Hepatoprotective potential of Cassia auriculata roots on ethanol and antitubercular drug-induced hepatotoxicity in experimental models

Abstract

Context: Tarvada [Cassia auriculata Linn. (Caesalpiniaceae)] is used against liver ailments in Indian folk medicine, but there is a lack of scientific evidence for this traditional claim.

Objective: The present study investigated the protective effect of methanol extract of tarvada (MECA) roots on ethanol and antitubercular drug induced hepatotoxicity in rats.

Materials and methods: In the therapeutic model, ethanol (40%, 4 g/kg b.w., p.o.) was administered to rats for 21 days and the intoxicated rats were treated with MECA (300 and 600 mg/kg, b.w.) and silymarin (100 mg/kg, b.w.) for next 7 days. In the prophylactic model, MECA and silymarin were administered simultaneously along with a combination of isoniazid (27 mg/kg, b.w.), rifampicin (54 mg/kg, b.w.) and pyrazinamide (135 mg/kg, b.w.) for 30 days. After the study duration, serum levels of AST, ALT, ALP, total bilirubin, total cholesterol, total protein, albumin were estimated along with hepatic catalase (CAT), reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) and liver histopathology in each group.

Results: Administration of tarvada root extract significantly (p < 0.01 and p < 0.05) lowered the elevated levels of serum AST, ALT, ALP, total bilirubin, total cholesterol, total protein and restored the abnormal levels of enzymatic antioxidants and MDA in liver due to toxicant administration in a dose-dependent manner. These results were confirmed by histopathological analysis.

Discussion and conclusion: Results suggest that tarvada root extract possess potent hepatoprotective activity against ethanol and antitubercular drug-induced hepatotoxicity in rats, which could be due to an inhibition of hepatic metabolizing enzymes and antioxidant activity.

• Antioxidant
• herb
• metabolic enzyme
• methanol extract
• prophylactic model
• silymarin
• therapeutic model

Introduction

Cassia auriculata Linn. (Caesalpiniaceae), commonly known as tarvada or tanner’s cassia (Nadkarni, 2002), is a fast growing, profusely branched, tall, and evergreen shrub. It is found throughout southern, western and central India and survives under adverse ecological conditions (Gaikwad et al., 2011). Natives of Andhra Pradesh state of India used the root extract against skin diseases, asthma, conjunctivitis and renal disorders (Annie et al., 2005). Extract of whole plant is used for liver ailments (Ayyanar & Ignacimuthu, 2008). The leaves are anthelmintic and good for ulcers, skin diseases and leprosy (Balakrishna et al., 2011). Previously reported activities of tarvada are antioxidant (Doshi et al., 2011), antidiabetic (Gupta et al., 2009), immunomodulatory (Chakraborthy, 2009), hepatoprotective (Dhanasekaran & Ganapathy, 2011), anthelmentic, antibacterial (Wadekar et al., 2011) and nephroprotective (Annie et al., 2005).

Chemical investigation of roots of the plant show the presence of anthraquinone glycosides such as 1,3-dihydroxy-2-methylanthraquinone; 1,3,8-trihydroxy-6-methoxy-2-methylanthraquinone; 1,8-dihydroxy-6-methoxy-2-methylanthraquinone-3-O-rutinoside, 1,8-dihydroxy-2-methylanthraquinone-3-O-rutinoside and a flavone glycoside, 7″,4′-dihydroxyflavone-5-O-d-galactopyranoside (Rai & Dasundhi, 1990). Isolated compounds from the root bark are a chalcone 3′,6′-dihydroxy-4-methoxychalcone, two leucoanthocyanins, viz. leucocyanidin-3-O-l-rhamnopyroside and leucopeonidin-3-O-l-rhamnopyroside (Balakrishna et al., 2011). While the hepatoprotective activity of the plant stem (Swathi et al., 2010) and leaf (Dhanasekaran & Ganapathy, 2011) have been proven experimentally, there is a lack of literature regarding hepatoprotective activity of root. Hence, it was decided to evaluate the hepatoprotective activity of methanol extract of tarvada (MECA) roots against ethanol and antitubercular drug-induced hepatotoxicity in rats with a view to validate its traditional claim.

Materials and methods

Plant material

Roots of tarvada were collected from the Satara district of Maharashtra (India) in August, 2011 and were authenticated by Mr. A.G. Diwakar, Director, Botanical Survey of India, Pune. A voucher specimen (CAAASJ4) has been deposited in the herbarium of the institute for future reference.

Animals

Wistar albino rats of either sex (150–180 g) were procured from the National Institute of Bioscience, Chaturshrungi, Pune. Animals were housed in polypropylene cages and the animal house was maintained under standard laboratory conditions (temperature 22 ± 2 °C, relative humidity 60%–70%) with dark and light cycle (12/12 h). The animals were provided with standard diet (Amrut Feeds, Pune, India) and water ad libitum. The animals were randomly assigned to different groups and a minimum period of one week was allowed for adaptation on each experiment. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) constituted in accordance with the rules and guidelines of CPCSEA, i.e. Committee for the Purpose of Control and Supervision on Experimental Animals (884/OP/05/ac/CPCSEA).

Preparation of extract

Roots of tarvada were washed thoroughly with distilled water and shade-dried at room temperature. The shade-dried roots of C. auriculata were ground to coarse powder and 1000 g of powder was exhaustively extracted by maceration using methanol as a solvent for 72 h at room temperature with intermittent shaking. The extract was filtered and the solvent was evaporated to obtain powdered residue. It was then dried in a vacuum desiccator and stored in an amber-colored bottle in a refrigerator for further use (Bandawane et al., 2011).

