aspartate aminotransferases (AST), alanine aminotransferases (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin along with other metabolites i.e., triglycerides, cholesterol, urea, glucose and creatinine. Lipid peroxidation and glutathione and were estimated in liver tissue. n-Hexane fraction was subjected to GC-MS analysis in order to identify potent compounds. The oral administration of n-hexane and methanol soluble fractions reduced the acetaminophen-augmented liver marker enzymes ALT, AST, ALP, LDH, along with bilirubin, urea, creatinine, glucose and triglycerides. The n-hexane and methanol soluble fractions also improved hepatic antioxidant level via enhancing hepatic glutathione and reversing lipid peroxidation. GC-MS spectroscopy of n-hexane fraction of S. ilicifolium revealed the presence of some new compounds. Among them, fatty acids were found to be in highest concentration followed by halogenated hydrocarbons, benzene derivatives, and sterols. Fatty acid in seaweed may be one of the factors for hepatoprotection from drug-induced hepatotoxicity. From the results, it is evident that n-hexane and methanol soluble fractions of S. ilicifolium have the ability to protect the liver against toxicity, which is comparable with silymarin used as a standard drug. Sargassum ilicifolium contains bioactive compounds with pharmaceutical importance.


Introduction
Acetaminophen (paracetamol) an analgesic-antipyretic drug is generally considered safe but its overdose may affect liver function [1][2][3] and causes severe hepatic necrosis to complete hepatic failure [4]. It also affects other organs and may trigger nephrotoxicity [5]. Since medicines used for managing liver diseases may have potential side effects thus progression of chronic liver diseases have not been prevented effectively with any therapy [6].
Marine macro-algae have shown great bioactivity potential including anti-inflammatory, antimicrobial, antiviral, antitumor, hypoglycemic and hypolipidemic activities [7][8][9], due to the presence of bioactive compounds that may be steroids, terpenoids, isoprenoids and sesquiterpenes [10]. The brown seaweeds, especially Sargassum species, are found in the shallow water of tropical and temperate regions [11][12][13] and contain a considerable amount of these bioactive compounds [14]. S. ilicifolium is one of the most commonly and abundantly found Sargassum species at Karachi coast. Several researchers evaluated the biological activities of S. ilicifolium [15][16][17]. Ambreen et al. [18] reported that S. ilicifolium contains calcium and ascorbic acid and also possesses antifungal activity. Similarly, Rebecca et al. [16] revealed antibacterial activity of ethanol extract of S. ilicifolium. The ethyl acetate fraction of S. ilicifolium has shown a prominent immune-stimulatory effect [15]. In our previous study, ethanol extract of S.ilicifolium (200 mg/kg b.w.) did not show any adverse effect on hepatic and renal function in rats [2,5]. Intraperitoneal administration of hexane, methanol or butanol extract of S. wightii did not cause mortality or showed toxic effect in mice [19]. The current report described the hepatoprotective role of solvent fractions and GC-MS profiling of n-hexane fraction of S.ilicifolium.

Collection of seaweed
Sargassum ilicifolium was collected from Buleji beach, Karachi and washed under tap water in the laboratory, dried under shade and grinded to powder in an electric miller. The seaweed powder was stored at room temperature until use. Voucher specimen and herbarium sheet of the seaweed was prepared and kept in the Seaweed Herbarium (KUH-SW-891), MAH Qadri Biological Research Center, University of Karachi for record. Dr. Aisha Begum, Associate Professor, Department of Botany, University of Karachi, Karachi identified the seaweed.
Ethanol extract and solvent fractions of S. ilicifolium Ethanol extract of S. ilicifolium was prepared by soaking (500 g), for 1 week in 2 L ethanol at room temperature. Then filtered over cotton wool and concentrated on a rotary vacuum evaporator (Buchi R-200 New Castle, DE) at 35°C to obtain a gummy mass that gives the yield of 4.8%. A portion (20 g) of ethanol extract of seaweeds was mixed with n-hexane (250 mL) in a separating funnel. The n-hexane soluble portion was separated and concentrated on a rotary vacuum evaporator at 35°C, that give an yield of 2.5%. The n-hexane insoluble residue was extracted with chloroform, that give an yield of 6.5%. The chloroform insoluble or remaining portion was mixed with methanol (91%). All three (n-hexane, chloroform and methanol) soluble fractions were stored in airtight vials at room temperature till used [20].

