Background

Certain medicinal agents can cause injury to the different organs, either used as directed or when taken in high doses [1]. Hepatotoxicity may induce by herbal products, chemical agents (chemicals used in industries and laboratories) and natural chemicals like microcystins. More than 900 drugs have been linked to cause liver injury and it is the most common reason for a drug to be withdrawn from the market [2, 3].

Acetaminophen is a widely used analgesic-antipyretic drug known to cause hepatotoxicity when overdosed [4]. Conversely, an unintentional or purposely overdose frequently causes acute liver failure [5]. Once the liver became injured, its efficient treatment with drug like glycyrrhizin is limited [6]. Consequently, interest in the utilization of alternative medicines for the treatment of hepatic disease has been increased [7, 8]. Seaweeds are also prosperous in bioactive compounds with antiviral, antioxidant, anti-inflammatory, antimicrobial and antitumor activities [9,10,11]. Marine macro-algae (seaweeds), despite their abundance, are poorly exploited, even though their total lipid content is usually low [12], but contain a high amount of polyunsaturated fatty acids (PUFA). The fatty acids have various biological activities [13,14,15].

Stokeyia indica is a brown alga, found abundantly at Karachi coast [16], possess various biological activities including cytotoxic [17, 18], hypolipidemic [19] and hypoglycemic [20]. In our previous studies, ethanol extract and polysaccharides fractions of S. indica showed strong antioxidant activity [10, 11]. In the present study we have evaluated its hepatoprotective role against damaged induced by acetaminophen and associated complications. Two oily fractions eluted from the ethanol extract of S. indica through column chromatography were characterized by using GC-MS.

Methods

Chemicals

Kits for the estimation of biochemical parameter including glucose, cholesterol, triglycerides, urea, creatinine, total-bilirubin, direct-bilirubin and liver enzymes (ALP, ASAT, LDH & ALAT) were purchased from Merck Limited, France, and Ecoline, Germany. Acetaminophen was purchased from Sigma Aldrich, U.S.A. In this study, other reagents were used also of analytical status.

Seaweed collection and preparation of ethanol extract

The seaweed, Stokeyia indica was collected from Buleji beach of Karachi coast at low tide and was identified. This seaweed was rinsed with water thoroughly in order to remove soil particles and adhered debris. For experimental use seaweed was dried, drudged in fine powder and stored. Stokeyia indica dried powder (500 g) was soaked in 2 l of distilled ethanol at room temperature for 1 week. The filtrate was collected and concentrated on a Buchi R-200 rotary vacuum evaporator at 40 °C. A final concentrated form of the extract was stored in a brown bottle at room temperature.

Experimental design

Male Wistar rats (140-180 g) were acquisitioned from Dow University of Health Sciences, Karachi, Pakistan. Rats were fed with a regular laboratory diet and tap water in animal house. They were separated into four groups, all group consists of six rats. Group I and II rats were orally administered distilled water daily for 14 days. Group III and IV rats pretreated daily for 14 days orally with S. indica ethanol extract (200 mg/kg). Group II and III rats were intraperitoneally injected with acetaminophen (1 g/kg) dissolved in 40% PEG 400 (Polyethylene Glycol 400) in 0.9% NaCl solution on day 14th. Before decapitation rats were fasted for 12 h. On day 15th rats were weighed and decapitated by cervical dislocation. Blood samples were collected and Serums were alienated after centrifugation for 10 min at 3000 rpm and stored at − 20 °C until used. Liver and kidney were rapidly dismembered, rinsed with ice-cold normal saline (0.9%), blotted dry, weighed and saved at − 20 °C for analysis.

Assessment of biochemical parameters

Hepatotoxicity was evaluated by the serum liver enzyme profile (alanine aminotransferases [ALAT), aspartate aminotransferases [ASAT), alkaline phosphatase [ALP), lactate dehydrogenase [LDH) and bilirubin (total and direct). Nephrotoxicity was evaluated by estimating serum kidney enzyme profile (urea, creatinine) and other parameters (glucose, cholesterol, triglycerides) were also estimated. All tests were performed using blood chemistry analyzer (Microlab-300, Merck, France); kits were purchased from Merck (France) and Ecoline (Germany).

Estimation of lipid peroxides and hepatic glutathione

Lipid peroxidation in liver was estimated by the formation of malondialdehyde (MDA). 10% homogenate was prepared by homogenizing liver tissues through 0.15 M Tris -HCl (PH 7.4) with Poly Tron (Kinematic) PT-MR 2100 homogenizer. The homogenates were used for the measurement of thiobarbituric acid reactive substance (TBARS) to evaluate lipid peroxidation [21] and also used for reduced glutathione [22].