Chemicals and drugs

Silymarin was purchased from Micro-Labs, Bangalore, India. Isoniazid, rifampicin and pyrazinamide were obtained as gift samples from Pharmalink Laboratories Ltd., Thane, India. Aspartate transaminase (AST), alanine transaminse (ALT) and bilirubin kits were purchased from Coral Diagnostics Ltd., Goa, India. Alkaline phosphatase (ALP), total protein, albumin and cholesterol kits were purchased from Pathozyme Diagnostics Ltd., Kolhapur, India. All the other chemicals and reagents used were of analytical grade and were purchased from Thomas Baker Ltd., Mumbai, India.

Preliminary phytochemical screening

The preliminary phytochemical analysis of the methanol extract of tarvada was carried out using the following tests: carbohydrates (Molisch’s, Fehling’s and Benedict’ tests), proteins (Biuret and Million’s test), steroids (Salkowski and Liebermann-Buchard’s test), anthraquinone glycosides (Borntrager’s test), saponins (Foam test), flavonoid glycosides (Shinoda and alkaline reagent tests), alkaloids (Dragendorff, Mayer’s, Hager’s and Wagner’s tests) tannins and phenolics (ferric chloride, lead acetate and potassium dichromate tests) (Khandelwal, 2006).

Determination of in vitro antioxidant activity

Determination of DPPH scavenging activity

The effect of MECA was estimated by using the method described by Dasgupta and Bratati (2004). A solution of 4 mg of 1,1-diphenyl-2-picrylhydrazyl (DPPH) in methanol (0.004%) was prepared and 3 ml of solution was mixed with 0.1 ml methanol solution of MECA each from concentrations ranging from 200 to 1000 µg/ml. The reaction mixture was mixed thoroughly and left in the dark at room temperature for 30 min. Absorbance of the mixture was determined spectrophotometrically at 517 nm. Ascorbic acid prepared at same concentration was used as the reference drug. The experiment was conducted in triplicate. The percent inhibition in scavenging DPPH radicals was calculated using the formula, where Ao = Absorbance without extract; Ae = Absorbance with extract (Kumaran & Karunakaran, 2007).

Determination of nitric oxide scavenging activity

Nitric oxide scavenging activity of MECA was determined by slightly modified procedure of Saha et al. (2008). In brief, 1 ml solution, each of sodium nitroprusside and phosphate-buffered saline was mixed with different concentrations of MECA and incubated at 25 °C for 150 min. The same reaction mixture without plant extract but with an equivalent amount of solvent, i.e., water or methanol, served as control. After the incubation period, the samples were mixed with 0.5 ml of Greiss reagent (1% sulphanilamide, 2% H₃PO₄ and 0.1% napthyl ethylenediamine dihydrochloride). The absorbance was read at 540 nm. Inhibition of nitrite formation by plant extracts, standard antioxidants, i.e., ascorbic acid and BHT were calculated relative to control. Inhibition data (% inhibition) was linearized against the concentrations of each extract and standard antioxidant. Butylated hydroxy tolune (BHT) was used as the standard drug.

Determination of reducing power

Reducing power of MECA was evaluated by the method of Kumaran and Karunakaran (2007). Various concentrations of plant extracts (100–1000 µg/ml) were mixed with 1 ml of 200 mmol/l Sodium phosphate buffer (pH 6.6) and 1 ml of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. After incubation, 1 ml of 10% trichloroacetic acid (w/v) was added and the mixture was centrifuged at 2000 g for 10 min using centrifuge (model no. C-24 BL; REMI Laboratory systems, India). The upper layer (2.5 ml) was mixed with 2.5 ml of deionized water, 0.5 ml of ferric chloride (0.1%) solution and the absorbance was measured at 700 nm.

Acute oral toxicity studies

Acute toxicity studies were performed according to the Organization for Economic Co-operation and Development (OECD) guidelines No. 423. Male/female Wistar albino rats selected by a random sampling technique were employed in this study. MECA was administered orally to different groups of overnight fasted rats at the dose levels of 300, 500, 2000 and 5000 mg/kg, body weight. After dosing, the animals were observed for 2 h, then intermittently for further 4 h and finally recording mortality up to 24 h until 14 days. The parameters observed were grooming, hyperactivity, sedation, loss of righting reflex and convulsions (Bandawane et al., 2011).

Experimental design

Ethanol-induced hepatotoxicity (therapeutic model)

Thirty-six healthy rats of either sex were divided randomly into six groups containing six rats in each group.

Group I: Normal control, received 1% gum acacia as suspension for 28 days.

Group II: Hepatotoxic control, received 40% ethanol (4 g/kg/day, p.o.) in 1% gum acacia for 21 days.

Group III: Animals were given ethanol for 21 days + self recovery for next seven days.

Group IV: Animals were given ethanol for 21 days +MECA (300 mg/kg/day, p.o.) for next seven days.

Group V: Animals were given ethanol for 21 days +MECA (600 mg/kg/day, p.o.) for next seven days.

Group VI: Animals were given ethanol for 21 days + silymarin (100 mg/kg/day, p.o.) for next seven days (Pramyothin et al., 2007).

All the treatments were given daily and orally in gum acacia (1%) as a suspending agent for the complete study duration.

