Hepatoprotective effect of ethanolic extract of Trichosanthes lobata on paracetamol-induced liver toxicity in rats

Background Trichosanthes lobata (family cucurbitaceae) is used to treat malarial fever and liver disorders. This study aims to investigate possible hepatoprotective activities of ethanolic extract of Trichosanthes lobata against paracetamol-induced hepatotoxicity. Methods Hepatotoxicity was induced in Wistar male rats by oral administration, 2 g/kg body weight on 7th day after the administration of ethanolic extract of Trichosanthes lobata and silymarin (100 mg/kg). Ethanolic extract of Trichosanthes lobata was administered orally at doses of 200 mg/kg and 400 mg/kg body weight daily for 7 days. Several serum markers, aspartate transaminase, alanine transaminase, alkaline phosphatase, bilirubin, total protein was measured to assess the effect of the extract on paracetamol (acetaminophen)-induced hepatic damage. The study included histopathological examination of liver sections. Results Blood samples from rats treated with ethanolic extract of Trichosanthes lobata (200 mg/kg body weight and 400 mg/kg body weight) had significant reductions in serum markers in paracetamol administered animals, indicating the effect of the extract in restoring the normal functional ability of hepatocytes. Silymarin (100 mg/kg, p.o.) was used as a reference drug. Conclusion The ethanolic extract of Trichosanthes lobata exhibits protective effects against paracetamol‒induced hepatotoxicity.

Results: Blood samples from rats treated with ethanolic extract of Trichosanthes lobata (200 mg/kg body weight and 400 mg/kg body weight) had significant reductions in serum markers in paracetamol administered animals, indicating the effect of the extract in restoring the normal functional ability of hepatocytes. Silymarin (100 mg/kg, p.o.) was used as a reference drug.

Conclusion:
The ethanolic extract of Trichosanthes lobata exhibits protective effects against paracetamol-induced hepatotoxicity.
Paracetamol (acetaminophen) is widely used as an antipyretic and analgesic, and it produces acute liver damage if administrated in excess [16,17]. Paracetamol is mainly metabolized in the liver to excretable glucuronide and sulphate conjugates [18,19]. However, the hepatotoxicity of paracetamol has been attributed to the formation of toxic metabolites when part of it is activated by hepatic cytochrome P-450 [20] to form the highly reactive metabolite N-acetyl-P-benzoquinone imine (NAPQI) [21]. NAPQI covalently binds to cysteine groups on proteins to form 3-(cystein-S-yl) acetaminophen adducts [22]. The glutathione protects hepatocytes by combining with the reactive metabolite of paracetamol, thus preventing covalent binding to liver proteins [23].
The experimental demonstration of the hepatoprotective activities is lacking. This study aims to investigate possible hepatoprotective properties of Trichosanthes lobata.

Chemicals
Paracetamol (acetaminophen) was purchased from S.D. Fine Chemicals, Ltd. (India). A gift sample of Silymarin was provided by Ranbaxy (Devas, India), and standard assay kits of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), bilirubin and total protein was obtained from Jain Scientific Industries (Moradabad, India). All other reagents were of analytical grade.

Plant materials
The plant Trichosanthes lobata was collected in Malapuram district, Kerala. The plant was authenticated (BSI/SRC/5/23/2010-11/Tech-1833 dt 4 February 2011) by Dr. Govindappa.Venkatesa Sundara Murthy, of the Botanical Survey of India, Coimbatore. Plants were authenticated with the existing herbarium specimens in central national herbarium (Botanical Survey of India, Coimbatore, India), after treatment of the plants with saturated solution of mercuric chloride and ethyl alcohol [24]. A voucher specimen (KMCH/COG/Tl/2011/02) was deposited at Department of Pharmacognosy, KMCH College of Pharmacy, Coimbatore, for future reference.

Preparation of extract
Extract of dried powdered Trichosanthes lobata was obtained with 70% ethanol (Merck, Germany) in a Soxhlet apparatus at 68°C for 72 h. The extract was filtered on Whatman No. 1 filter paper (Scientific Furnishings, Ltd., Chichester, UK) and concentrated using a rotary vacuum evaporator (N-1001 T-WD, Eyele, Japan) at 40°C -45°C (0.9% w/w).

Experimental animals
Swiss female mice (20-25 g) and albino adult Wister male rats (150-200 g) were obtained from the animal house of KMCH College of Pharmacy, Tamilnadu, India. The study protocol was approved by the institutional animal ethics committee, Committee for the Purpose of Control and Supervision on Experimental Animals (CPCSEA), New Delhi, India, as per approval no. 509/ 01/C/CPCSEA dt, 10 January, 2009. Both rats and mice were housed in plastic cages (47 × 34 × 18 cm) in an airconditioned environment, with 10 mice per cage or 6 rats per cage. The floor of the cages was lined with saw dust, which was replaced every 48 h. Both rats and mice were fed with standard pellet diet (Kamadenu Enterprises, Bangalore, India), and they had free access to water.

Acute toxicity test
Acute oral toxicity was determined according to method described by Litchfield [27]. Female mice were divided into 8 groups of 6 animals each. The control group received 0.5 mL of 0.5% w/v sodium carboxymethyl cellulose (Qualigens, India) orally. The other groups received 100, 200, 400, 800, 1000, 2000, and 3000 mg/kg body weight ethanol extract of Trichosanthes lobata in 0.5% sodium carboxymethyl cellulose orally. Immediately after dosing, the mice were continuously observed for at least 4 h, and occasionally up to 6 h. They were then observed for up to 14 days (frequency of 12 h/day) for signs of toxicity and mortality.