Histopathological analysis

For histopathological analyses liver samples were preserved in 5% phosphate-buffered neutral formalin, dehydrated in graded (30–100%) ethanol and fixed in paraffin. Sections were cut down at 3-μm thickness and stained with hematoxylin and eosin stain and then observed under a light microscope at 40x magnification to studied histopathological alteration in liver structural design, and were taken photomicrographs of all samples.

Column chromatography and GC-MS analysis

Ethanol extract of Stokeyia indica was loaded on pre-packed silica gel column (Column filled through silica gel, G 60 in n-hexane). Two fractions (F46 and F47) eluted from 15% methanol in chloroform (15:85) were oily in nature. They were analyzed on Gas Chromatograph (Agilent Model 6890 N) in combination with a Mass Spectrometer (JMS-600 H Jeol). GC temperature planned as of 70 °C to 260 °C with a rate of 5 °C/min. The injection port temperature held at (260 °C) and the mover gas He (helium) was run at the speed of (1 ml/min).

Statistical analysis

Statistical analysis for comparing the data was performed by using IBM SPSS Statistics software, version 20 (IBM Corp., Pakistan). Results are showed as mean ± SD by using the analysis of variance (one-way ANOVA) followed by Duncan’s multiple range test to find out the differences between the averages at (P < 0.05) significance value.

Results

The absolute and relative weight of the liver and kidney were significantly (P > 0.05) elevated by acetaminophen intoxication as compared to normal control group rats. Whereas pretreatment with S.indica ethanol extract before acetaminophen intoxication prevent the negative effect of the toxicant on organ weight (Table 1). Group II rats treated with a single injection of acetaminophen developed hepatic damage as compared to the normal control rats. It was manifest from a distinct increased within the concentration of hepatic and cardiac enzymes (ALAT, ASAT, ALP, and LDH) and other biochemical parameters including bilirubin (total & direct). Acetaminophen intoxication also affects kidney function, glucose, and lipid metabolism by elevating the level of creatinine and urea, glucose and triglyceride and decreasing the total cholesterol (Table 2). Stokeyia indica pretreated group rats showed a significant (P < 0.05) improvement in the level of the altered biochemical parameter as compared to the acetaminophen control rats. Whereas elevated levels of bilirubin, creatinine, urea, glucose, and triglyceride were found decreased towards normal range with increased in total cholesterol. The increased concentration of cardiac and liver enzymes was also found decreased towards normal range in rats pre-treated with seaweed as compared to rats intoxicated with acetaminophen only (Table 2). Stokeyia indica control group rats showed the non-toxic effect on liver enzymes and biochemical markers.

Table 1 Absolute weights (g) and relative weights (%) of liver and kidney tissues in different groups of rats
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Table 2 Effect of Stokeyia indica ethanol extract on serum biochemical parameters in normal and acetaminophen intoxicated rats
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Acetaminophen administration increased the concentration of MDA and decreased glutathione concentration. Stokeyia indica pre-treated rats alleviated these changes via decreasing the concentration of MDA and escalating the concentration of reduced glutathione (Table 3).

Table 3 Effect of Stokeyia indica on hepatic malondialdehyde (MDA) and glutathione concentration in acetaminophen intoxicated rats
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The histopathological analysis of liver confirmed the observed modification of serum enzymatic levels to liver injury and their attributes on health. Normal hepatic architecture was observed in the liver sections of control rats with a central vein and hepatocytes radiating from it (central vein) (Fig. 1a), while liver section of acetaminophen treated animals showed centrilobular necrosis with vacuolar cytoplasmic deterioration around the central vein, hepatic degeneration, destruction of lobular architecture and nuclear degeneration in certain areas, (Fig. 1b). These were markedly diminished by administration of S. indica ethanol extract (200 mg/kg) and showed improvement in the liver architecture (Fig. 1c and d).

Fig. 1
figure 1

(a, b, c, d) Histopathology analysis of liver sections in normal, acetaminophen treated and Stokeyia indica (40X,10X) treated rats model. a Histopathological photomicrograph of normal (control) rats showed normal cell structure at 40 x magnification. b Histopathological photomicrograph of acetaminophen (control) rats showed extensive hepatocellular damage with the presence of centrizonal necrosis at 40x magnification. c Histopathological photomicrograph of rat liver section pre-treated with crude ethanol extract of Stokeyia indica (200 mg/kg) intoxicated with acetaminophen showed improvement in the liver architecture at 40x magnification. d Histopathological photomicrograph of rat liver section pre-treated with crude ethanol extract of Stokeyia indica (200 mg/kg) intoxicated with acetaminophen showed improvement in the liver architecture at 10x magnification

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Fractions F46 and F47 of ethanol extract of Stokeyia indica eluted from column chromatography were analyzed by GC-MS to detect various compounds. The constituents identified in the fraction F46 were identified as Methyl-n-Nonadecanoate (C20H40O2), Methyl-Hexeicosanoate (C22H44O2) (Table 4).