Antitubercular drug-induced hepatotoxicity (prophylactic model)

Thirty healthy rats of either sex were divided into five groups, six in each group.

Group I: Normal control, received 1% gum acacia as suspension for 30 days.

Group II: Hepatotoxic control, received antitubercular drug combination (ATC), namely isoniazid (27 mg/kg, p.o.), rifampicin (54 mg/kg, p.o.) and pyrazinamide (135 mg/kg, p.o.) as a suspension in 1% gum acacia for 30 days.

Group III: Animals were given ATC + MECA (300 mg/kg, p.o.) simultaneously for 30 days.

Group IV: Animals were given ATC + MECA (600 mg/kg, p.o.) simultaneously for 30 days.

Group V: Animals were given ATC + silymarin (100 mg/kg, p.o.) simultaneously for 30 days.

All the treatments were given daily and orally in gum acacia (1%) for 30 days. MECA and silymarin in their respective groups were administered 45 min after the antitubercular drug challenge (Kale et al., 2003; Ubaid et al., 2003).

Biochemical determinations

On the last day of the studies, blood was withdrawn from each group after an overnight fast by cardiac puncture under light ether anesthesia, collected in sterile centrifuge tubes and allowed to clot. Collected blood was centrifuged at 10,000 g for 10 min in cooling centrifuge (REMI, C-24 BL) and the separated serum was used for the estimation of biochemical parameters, such as aspartate transaminase (AST), alanine transaminse (ALT), alkaline phosphatase (ALP), total bilirubin, total protein, albumin and total cholesterol using standard assay kits (Burstein et al., 1970; Jendrassik & Grof, 1938; Kind & King, 1954; Kingsley & Frankel, 1939; Reitman & Frankel, 1957).

Livers were isolated and immediately washed with ice-chilled saline and blotted dry between filter papers. Livers were weighed and 10% homogenate was prepared with 0.01 M phosphate buffer (pH 7.0) in a tissue homogenizer (model no. C-24 BL; REMI Laboratory systems, India). The supernatant of liver homogenates were used for the estimation of MDA lipid peroxidation, catalase (CAT), superoxide dismutase (SOD) and reduced glutathione (GSH) using methods described by Kumar et al. (2010), Sahreen et al. (2011), Marklund et al. (1985) and Kaur et al. (2006), respectively.

Histopathological studies

A portion of the liver was cut into fine pieces of approximately 6 mm size and fixed in phosphate-buffered 10% formaldehyde solution. After embedding in paraffin wax, thin sections of 5 μm thickness were cut and stained with haematoxylin-eosin dye. The thin sections of liver were made into permanent slides and examined under high-resolution microscope with photographic facility and photomicrographs were taken (Luna, 1960).

Statistical analysis

All the data are presented as mean ± SEM of measurements made on six animals in each group. Statistical analysis was performed with one-way analysis of variance (ANOVA) followed by Dunnet’s multiple test using GraphPad Instat (version-3) software (GraphPad Software, CA). A value of p < 0.05 was considered to be statistically significant when compared with the respective control (Woolson, 1987).

Results

Preliminary phytochemical screening

On preliminary phytochemical analysis, methanol extract of tarvada roots revealed the presence of alkaloids, tannins, flavonoids, carbohydrates, glycosides, amino acids and steroids. The results were confirmed by thin layer chromatography, which showed the presence of tannins and flavonoids.

Acute oral toxicity studies

In the present study, results of acute toxicity study revealed high margin of safety and the absence of mortality up to a dose of 5000 mg/kg, b.w. Hence, the minimum lethal dose (LD₅₀) could not be calculated and is greater than 5000 mg/kg b.w. Hence, we selected two submaximal doses (300 and 600 mg/kg) for MECA based on previously reported studies (Annie et al., 2005).

Ethanol-induced hepatotoxicity

Effect of MECA on serum biochemical parameters

The results of hepatoprotective effect of MECA on ethanol intoxicated rats are shown in Tables 1 and 2. Administration of ethanol caused a significant (p < 0.05 and p < 0.01) elevation in serum aspartate transaminase (AST), alanine transaminse (ALT), alkaline phosphatase (ALP), total bilirubin and total cholesterol levels and also a significant (p < 0.05) decrease in total protein and albumin levels. Treatment of the ethanol intoxicated rats with MECA at 300 and 600 mg/kg body weight reduced the levels of aspartate transaminase (AST), alanine transaminse (ALT), alkaline phosphatase (ALP), total bilirubin and total cholesterol significantly (p < 0.05 and p < 0.01) as shown in Figure 1. Also, the administration of MECA (p < 0.05) significantly elevated the levels of total proteins. Results of administration of MECA (600 mg/kg) were comparable to standard control group. Self recovery group did not show any significant (p > 0.05) decrease in serum enzymatic levels except aspartate transaminase levels (p < 0.05). Administration of MECA at any of the dose levels did not elevate the albumin levels, which were depleted in the ethanol control group (Figure 2).