Animals
Male rats (140-170 g) of Wistar strain, obtained from Dow University of Health Sciences, Karachi were used in this study. They were kept in polyethylene cages, fed with standard pellet diet [21] and water. Animals were kept in the laboratory for 1 week to allow the animals to acclimatize with laboratory conditions. Experiments were conducted with the permission of Institutional Animal Research Ethical Committee.

Induction of hepatotoxicity
Acetaminophen (Sigma Aldrich, U.S.A.) was administered intraperitoneally at 600 mg/kg, body weight (b.w.) and the dose was prepared in 40% polyethylene glycol (Sigma Aldrich, U.S.A.) with constant stirring and mild heating.

Experimental design
Rats were kept in pre bedded polyethylene cages (6 rats/ cage) with standard laboratory conditions (temperature 25 ± 2°C and 12 h light/dark cycle), fed with standard pellet diet prepared in the laboratory following the procedure of NRC (1995) [21] and tap water ad libitum. The animals were kept in the laboratory for 1 week before starting the experiment to acclimatize animals with laboratory conditions. Experiments were conducted with the permission of the Institutional Animal Research Ethical Committee.

Effect of solvent fractions of S. ilicifolium
Rats were divided into six major groups and each group consisted of 6 rats to determine the effect of seaweed fractions in acetaminophen intoxicated rats.

Normal control
Rats were orally administered with distilled water (1 ml/ kg b.w.) for 14 days along with the normal diet (n = 6).

Sargassum ilicifolium fractions treated group
This group was further divided into 3 subgroups viz.; I, II, III (n = 6 in each sub-group) and rats were administered orally (p.o.) by solvent fractions (hexane, chloroform and methanol separately) of ethanol extract of the S. ilicifolium @ 150 mg/kg b.w., dissolved in water, daily for 14 days.

Acetaminophen (AAP) control group
Rats were administered with distilled water (p.o., 1 ml/kg b.w.) for 14 days along with the normal diet. On day 14 rats were intoxicated by a single intraperitoneal injection of acetaminophen (i.p., 600 mg/kg b.w., in saline).

Sargassum ilicifolium fractions + acetaminophen treated group
This group was further divided into 3 subgroups viz.; I, II, III. Each group (n = 6) was administered orally with n-hexane, chloroform and methanol fractions of S. ilicifolium at the dose of 150 mg/kg b.w., daily for 14 days. On day 14th rats were intoxicated by a single intraperitoneal injection of acetaminophen (i.p., 600 mg/kg b.w., in saline) in each subgroup.

Silymarin treated group
Treated with silymarin at the dose of 50 mg/kg b.w., suspended in distilled water and given orally to rats daily for 14 days.

Silymarin treated group + acetaminophen treated group
Treated with silymarin at the dose of 50 mg/kg daily for 14 days and injected with AAP on the last day.
On the 15th day all animals were decapitated after 12 h fasting and blood was collected for assessment of liver marker enzymes and other biochemical parameters.

Assessment of hepatic damage
Liver enzymes ALP, ALT, LDH, AST and metabolites bilirubin, creatinine, urea, triglycerides, cholesterol and glucose in serum were determined by using kits from Merck (France) and Ecoline (Germany). Glutathione and lipid peroxidation (MDA) in liver tissue was determined according to the method of Samarth et al. [22] and Ohkawa et al. [23] respectively.
Characterization of n-hexane fraction of S. ilicifolium by gas chromatography and mass spectrometry (GC-MS) n-Hexane fraction was subjected to GC-MS analysis on Agilent 6890 Gas Chromatograph hyphenated with Mass Spectrometer, Jeol, JMS-600H. The peak of each compound was identified by comparing their retention indices and mass spectra against National Institute of Standards and Technology (mainlib) USA and compared with Science finder [24].

Statistical analysis
Data were analyzed and their means were compared at a significant level (p < 0.05) using Duncan's multiple range test [25].

