Hepatoprotective effects of methanolic extract of Alcea rosea against acetaminophen-induced hepatotoxicity in mice

The aim of this study was to evaluate the hepatoprotective effects of Alcea rosea against acetaminophen-induced hepatotoxicity in mice. Aqueous methanolic extract of A. rosea were given orally for 7 consecutive days followed by daily toxic dose of acetaminophen. At the end of treatment period, evaluation of hepatoprotective activity of A. rosea was done on basis of levels of liver enzyme markers (aminotransferases, alkaline phosphatase and bilirubin) and histopathological examination of liver tissues. Acetaminophen significantly increased serum levels of liver enzyme markers whereas, the extract of A. rosea significantly reduced serum levels of elevated liver enzyme markers in dose-dependent manner compared to acetaminophen treated mice group. Histopathological examination of liver tissues also supported the protective effects of A. rosea on liver enzyme markers. We conclude that extract of A. rosea has strong hepatoprotective effects against acetaminophen-induced hepatotoxicity; thereby, affirming its traditional therapeutic role in liver injury.

mice (Kulkarni et al., 2002), immunomodulatory property (Ghaoui et al., 2008), analgesic and anti-inflammatory activity (Mert et al., 2010;Wang et al., 1989). Roots of A. rosea have also been used for the treatment of inflammation, diarrhea, constipation, bronchitis, severe coughs and angina (Ahmadi et al., 2012). The dry extracts of A. rosea stems and roots contain monosaccharides, oligosachharides, mucus, microelements and proteins. The aim of this study was to investigate the possible hepatoprotective activity of A. rosea extract against the hepatotoxicity induced by acetaminophen overdose in mice used as an experimental animal model.

Materials and Methods
Collection and authentication of plant: Aerial parts of A. rosea were collected from the fields in Faisalabad, Pakistan during July 2012. The plant was verified and given the voucher no: 617-2-13 at University of Agriculture Faisalabad, Pakistan. The voucher was kept in University of Agriculture Faisalabad for future reference.
Preparation of plant extract: The aerial parts of A. rosea were washed by tap water and then air dried. The dried plant was pulverized into coarse powder by mechanical grinder and stored in air tight container. Dried powder (1 kg) was soaked in aqueous methanol (30:70) for seven days with daily shaking. After soaking for seven days, the extract was separated by filtration and concentrated using rotary evaporator at 70ºC. The crude extract was stored in air tight container.
Experimental animals: Swiss albino mice, weighing between 20-30 g, were used for the study. The animals were procured from animal house of University of Agriculture Faisalabad, Pakistan and were kept at animal house in College of Pharmacy, Government College University, Faisalabad, Pakistan. After randomization into various groups, the mice were acclimatized for period of 7 days under standard husbandry conditions at room temperature (25 ± 3 ºC) and 12 hours light/ dark cycle. All the animals were fed under strict hygienic conditions with rodent pellet diet and water ad libitum. The experiments on animals were performed in accordance to the guidelines of Ethical committee Government College University, Faisalabad and were approved by Advanced Studies and Research Board.
Experimental design: In this experimental protocol, Swiss albino mice were selected and divided into five groups of five animals in each group. The test substances were administered by oral gavage using a stomach tube. Prior to dosing, animals were kept for 12 hours of fasting. After giving the dose, food was withheld for further 3-4 hours.
The experimental protocol was designed for seven days. Group A was maintained as control group and received 1 mL/kg normal saline daily. Group B received acetaminophen 250 mg/kg orally for seven consecutive days. Group C animals were treated with silymarin (50 mg/kg, p.o) which served as standard group. Groups D and E animals were treated with two different doses of aqueous methanolic extract of A. rosea (200 mg/kg and 400 mg/kg) respectively. Group C, D and E were also intoxicated with acetaminophen (250 mg/kg) 3 hours after the administration of silymarin and extracts of A. rosea for seven days.
Biochemical investigation: After 24 hours following the last dose, all the animals were anesthetized with chloroform and sacrificed. The blood was collected in eppendorf tubes and allowed to clot and serum was separated with the help of centrifuge at 4,000 rpm for 20 min. Biochemical parameters like aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, direct and indirect bilirubin were measured using commercially available kits (Merck, and DiaSys Diagnostic systems Gmbh, Germany) according to the standard protocol.
Histopathological studies: Histopathological analysis was performed as described previously (Hussain et al., 2014). All the mice were killed mercifully and their liver tissues were dissected out, washed with ice-cold normal saline. A small cross section of the liver was separated out and embedded in paraffin after fixing with 10% neutral-buffered formalin. These tissues were dehydrated with graded ethanol and cleaned by xylene following paraffin infiltration. Finally, tissue sections were cut in size of 4-5 µm, deparaffinised with xylene and rehydrated with graded isopropyl alcohol and a drop of water. Water was removed and slides were oven dried. After tissue fixation, staining was done with hematoxylin and eosin. The stained sections of slides were examined under high-resolution microscope by blind observer and photographs were taken.
Statistical analysis: Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison test using Graph Pad Prism 5 (Graph Pad, Software Inc., USA). The value of significant difference was considered at p< 0.05.