Figure 1. Effect of methanol extract of Cassia auriculata (MECA) roots on serum AST, ALT, ALP and total cholesterol in ethanol-induced hepatotoxic rats. N = 6; Values are mean ± SEM. NC: normal control, EC: ethanol control, AST: aspartate transaminase, ALT: alanine transaminase, ALP: alkaline phosphatase, #p < 0.05, ##p < 0.01 as compared to normal control group. *p < 0.05, **p < 0.01, as compared to ethanol control group. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

Figure 2. Effect of methanol extract of Cassia auriculata (MECA) roots on serum total proteins, albumin and total bilirubin in ethanol induced hepatotoxic rats. N = 6; Values are mean ± SEM. NC: normal control; EC: ethanol control. #p < 0.05, ##p < 0.01 as compared to normal control group. *p < 0.05, **p < 0.01 when compared to ethanol control group. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

Table 1. Effect of methanol extract of Cassia auriculata (MECA) roots on serum AST, ALT, ALP and total bilirubin in ethanol-induced hepatotoxic rats.

Groups	AST (IU/l)	ALT (IU/l)	ALP (KA units/l)	Total bilirubin (mg/dl)
NC	203.73 ± 5.90	53.00 ± 5.58	176.4 ± 4.00	1.05 ± 0.29
EC	308.25 ± 5.44#	135.53 ± 21.35##	314.16 ± 18.26##	2.54 ± 0.19##
EC + Self-recovery	252.75 ± 12.18*	101.87 ± 3.96	268.66 ± 19.13	2.53 ± 0.27
EC + MECA 300 mg/kg	289.26 ± 19.63	70.70 ± 4.80**	253.6 ± 16.16	1.87 ± 0.21
EC + MECA 600 mg/kg	242.18 ± 9.49*	68.50 ± 14.45**	176.4 ± 12.52**	1.42 ± 0.30**
EC + Silymarin 100 mg/kg	225.90 ± 15.35*	75.60 ± 10.07**	186.01 ± 21**	1.41 ± 0.18**

Data are expressed as mean ± S.E.M. (n = 6). NC: normal control, EC: ethanol control. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

#p < 0.05, ##p < 0.01 as compared to normal control group.

*p < 0.05, **p < 0.01 as compared to ethanol control group.

Table 2. Effect of methanol extract of Cassia auriculata (MECA) roots on serum total proteins, albumin, total cholesterol, liver thiobarbituric acid reactive substances (TBARS) and liver catalase in ethanol-induced hepatotoxic rats.

Groups	Total proteins (g/dl)	Albumin (g/dl)	Total cholesterol (mg/dl)	TBARS (n moles/mg wet liver)	CAT (units/min)
NC	7.85 ± 1.57	6.83 ± 0.23	33.086 ± 3.33	15.423 ± 3.24	5.96 ± 1.07
EC	5.51 ± 0.14#	4.82 ± 0.38##	64.39 ± 5.55#	35.26 ± 3.48#	1.45 ± 0.67##
EC + Self-recovery	3.64 ± 0.62	3.03 ± 0.25	64.39 ± 11.64	51.10 ± 6.16	0.44 ± 0.01
EC + MECA 300 mg/kg	7.28 ± 0.33*	2.95 ± 0.33	76.65 ± 8.54	57.21 ± 8.24	0.44 ± 0.01
EC + MECA 600 mg/kg	7.81 ± 0.77*	3.87 ± 0.41	47.92 ± 7.00*	51.10 ± 6.16*	0.98 ± 0.13
EC + Silymarin 100 mg/kg	7.70 ± 0.25**	4.01 ± 0.35	35.75 ± 5.11*	22.98 ± 3.74*	0.75 ± 0.01

Data are expressed as mean ± S.E.M. (n = 6). NC: normal control, EC: ethanol control. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

#p < 0.05, ##p < 0.01 as compared to normal control group.

*p < 0.05, **p < 0.01 as compared to ethanol control group.

Effect of MECA on hepatic antioxidant parameters

Hepatic thiobarbituric acid reactive substances (TBARS) level was significantly (p < 0.05) elevated in hepatotoxic (ethanol) control group as compared to normal control group. Administration of MECA (600 mg/kg) caused a significant (p < 0.05) decrease in ethanol-induced rise in TBARS levels. However, depleted levels of catalase were not increased by MECA at any of the dose levels as compared to the ethanol control group (Figure 3).

Figure 3. Effect of methanol extract of Cassia auriculata (MECA) roots on liver tissue TBARS levels in ethanol-induced hepatotoxic rats. N = 6; Values are mean ± SEM. NC: normal control, EC: ethanol control, TBARS: Thiobarbituric acid reactive substances. #p < 0.05, ##p < 0.01 as compared to normal control group. *p < 0.05 when compared to ethanol control group. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

Effect of MECA on liver histopathology

As shown in Figure 4, in normal control rats, liver histological sections showed hepatocytes with prominent nucleus and nucleolus with no evidence of inflammation, necrosis and central vein dilatation. As compared to normal control group, liver of ethanol control group showed extensive liver injuries characterized by sinusoidal infiltration in hepatocytes with necrosis of single-cell, piecemeal and bridging type. Central vein showed dilatation and there was evidence of Kupffer cell hyperplasia and focal fatty changes. Administration of MECA at 300 and 600 mg/kg dose levels reduced the severity of hepatic damage as indicated by minimal single cell necrosis, central vein dilatation and Kupffer cell hyperplasia with no evidence of focal fatty changes and bridging necrosis. Hepatocytes also showed regenerative activity at some places. The result of MECA administration was comparable with the silymarin-treated group, which showed almost normal hepatic architecture.

Figure 4. Effect of MECA on liver histopathology of ethanol treated rats. A = Normal control; B = Ethanol control: 40% Ethanol – 4 g/kg; C = Ethanol control + silymarin (100 mg/kg); D = Ethanol control + self recovery; E = Ethanol control + MECA (300 mg/kg); F = Ethanol control + MECA (600 mg/kg).