Effect on serum biochemical markers
Acetaminophen intoxication significantly (p < 0.005) raised liver function markers like ALP, ALT, LDH, AST and bilirubin level in serum. n-Hexane fraction of S. ilicifolium showed improvement as evident by considerable decline of serum AST, ALT, ALP, LDH and bilirubin levels. The methanol soluble fraction also significantly (p < 0.005) reversed the increased level of ALP, ALT, LDH, AST and bilirubin. Chloroform soluble fraction was found less effective. The acetaminophen also affected kidney function and increased creatinine and urea many fold as compared to control. The creatinine and urea were decreased significantly in n-hexane and methanol fraction in comparison with AAP control. The glucose concentration was reached to (169.6) after acetaminophen intoxication and it was reduced up to (− 22%) and (− 30.4%) in n-hexane and methanol fractions pretreated rats respectively. The cholesterol level was restored towards normal value in rats pretreated with nhexane fraction (+ 32.5%) of S. ilicifolium (Tables 1 & 2).
Administration of chloroform soluble fraction of S. ilicifolium to normal rats for 14 days did not influence the liver enzymes except for AST and LDH, while other liver metabolites like bilirubin, creatinine and urea remain unchanged when compared with normal rats. Similarly, n-hexane fraction did not alter the ALT, ALP, glucose level and other biochemical markers. The rats pretreated with a methanol fraction of S. ilicifolium also not shown any adverse effect on enzymes, but reduced glucose level than control rats. Creatinine, cholesterol and triglyceride level were found increased by the methanol fraction of S. ilicifolium (Tables 1 & 2).
Administration of silymarin in AAP intoxicated rats significantly decreased the liver enzymes and bilirubin level as compared to AAP intoxicated rats. On the other side, kidney profile; creatinine and urea, were also reduced in silymarin + AAP treated group as compared to AAP control rats. Similarly, silymarin in AAP intoxicated rats also showed protective effect by lowering the glucose level and lipid parameters (cholesterol and triglyceride) as compared to AAP control group (Tables 3 & 4).

Effect on solvent fractions of S. ilicifolium on glutathione (GSH) and lipid peroxidation (TBARS)
Glutathione (GSH) level was significantly (p < 0.05) decreased in acetaminophen (AAP) treated rats as compared to normal control rats. However, glutathione level was improved and increased in rats pretreated with nhexane, methanol and chloroform fractions of S. ilicifolium in acetaminophen intoxicated rats as compared to acetaminophen (AAP) control group. Methanol fraction improved this level and brought almost to the normal control rats, while n-hexane fraction increased this level higher than the normal control ( Fig. 1).
AAP intoxicated rats significantly elevated TBARS [6.16 μmole/gm wet liver tissue (w.l.t)] level as compared to normal control (2.4 μmole/gm). n-hexane (3.19μmole/gm) and methanol (4.1μmole/gm) fraction of S. ilicifolium significantly reduced TBARS level as compared to AAP intoxicated rats (Fig. 1). The overall highest hepatoprotective activity was found in n-hexane soluble fraction of S. ilicifolium followed by methanol fraction. The n-hexane fraction was thus characterized on GC-MS considering its hepatoprotective activity.
Glutathione level was significantly improved and TBARS level decreased in rats treated with silymarin (50 mg/kg b.w.) in acetaminophen intoxicated rats as  compared to acetaminophen (AAP) control group (Fig. 2).
GC/MS profiling of n-hexane fraction of S. ilicifolium The GC/MS characterization of n-hexane soluble portion of S. ilicifolium revealed the presence of different volatile compounds like normal hydrocarbon, alcohols, fatty acid, aliphatic compounds, benzene derivatives, aldehyde and terpenoid. Total fifty one compounds were isolated and identified in which forty six compounds were found new from this source ( Table 5). According to the results the highest concentration of hexadecanoic acid was found in our extract followed by octadecenoic acid. However the hexadecanoic is already a known compound from this source while octadecenoic acid is a new compound from S. ilicifolium. Our results showed the presence of steroids; spiro (1, 3-dioxolane)-2, 3′-(5′androsten-16′-ol) and estra-1, 3, 5(10)-trien-17β-ol in S. ilicifolium (Table 5