Results
Acetaminophen produced a higher concentration of AST up to 313.6 ± 16.4 U/L (p<0.01) in Group B animals compared to the levels in Group A animals (77.6 ± 6.4 U/L) (Figure 1). A. rosea at the dose of 200 mg/kg showed no significant effect on the level of AST (287.3 ± 13.9 U/L) in Group D animals as non-significant difference (p>0.05) was observed when directly compared with the levels in Group B animals. Administration of methanolic extract of A. rosea at the dose of 400 mg/kg significantly (p<0.01) decreased the AST level in Group E when directly compared to the levels in Group B proving its hepatoprotective nature. A. rosea at the dose of 400 mg/kg reduced the AST level (176.6 ± 3.8 U/L) more significantly in Group E and its results were more comparable to the AST levels (161.3 ± 17.0 U/L) in Group C animals as compared to AST levels obtained at 200 mg/kg dose of A. rosea in Group D animals.
In Group A animals, ALT level was found to be 30.0 ± 1.1 U/L, which showed that liver was functioning properly but this level was highly increased (105.6 ± 3.9 U/L) in Group B (p<0.01) indicating a decline in hepatic function leading to liver damage (Figure 1). Silymarin decreased the level of ALT (43.6 ± 2.7 U/L) in Group C animals nearly to the levels in Group A animals. The methanolic extract of A. rosea at the dose of 400 mg/kg also decreased the ALT levels (64.3 ± 6.6 U/L) that were comparable to the levels of Group A and C animals. Ostensibly, the results obtained from A. rosea with the dose of 200 mg/kg showed only a slight decrease of ALT levels (87.6 ± 3.3 U/L) in comparison to the levels of ALT in Group B.
Similarly, ALP levels in Group A animals were 61.3 ± 3.5 U/L. These levels were highly increased in Group B (207.3 ± 14.5 U/L) (Figure 1). Group D showed nonsignificant (p>0.05) change in ALP levels compared to ALP levels in Group B. A. rosea at dose 400 mg/kg showed that plant extract significantly decreased the levels of ALP (120.6 ± 5.9 U/L) in serum same as observed in Group C animals treated with standard drug silymarin (106.6 ± 4.6 U/L). A. rosea administered at the dose of 200 mg/kg has not shown any satisfying results giving less or no hepatoprotective activity against acetaminophen as it did not declines ALP Acetaminophen, in part of its toxic effects, leads to the fulminant liver damage which can be seen by the elevated levels of bilirubin. The levels of total bilirubin were highly increased in Group B animals (1.6 ± 0.1 mg/dL) treated with acetaminophen than in Group A animals (0.3 ± 0.1 mg/dL) (Figure 2). Groups C, D and E pretreated with silymarin, AR-200 and AR-400 respectively showed improved liver function by lowering the serum levels of total bilirubin compared to acetaminophen-intoxicated animals in Group B. Silymarin showed its standard activity by decreasing the levels of total bilirubin up to 0.6 ± 0.1 mg/dL that were comparable to the levels in Group A. The tested plant Groups D and E displayed the mean values of total bilirubin 0.9 ± 0.1 mg/dL and 0.5 ± 0.1 mg/dL, respectively showing hepatoprotective activity in dosedependent manner.
Toxic dose of acetaminophen in Group B animals led to the remarkable increase in serum levels of direct bilirubin (0.9 ± 0.1 mg/dL) and indirect bilirubin (0.7 ± 0.1 mg/dL) indicating the liver damage. While in Group A animals, the mean values of direct and indirect bilirubin were 0.2 ± 0.0 mg/dL and 0.1 ± 0.0 mg/dL respectively. Evidently, silymarin displayed the values of direct and indirect bilirubin 0.3 ± 0.0 mg/dL and 0.2 ± 0.0 mg/dL, respectively in Group C that were more or less comparable to Group A. A. rosea decreased the levels of direct bilirubin from 0.5 ± 0.1 mg/dL to 0.2 ± 0.0 mg/dL and indirect bilirubin from 0.3 ± 0.0 mg/ dL to 0.2 ± 0.0 mg/dL in dose-dependent manner. Figure 3 shows the histopathological examinations of liver tissues after the treatment period. The liver tissue Group A exhibiting all the hepatocytes arranged in cord fashion with eosinophilic cytoplasm and prominent nucleus containing 1-2 nucleoli. Portal areas showed normal lining of bile duct and hepatic artery. The tissue section intoxicated with acetaminophen in Group B animals. Photomicrograph reveals that the hepatocytes were swollen and lost their structural integrity. It showed degenerative (cytoplasmic vacuolation) and necrotic changes in hepatocytes. Capillaries were engorged with RBCs (Congestion). The liver section of Group C animals treated with standard drug silymarin. Photomicrograph showed hepatocytes in normal shape and size with normal portal area. There are mild to moderate degenerative and congestive changes. Regenerative activity is maximum. The liver tissues of Group D treated with A. rosea with the dose of 200 mg/ kg. Hepatocytes showed mild to moderate cytoplasmic vacuolation. Nucleus is vesicular (prominent nucleus). Liver sections from mice treated with A. rosea with the dose of 400 mg/Kg. Regenerative activity is seen with normal hepatocytes with prominent nucleus. Mild congestion is seen but cytoplasm is clear (no vacuolation). The histological examination proved the protective role A. rosea against acetaminophen toxicity.
The plant A. rosea of family Malvaceae used for this research is traditionally used for a number of remedies. A. rosea has been used in homeopathy for the treatment of jaundice. In the present study, A. rosea was tested to validate its use as hepatoprotective agent. The extract of A. rosea, depicted its hepatoprotective effect maintaining the low levels of liver enzyme markers. Thus it gives a plausible explanation that this plant showed hepatoprotective activity attributed to its phytochemical constituents.
To conclude, A. rosea exhibited hepatoprotective effects in mice intoxicated with acetaminophen. This protective effect can be credited to the phytoconstituents possessed by the plant.