Antitubercular drug-induced hepatotoxicity

Effect of MECA on serum biochemical parameters

The results of hepatoprotective effect of MECA on antitubercular combination (ATC) intoxicated rats are shown in Tables 3 and 4. Administration of ATC caused a significant (p < 0.05 and p < 0.01) elevation in serum AST, ALT, ALP, total bilirubin and total cholesterol levels and a significant (p < 0.05) decrease in total protein levels. Administration of MECA at 300 and 600 mg/kg body weight caused a significant (p < 0.05 and p < 0.01) decrease in antitubercular drug-induced rise in serum AST, ALT, ALP, total bilirubin and total cholesterol levels (Figure 5). MECA (300 and 600 mg/kg, body weight) treated groups also showed a significant (p < 0.05) increase in total protein levels as compared to antitubercular combination control group (Figure 6). Results of administration of MECA (600 mg/kg) were comparable to standard control group. Administration of MECA did not elevate the albumin levels which were depleted in antitubercular combination control group (Table 4).

Figure 5. Effect of methanol extract of Cassia auriculata (MECA) roots on serum AST, ALT, ALP and total cholesterol in antitubercular drug-induced hepatotoxic rats. N = 6; Values are mean ± SEM. NC: normal control, ATC: antitubercular combination control; AST: aspartate transaminase, ALT: alanine transaminase, ALP: alkaline phosphatase, #p < 0.05, ##p < 0.01 as compared to normal control group. *p < 0.05, **p < 0.01 as compared to antitubercular combination control group. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

Figure 6. Effect of methanol extract of Cassia auriculata (MECA) roots on serum total proteins, albumin and total bilirubin in antitubercular drug-induced hepatotoxic rats. N = 6; Values are mean ± SEM. NC: normal control, ATC: antitubercular combination control. #p < 0.05, ##p < 0.01 as compared to normal control group. *p < 0.05 when compared to antitubercular combination control group. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

Table 3. Effect of methanol extract of Cassia auriculata (MECA) roots on serum AST, ALT, ALP and total bilirubin in antitubercular drug-induced hepatotoxic rats.

Groups	AST (IU/l)	ALT (IU/l)	ALP (KA units/l)	Total bilirubin (mg/dl)
NC	193.73 ± 5.90	36.003 ± 5.20	146.40 ± 8.15	0.345 ± 0.09
ATC	262.53 ± 11.53#	77.895 ± 6.36##	278.53 ± 15.85##	1.79 ± 0.15#
ATC + MECA 300 mg/kg	208.08 ± 13.64*	61.30 ± 2.78*	190.17 ± 22.53**	1.82 ± 0.70
ATC + MECA 600 mg/kg	196.6 ± 11.50*	55.50 ± 2.79**	181.84 ± 13.42**	0.51 ± 0.14*
ATC + Silymarin 100 mg/kg	168.33 ± 3.70**	54.92 ± 4.47**	177.16 ± 18.72**	0.47 ± 0.07*

Data are expressed as mean ± S.E.M. (n = 6). NC: normal control, ATC: antitubercular combination control. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

#p < 0.05, ##p < 0.01 as compared to normal control group.

*p < 0.05, **p < 0.01 as compared to antitubercular combination control group.

Table 4. Effect of methanol extract of Cassia auriculata (MECA) roots on serum total proteins, albumin and total cholesterol in antitubercular drug-induced hepatotoxic rats.

Groups	Total proteins (g/dl)	Albumin (g/dl)	Total cholesterol (mg/dl)
NC	9.03 ± 0.56	3.92 ± 0.46	42.54 ± 2.44
ATC	4.56 ± 0.13##	3.75 ± 0.16	65.96 ± 3.49#
ATC + MECA 300 mg/kg	6.66 ± 0.50*	3.73 ± 0.24	52.035 ± 2.80
ATC + MECA 600 mg/kg	6.93 ± 0.59*	3.19 ± 0.19	44.79 ± 5.37*
ATC + Silymarin 100 mg/kg	6.63 ± 0.39**	4.09 ± 0.14	44.75 ± 5.37*

Data are expressed as mean ± S.E.M. (n = 6). NC: normal control, ATC: antitubercular combination control. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

#p < 0.05, ##p < 0.01 as compared to normal control group.

*p < 0.05, **p < 0.01 as compared to antitubercular combination control group.

Effect of MECA on hepatic antioxidant parameters

The results of hepatic SOD, GSH, CAT and TBARS in antitubercular drug-induced hepatotoxicity are given in Table 5. The SOD, CAT and GSH levels from the liver homogenate decreased significantly (p < 0.05, p < 0.01 and p < 0.01, respectively) in toxic (antitubercular combination) control group. A significant (p < 0.05) rise in depleted levels of SOD and GSH were observed in groups treated with MECA (300 and 600 mg/kg) as compared to antitubercular combination control group (Table 5). Antitubercular drug combination also caused a significant (p < 0.05) rise in liver TBARS levels. Administration of MECA (600 mg/kg) caused a significant (p < 0.05) decrease in antitubercular drug-induced rise in liver TBARS levels (Figure 7). In all the antioxidant parameters, MECA showed better activity at a higher dose level (600 mg/kg), which was comparable to the silymarin-treated group.