Table 4 GC-MS of Fraction F46 from Stokeyia indica
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Fraction F47 revealed the existence of Methyl-n-Nonadecanoate (C20H40O2,), Methyl-n-Hexadecanoate (C17H34O2), Methyl Octadecanoate (C19H38O2), Octadecanoic acid, Butyl ester (C22H44O2) (Table 5).

Table 5 GC-MS of Fraction F47 from Stokeyia indica
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Discussion

Liver diseases are important problem all over the world and it is increasing day after day. The drug induced liver injury (DILI) may leads in increased concentrations of serum enzymes like, ASAT, ALAT, ALP, along with increase in serum bilirubin, glucose, triglycerides, urea and creatinine [4]. In the current study, seaweed, S.indica ethanol extract efficiently attenuates the acetaminophen-induced hepatotoxicity by ameliorating the activities of these serum enzymes and also improved total and direct bilirubin concentration. Compernolle et al. [23] reported that increasing concentration of bilirubin metabolite in blood serum is utilized as an indicator of liver damage. It has been reported that bilirubin concentration increased as a consequence of enlarged production, reduced uptake through the liver, decreased conjugation, decreased discharge from the liver or obstruction of the bile duct [24, 25]. These results confirmed prior reports that acetaminophen has an unsafe and hectic pressure on liver tissues [26, 27].

In the current study, acetaminophen intoxication also produced an adverse effect on kidney function as manifested in the increased level of creatinine and urea in the blood. Overdose of paracetamol become the reason for many metabolic disorders as well as an increase in serum urea and creatinine [28]. The enhancement in serum urea and creatinine concentrations provides a significant indication of renal toxicity [29]. Seaweed pretreated intoxicated rats showed decreased level of creatinine and urea concentration towards normal range as compared to acetaminophen intoxicated rats, which showed elevated level of kidney parameters.

Lipid metabolism and kidney functions may be affected by liver damage [30]. In this study, rats intoxicated with acetaminophen also showed a disturbance in lipid metabolism, by increasing triglyceride level and decreasing total cholesterol as compared to acetaminophen intoxicated rats. An interruption within cholesterol metabolism may possibly due to liver parenchymal cell deaths in due course directed toward the interruption of lipid metabolism in the liver [31]. Khan et al. [32] reported that as a result of the reduction of lipase activity, triglyceride levels increased which possibly directed to reduce triglyceride hydrolysis. It has also been observed that rats pre-treated with ethanol extract of S. indica reinstate the cholesterol concentration in acetaminophen intoxicated rats; these consequences are in conformity with the results of Bigoniya and Rana [33].

There are reports that hepatic lipid peroxidation elevated in acetaminophen toxicity [34, 35]. In this study, acetaminophen intoxication also consequences in augmentation in lipid peroxidation, it is specified through the considerable augmentation in MDA. Extreme reactive oxygen specie (ROS) production activates the procedure of lipid peroxidation in cell membranes and results in obliteration of cells component and cells death. Whereas pretreatment of S. indica reinstate the hepatic GSH concentration and decreased the formation of MDA in acetaminophen-intoxicated rats which are in conformity by prior reports [36, 37]. The histopathology study of the liver also showed hepatoprotective effect of S.indica. The acetaminophen treated animals showed centrilobular necrosis with vacuolar cytoplasmic deterioration around the central vein, hepatic degeneration, destruction of lobular architecture and nuclear degeneration, while S. indica treated rats showed more or less normal hepatic architecture.

In this study, GC-MS analysis of two oily fractions of ethanol extract of S.indica eluted from column chromatography revealed the presence of several fatty acids including methyl palmitate which possessed antioxidant property. Fatty acid from a brown alga Spatoglossum asperum is known to possess various biological activity [38]. Hepato-protective potential of S.indica may be due to presence of methyl palmitate, that has been reported to ameliorate Aspartate Aminotransferase (ASAT), LDH (lactic dehydrogenase), serum glucose concentration in galactosamine intoxicated rats alongwith decresing the liver necrosis and inflammatory reactions [39]. Another report also supports this result that toxicity of carbon tetrachloride (CCl4) to rats was decreased by methyl palmitate prior to intragastric CCl4 administration. Methyl palmitate decreases the CCl4-induced elevation in hepatic triglyceride content and in GPT (serum glutamic-pyruvic transaminase) activity and attenuate the CCl4 induced adverse effects on the liver tissue [40].

Conclusion

Results of our study indicate that seaweed, S.indica was effective in suppressing acetaminophen induced damage to liver. It was able to reduce all the elevated biochemical parameters and had therapeutic and preventive effect against acetaminophen induced hepatic and renal damage. The preventive activity of S. indica against oxidative stress may be due to the presence of fatty acids methyl palmitate, which is reported to possess antioxidant and hepato-protective properties.