Discussion
In this study, pretreatment with n-hexane fractions of S. ilicifolium significantly decreased the toxin (acetaminophen) induced raised in serum transaminases, alkaline phosphatase, lactate dehydrogenase and bilirubin level. The methanol soluble fraction also reversed the increased level of ALT, AST, ALP and LDH but to a lower extent than n-hexane fraction. Raghavendran et al. [26] and Raghavendran and Srinivasan [27] have reported the protective role of ethanol and water extracts of S. polycystum against AAP induced hepatic damage. The drug induced hepatotoxicity may trigger nephrotoxicity as well [5]. An increase in urea and creatinine concentration in serum is indicative of nephrotoxicity [28]. In this study, adverse effect of acetaminophen was also found in AAP dosed rats, which was attenuated in rats pretreated with n-hexane fraction of S.ilicifolium evident from decreased concentration of urea and creatinine. Hepatotoxicity may affect lipid and glucose metabolism. In our study, n-hexane and methanol fractions of S. ilicifolium lowered the raised triglycerides level and improved decreased level of cholesterol in AAP dosed rats. Taj et al. [29] reported attenuation of adverse effect of AAP on liver and kidney function and glucose metabolism by the ethanol extract of a brown alga Stokeyia indica. Similarly Sohail et al. [5] reported the hepatoprotective and nephroprotective role of ethanol extract of S.ilicifolium in drug induced hepatotoxicity and nephrotoxicity in Table 3 Effect of silymarin at dose of 50 mg/kg b.w., on liver enzymes and bilirubin in normal and acetaminophen (AAP) dosed rats      (17)  rats. The present findings further investigated and confirmed the hepatoprotective and nephroprotective role of n-hexane fraction of S.ilicifolium. In the biological system oxidative stress may result in increased lipid peroxidation thus indicating cellular damage [30]. Excess production of free radicals may damage cell membranes and lipoproteins by a process called lipid peroxidation, resulting in the production of malondialdehyde (MDA). MDA is an end product of membrane damage, can bind with thiobarbituric acid, hence also called thiobarbituric acid reactive substances (TBARS). Glutathione can help in reducing the free radicals [31]. A decrease in GSH and increase in MDA is considered as a sign of liver dysfunction [3]. Reduction in GSH level of kidneys and lungs was reported by Taye and Abdel-Raheem [32], along with CCl 4 intoxication. Ohta et al. [33] also reported GSH reduction in various organs of rats injected with CCl 4 . In this study glutathione (GSH) was improved and TBARS (MDA) was decreased in rats pretreated with n-hexane and methanol fractions of S. ilicifolium in AAP intoxicated rats as compared to AAP control group.

Rat groups ALT (U/L) AST (U/L) ALP (U/L) LDH (U/L) Bilirubin (mg/dl)
The hepatoprotective activity of different phytoconstituents like flavonoides, triterpenes, saponins and alkaloids and fatty acids has been reported earlier [31,34]. In present study GC-MS analysis of n-hexane extract showed that hexadecanoic acid was found in highest concentration followed by cis-13-octadecenoic acid, hexanoic acid 2-ethyl-oxybis (2,1-ethanediyloxy-2,1ethanediyl) ester, 6-octadecenoic acid respectively. The fatty acids related compounds present in Sargassum fulvellum and S. thunbergii possess anti-inflammatory activities, as these are competitive inhibitors of cyclooxygenase and/or lipoxygenase, hence decrease the production of prostaglandins and leukotrienes [35,36]. Biological activities of fatty acids from a brown alga Spatoglossum asperum has been reported earlier [37]. Hepatoprotective potential of S. ilicifolium may be due to presence of fatty acids that have been reported to ameliorate liver function enzymes in rats, which ultimately lead to reduced liver necrosis and inflammations [29,38]. The n-hexadecanoic acid is known as an antiinflammatory compound [34]. Seaweeds also contain diterpenes, triterpenes and halogenated compounds with diverse biological activities as antibacterial, antioxidant, insecticidal and cytotoxic activities [39][40][41]. In this study, besides fatty acids, hydrocarbons, alcohols, aliphatic compounds, benzene derivatives, aldehyde and terpenoid were also found in n-hexane fraction of S.ilicifolium. Hepatoprotective activity of S.ilicifolium might be due to the presence of individual compounds or combinations of more than one compound. Like other natural products, presumably, seaweed acts as an antioxidant agent, increasing intracellular concentration Fig. 3 Compounds identified from n-hexane soluble fraction of Sargassum ilicifolium of glutathione [42,43]. It may enhance protein synthesis and regeneration of liver cells [44]. Polysaccharide from Sargassum sp., has been reported to decrease the MDA and increased glutathione in acetaminophen intoxicated rats [3].

Conclusion
Present study described that n-hexane and methanol soluble fraction of a brown seaweed S. ilicifolium, exhibited hepatoprotective activity via reducing liver marker enzymes and enhancing hepatic antioxidant level. Characterization of n-hexane soluble fraction of S. ilicifolium confirmed the presence of different volatile compounds, in which fatty acids were found to be in highest concentration followed by halogenated hydrocarbons, fatty acid derivatives and sterols. Further investigation is needed to examine the hepatoprotective effect of individual compounds identified from n-hexane soluble fraction of S. ilicifolium.