Figure 7. Effect of methanol extract of Cassia auriculata (MECA) roots on liver tissue TBARS levels in antitubercular drug-induced hepatotoxic rats. N = 6; Values are mean ± SEM. NC: normal control, ATC: antitubercular combination control, TBARS: Thiobarbituric acid reactive substances. ##p < 0.01 as compared to normal control group. *p < 0.05 when compared to antitubercular combination control group. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

Table 5. Effect of methanol extract of Cassia auriculata (MECA) roots on liver SOD, GSH, CAT and TBARS in antitubercular drug-induced hepatotoxic rats.

Groups	SOD (U/mg protein used)	GSH (µ moles/mg wet liver)	CAT (U/min)	TBARS (n moles/mg wet liver)
NC	25.82 ± 1.47	0.20 ± 0.02	2.8 ± 0.35	2.57 ± 0.48
ATC	9.93 ± 1.14##	0.13 ± 0.00#	0.53 ± 0.25##	6.27 ± 0.18##
ATC + MECA 300 mg/kg	20.69 ± 3.71*	0.17 ± 0.01	1.18 ± 0.26	7.07 ± 1.20
ATC + MECA 600 mg/kg	27.50 ± 2.29**	0.18 ± 0.00*	0.874 ± 0.37	3.81 ± 0.21*
ATC + Silymarin 100 mg/kg	21.42 ± 2.23**	0.19 ± 0.00*	1.66 ± 0.13*	3.44 ± 0.35*

Data are expressed as mean ± S.E.M. (n = 6). NC: normal control, ATC: antitubercular combination control. Data analyzed by one-way ANOVA followed by Dunnet’s multiple test for comparison.

#p < 0.05, ##p < 0.01 as compared to normal control group.

*p < 0.05, **p < 0.01 as compared to antitubercular combination control group.

Effect of MECA on liver histopathology

As shown in Figure 8, administration of antitubercular drug combination caused toxic changes in the hepatic architecture as evidenced by changes such as portal triaditis, eosinophilic infilteration around central vein, dilatation of central vein and necrosis of single cell and piecemeal type as compared to liver of normal control group. MECA-treated groups at 300 and 600 mg/kg dose levels showed decreased evidence of portal triaditis, eosinophilic infilteration, central vein dilatation, scanty necrosis and hepatocyte regenerative activity as compared to the toxic liver.

Figure 8. Effect of MECA on liver histopathology of antitubercular drug treated rat. A = Normal control; B = Antitubercular combination control; C = Antitubercular combination control + silymarin (100 mg/kg); D = Antitubercular combination control + MECA (300 mg/kg); E = Antitubercular combination control + MECA (600 mg/kg).

Results were comparable to the standard control group, which showed almost normal hepatic architecture. The results were in good agreement with the results of serum enzyme levels and hepatic oxidative stress levels.

In vitro antioxidant studies

As evident from Table 6, MECA showed a concentration dependent increase in scavenging of DPPH. IC₅₀ values of MECA, was found to be 774 µg/ml, which was comparable with standard antioxidant butylated hydroxyl toluene (BHT) (IC₅₀ = 583 µg/ml). As shown in Table 7, the scavenging of nitric oxide by MECA was found to be concentration dependent. Ascorbic acid was used as standard compound. IC₅₀ value of MECA, was found to be 144 µg/ml, which was comparable with ascorbic acid (IC₅₀ = 70 µg/ml). As shown in Figure 9, reducing power increased with an increase in extract concentration as evident by the concentration-dependant increase in optical density at 700 nm. The highest reducing power of MECA was shown at a concentration of 1000 µg/ml in the following order: ascorbic acid (2.1097) > MECA (1.3022). The results obtained were comparable with standard antioxidant ascorbic acid.

Figure 9. Reducing power of methanol extract of Cassia auriculata (MECA) roots. MECA: Methanolic extract of Cassia auriculata, ASC ACID: Ascorbic acid.

Table 6. DPPH free radical scavenging activity of methanol extract of Cassia auriculata (MECA) roots.

% Inhibition
Concentration (µg/ml)	MECA	BHT
200	12.52 ± 0.01	21.95 ± 0.01
400	26.3 ± 0.02	29.33 ± 0.01
600	38.76 ± 0.02	51.43 ± 0.02
800	47.28 ± 0.02	52.54 ± 0.01
1000	68.62 ± 0.02	60.42 ± 0.02

Data are expressed as mean ± S.E.M. (n = 3). MECA: Methanol extact of Cassia auriculata roots, BHT: Butylated hydroxy toluene.

Table 7. Nitric oxide scavenging activity of methanol extract of Cassia auriculata (MECA) roots.

% Inhibition
Concentration (µg/ml)	MECA	ASCORBIC ACID
20	8.86 ± 0.02	27.86 ± 0.01
40	12.53 ± 0.03	36.16 ± 0.01
60	20.84 ± 0.01	43.04 ± 0.01
80	33.44 ± 0.02	55.69 ± 0.02
100	41.86 ± 0.02	69.42 ± 0.02

Data are expressed as mean ± S.E.M. (n = 3). MECA: Methanol extact of Cassia auriculata roots.