Hepatoprotective activities
Paracetamol induced hepatotoxicity model was adopted for the study [28]. The rats were divided into 5 groups of 6 animals each. Group I served as a control and received normal saline, 5 mL/kg body weight, daily for 7 days. Group II constituted the hepatotoxic group and were treated similarly to group I. Group IV and Group V received ethanolic extract (200 and 400 mg/kg body weight per day, respectively) suspended in 0.5% sodium carboxymethylcellulose for 7 days. Group III received the reference drug, silymarin (100 mg/kg body weight daily) for 7 days.
On the 7th day, paracetamol suspension was given orally, 2 g/kg body weight, to all the rats except those in Group I. At the end of the experimental period, the rats

Histopathological studies
Paraffin sections (7 μm thick) of buffered formalin-fixed liver samples were stained (nuclei in blue and cytoplasm in pink) with hematoxylin-eosin [29] to identify the histological changes under the microscope (Vision micro systems, India).

Biochemical studies
Blood was obtained from all animals by puncturing the retro-orbital plexus. Blood samples were allowed to clot for 45 min at room temperature. Serum was separated by centrifugation [Model No.LAC 10370, Remi, India] at 2.5 x g at 30°C for 15 min and assayed for AST, ALT, ALP [30], bilirubin [31] and total protein [32], as shown in Table 2.

Statistical analysis
For determination of significant inter-group differences of each parameter one-way analysis of variance (ANOVA) was carried out. Dunnet test was used for individual comparisons after significant ANOVA results. The differences with P < 0.05 were considered statistically significant. GraphPad prism 4 software (GraphPad Software, Inc. California, USA) was used for the statistical analysis.

Phytochemical investigation and TLC study
Phytochemical screening of the ethanolic extract of Trichosanthes lobata confirmed the presence of proteins, steroids, tannins, carbohydrates, glycosides, saponins, and flavonoids. The spots obtained after TLC development revealed that the ethanolic extract possesses flavonoids, saponins, and tannins.

Acute toxicity test
The ethanolic extract of Trichosanthes lobata did not result in any mortality of mice up to the dose of 3000 mg/ kg body weight. Hence, doses of 200 and 400 mg/kg body weight were selected.

Histopathological studies
Histopathological studies of rat liver tissue from the control group (Group I) showed normal hepatic cells with central vein and sinusoidal dilation (Figure 1). In the paracetamol group (Group II), severe hepatotoxicity was observed in the form of severe necrosis and disappearance of nuclei ( Figure 2). Histopathological analysis showed that the pathological lesions caused by paracetamol were very minimal in groups pretreated with ethanolic extract of Trichosanthes lobata (Group IV and V). Normal hepatocytes with regenerating hepatocytes and mild inflammation in the portal area were observed in groups IV and V, treated with ethanolic extract of Trichosanthes lobata, 200 and 400 mg/kg body weight, respectively (Figures 3 and 4). Liver tissue from paracetamol + silymarin group (Group III) had normal hepatic cells with portal vein and portal artery ( Figure 5).

Biochemical studies
The effects of ethanolic extract of Trichosanthes lobata on AST, ALT, ALP, bilirubin, and total protein levels in rats with paracetamol-induced liver damage were summarized in Table 2. Administration of paracetamol (2 g/kg body weight, orally) after 18 h resulted in a significant (P < 0.05) elevation of hepatospecific serum markers such as AST, ALT, ALP, bilirubin, and total protein in the paracetamol group (Group II) in comparison with the control group (Group I). On administration of ethanolic extract of Trichosanthes lobata (Groups IV and V) and paractamol + silymarin group (Group III), the serum markers were restored to the normal levels.

Discussion
Histopathological studies of rats administered paracetamol showed severe necrosis and disappearance of nuclei. This could be due to the formation of highly reactive metabolites (e.g. NAPQI), because of excessive administration of paracetamol. All these histopathological changes were significantly reduced in rats treated with ethanolic extract of Trichosanthes lobata.
The study of serum markers such as AST, ALT, ALP, and bilirubin, and total protein has been found to be of great value of assess to clinical and experimental liver damage [33]. In the present investigation, the rats suffered significant hepatic damage from treatment with paracetamol, as indicated by elevated levels of serum markers ( Table 2). A rise in AST is usually accompanied by an increase in ALT, which plays a vital role in the conversion of amino acids to keto acids [34]. Pretreatment with ethanolic extract of Trichosanthes lobata, both at 200 mg/kg body weight and 400 mg/kg body weight, significantly attenuated elevated levels of serum markers. This suggests that ethanolic extract of Trichosanthes lobata conditions the hepatocytes so as to protect the integrity of the membrane from paracetamol-induced leakage of serum markers into    circulation. These changes can be considered a functional improvement of hepatocytes and may be caused by accelerated regeneration of parenchyma cells. Serum ALP and bilirubin are related to hepatic cell damage [28]. Increase in serum ALP is due to increased synthesis in the presence of increasing biliary pressure [35]. The decrease in the levels of ALP and bilirubin may be due to the presence of flavonoids and their antioxidant effects which may protect the hepatic cell damage induced by paracetamol.
A potential of hepatoprotective property underlying Trichosanthes lobata may be attributed to the antioxidative constituents. The plants most commonly used to treat liver disorders are Curcuma longa (turmeric), Glycyrrhiza glabra (licorice), and Camellia sinensis (green tea), and they are all reported to be hepatoprotective due to the powerful anti-oxidative properties [36][37][38][39]. Also, the antioxidant properties of Trichosanthes cucumerina are attributed to flavonoids, carotenoids, lycopene, phenolics, and β-carotene [40]. The presence of flavonoids, saponins, and tannins in ethanolic extract of Trichosanthes lobata was confirmed by phytochemical analysis and TLC, and these compounds are reported to have antioxidant properties [41].