Discussion

Because of the strategic placement of liver in human body, it is predisposed to numerous disorders owing to continuous interaction with chemicals, drugs and xenobiotics (Arthika & Shanthammal, 2011). Currently available allopathic treatment for liver diseases is only symptomatic and does not treat the root cause of the disease. Their side effects further limit their use (Ram & Goel, 1999). Thus, an actual curative therapeutic agent has not yet been found and management of liver disease is still a challenge to the modern health care system. Scientific studies available on medicinal plants indicate that promising phytochemicals can be developed for many health problems. A number of medicinal preparations have been advocated especially in Ayurvedic system of Indian medicine for the treatment of liver disorders. Ethanol-induced hepatotoxicity and antitubercular drug-induced hepatotoxicity are commonly used screening models for evaluation of new hepatoprotective drugs (Gupta, 1994; Pralhad & Ganesh, 2009). Reactive oxygen species (ROS) are capable of damaging biological macromolecules such as DNA, carbohydrates or proteins. Hence, it was decided to evaluate in vitro antioxidant activity of MECA on DPPH scavenging, nitric oxide scavenging and ferric reducing power assays. MECA showed potent nitric oxide scavenging activity, which may be due to the greater presence of flavonoids in the particular extract. DPPH (2,2-dipheny-lpicrylhydrazyl) is a stable free radical at room temperature, which produces a violet solution in ethanol. It shows a strong absorption band at 517 nm in visible spectrum (deep violet color). The decrease of DPPH molecules by the action of test extracts can be correlated with the number of available hydroxyl groups in methanol extract of Cassia auriculata (Annie et al., 2005). The reducing property activity shown by C. auriculata further supports the in vitro antioxidant activity of the extract.

Alcohol was recognized to be a cause of liver damage by the ancient Greeks and it is currently the most common cause of liver disease other than paracetamol (Lelbach, 1975). In a developing country like India, the number of alcohol addicts is increasing day by day. Hence, we decided to evaluate hepatoprotective potential of tarvada roots against ethanol-induced hepatotoxicity in rats and to determine the possible mechanism of action for hepatoprotection. Consumption of ethanol leads to the formation of acetaldehyde via metabolism through alcohol dehydrogenase enzyme. Acetaldehyde can form adduct with substrates and these adducts act on proteins or small molecules such as cysteins, which mediate lipid peroxidation and free radical generation in mitochondria. This in turn leads to cell damage, necrosis and steatosis (Gramenzi et al., 2006).

In this model, we administered ethanol at the selected dose level to generate hepatotoxicity and was followed by treatment with MECA. Our results confirmed the liver toxicity caused by ethanol at the selected dose level as manifested by increased serum levels of AST, ALT, ALP, total bilirubin, total cholesterol and depleted levels of total proteins and albumin as compared to normal rats. The fact that MECA at both the dose levels and silymarin decreased AST, ALT, ALP levels significantly suggest that MECA may have a role in the improvement of hepatocellular damage caused by ethanol (Thapa & Walia, 2007). The fact that MECA restored ethanol induced rise in serum alkaline phosphatase (ALP) and bilirubin to near normal levels indicates that MECA contains phytochemicals capable of treating ethanol-induced cholestasis (Deepa & Varalakshmi, 2003). Also our finding that MECA at higher dose level reversed the elevated total cholesterol and depleted total protein levels indicate the role of tarvada in repairing the functional damage of the liver caused by alcohol. Histopathological analysis supported the above findings by the appearance of minimum sinusoidal infilteration and fatty changes in MECA-treated livers. But the experimental group left for self-recovery after alcohol intoxication failed to show any significant improvement in liver function. Thus, our results indicate that MECA has a potential in speeding up the healing process of liver after alcohol intoxication. Previous study on Chinese medicinal herb Hu-chang and its anthraquinone glycosides stated that hepatocyte regeneration is due to an increased synthesis rate of ribosomal RNA (rRNA) for the activation of RNA polymerase thereby stimulating RNA synthesis (Dharmananda, 2012). Thus, the improvement in hepatocyte functioning by tarvada root extract may be due to the stimulation of RNA synthesis. Previous studies have shown that flavonoids from Radix puerariae (Fabaceae) inhibited alcohol dehydrogenase enzymes in mammals. So it can also be stated that tarvada may inhibit alcohol dehydrogenase enzyme (Middleton et al., 2000), thus decreasing the formation of acetaldehyde which is responsible for ethanol toxicity.

Further, we evaluated the effect of tarvada roots on antitubercular drug-induced hepatotoxicity in rats with a view that Cassia auriculata might be beneficial to a number of patients suffering from tuberculosis. About one-third of world's total population has latent tuberculosis and approximately nine million new cases of active tuberculosis emerge annually resulting in two to three million deaths (Naik & Panda, 2008). Currently used antitubercular chemotherapy includes a combination of three drugs, viz., isoniazid (INH), rifampicin (RMP) and pyrazinamide (PZA). Each of them has side effect of liver damage and their toxicity increases in a synergistic manner when they are given together. In the present study, we administered a combination of isoniazid, rifampicin and pyrazinamide for 30 days to induce hepatotoxicity in rats. Our results of biochemical parameters such as AST, ALT, ALP, total bilirubin, total cholesterol and total proteins clearly indicate hepatotoxicity induced by antitubercular drugs as compared to normal control group and were consistent with the previous studies (Kale et al., 2003). The fact that AST, ALT, ALP and total bilirubin levels in antitubercular control group increased in comparison with normal rats indicate that the toxicity is of both hepatocellular as well as cholestatic type. Simultaneous administration of MECA to rats significantly reversed the antitubercular drug-induced elevated levels of serum marker enzymes, total bilirubin, total cholesterol and depleted levels of total protein. These findings suggest that tarvada root contains phytochemicals capable of protecting the liver against hepatotoxicity of mixed type.

These findings have been strengthened by histopathological improvement in the MECA treated liver tissue in comparison with antitubercular control group. Thus, our results indicate that the methanol extract of tarvada possess potent hepatoprotective activity against antitubercular drug-induced hepatotoxicity in rats. The reason behind the observed protective effect may be the inhibition of isoniazid hydrolase enzyme by MECA, thereby reducing the formation of toxic metabolite of isoniazid known as hydrazine (Tondon et al., 2008). It is also possible that the extract may reduce the rifampicin-mediated induction of cytochrome P-450 enzyme by allosteric modification of the enzyme thereby reducing the synergistic effect of the combination of antitubercular drugs.

Previous studies suggest that oxidative stress is a mediator of liver toxicity in various experimental models such as paracetamol, carbon tetrachloride, antitubercular drug and ethanol-induced hepatotoxicity (Adewusi & Afolayan, 2010). Hence, we decided to evaluate liver tissue of all experimental groups for thiobarbituric acid reactive substances (TBARS) level and catalase levels in the ethanol-induced hepatotoxicity model. The ethanol control group showed a significant rise in TBARS levels as compared to the normal control group indicating that induction of oxidative stress is involved in ethanol-induced hepatotoxicity. This indication was supported by depleted catalase levels in the ethanol control group. Our findings that MECA restored the TBARS levels suggest that MECA can reduce the lipid peroxidation induced by ethanol.

However, MECA at any of the dose levels failed to increase the depleted catalase levels at both the dose levels. Phytochemicals are known to stimulate synthesis of antioxidant enzymes at the transcriptional level through antioxidant response elements (Duh et al., 2011). Thus, tarvada roots do not contain any phytochemicals, which are capable of upregulating the synthesis of liver catalase enzyme. This suggest that there are some other enzymatic antioxidants whose levels are modified by MECA such as reduced glutathione (GSH), glutathione peroxidase (GPX) or superoxide dismutase (SOD).

Therefore, in the antitubercular drug-induced hepatotoxicity model, in addition to estimation of TBARS and catalase, we evaluated the levels of SOD and GSH in the liver tissue. Our results showed that SOD, GSH and CAT levels were depleted and TBARS levels were increased in intoxicated rats as compared to normal rats indicating that oxidative stress is the mediator of antitubercular drug-induced liver toxicity. SOD is an important defense enzyme and catalyzes the dismutation of harmful superoxide anions. CAT is also an antioxidant enzyme, which catalyzes the reduction of hydrogen peroxide and prevents the tissue from oxidative damage. GSH along with SOD and CAT plays a vital role in maintaining the antioxidant status of the liver by scavenging reactive toxic metabolites of INH and RMP (Tondon et al., 2008). Our study revealed that MECA administration at a higher dose level significantly increased hepatic GSH and SOD levels that were depleted in the antitubercular control group. This may be due to stimulation of de novo synthesis of the antioxidant enzymes, which may be because of capability of MECA to cause upregulation of transcriptional factors (Hussain et al., 2012). Thus, MECA indirectly prevents the formation of toxic metabolites of INH by stimulating the synthesis of hepatic antioxidant enzymes. This potential of increase in the levels of enzymatic antioxidants may be a mechanism for the improvement in serum biochemical parameters caused by tarvada.

Comparison of the two experimental models showed that tarvada roots showed better improvement in the serum parameters in antitubercular drug-induced hepatotoxicity model. The reduction caused by the extract for liver thiobarbituric acid substances level was also more pronounced in the antitubercular drug-induced hepatotoxicity model as compared to the ethanol-induced hepatotoxicity model, which shows that antioxidant activity exerted by MECA was greaten in the prophylactic model. Thus, it can be stated that tarvada roots have shown more potent hepatoprotective activity as a prophylactic and anti-hepatotoxic agent than as a therapeutic agent. Review of the literature revealed that anthraquinone glycosides, such as rubiadin (Rao et al., 2006) and emodin (Bhadauria, 2010), tannins and phenolic compounds (Atta et al., 2006) have potent hepatoprotective activity. Flavone glycosides such as hirsutrin, avicularin, quercetin, rutin (Janbaz et al., 2002) and apigenin (Sayed et al., 2011) have significant antioxidant and hepatoprotective activities. The fact that previous reports (Balakrishna et al., 2011) and our results of phytochemical analysis detected the presence of tannins, flavonoids, phenolic compounds and anthraquinone glycosides in tarvada root extract strongly indicate that the hepatoprotective mechanism in the present study can be attributed to the unique classes of phytochemicals, such as tannins, flavonoids, phenolic compounds and anthraquinone glycosides.

Conclusions

Hence, from the present study we conclude that the methanol extract of tarvada roots have significant hepatoprotective activity against ethanol and antitubercular drug-induced hepatotoxicity. The mechanism for the observed hepatoprotection may be inhibition of drug metabolising enzymes, such as alcohol dehydrogenase and cytochrome P-450, in addition to the antioxidant activity of the extract. However, further studies are obligatory for the identification and separation of hepatoprotective components from the extract and to reveal the exact mechanism of action for the observed hepatoprotection.

Declaration of interest

The authors report no declarations of interest.

Acknowledgements

The authors are thankful to the Principal, Modern College of Pharmacy, Nigdi, Pune, for providing the necessary facilities to carry out research work. The authors are thankful to Mr. B.P. Pimple for his valuable suggestions on phytochemical aspects of the study.