Hepatoprotective activity of punarnavashtak kwath, an Ayurvedic formulation, against CCl₄-induced hepatotoxicity in rats and on the HepG2 cell line

Abstract

Objective: Punarnavashtak kwath (PNK) is a classical Ayurvedic formulation, mentioned in Ayurvedic literature Bhaishajya Ratnavali, for hepatic disorders and asthma. This study investigated the hepatoprotective activity of PNK to validate the traditional use of this formulation.

Materials and methods: PNK was prepared in the laboratory according to the method given in Ayurvedic literature. Phytochemical screening was performed to determine the presence of phytoconstituents. Hepatoprotective activity was evaluated against CCl₄-induced hepatotoxicity in rats and by its effect on the HepG2 cell line.

Results: Preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, saponins, and a bitter principle in PNK. Administration of PNK produced significant hepatoprotective effect as demonstrated by decreased levels of serum liver marker enzymes such as aspartate transaminase, serum alanine transaminase, serum alkaline phosphatase, and serum bilirubin and an increase in protein level. Thiopentone-induced sleeping time was also decreased in the PNK-treated animals compared with the CCl₄-treated group. It also showed antioxidant activity by increase in activity of glutathione, superoxide dismutase, and catalase and by a decrease in thiobarbituric acid reactive substance level compared with the CCl₄-treated group. Results of a histopathological study also support the hepatoprotective activity of PNK. Investigation carried out on the HepG2 cell line depicted significant increase in viability of cells exposed to PNK as compared with CCl₄-treated cells.

Discussion and Conclusion: It can be concluded that PNK protects hepatocytes from CCl₄-induced liver damages due to its antioxidant effect on hepatocytes. An in vitro study on HepG2 cell lines also supports its protective effect.

Keywords::

• Antioxidant activity
• hepatoprotective
• HepG2 cell line
• histopathological study
• liver enzyme
• sleeping time

Introduction

The liver is an important organ actively involved in many metabolic functions and is a frequent target for a large number of toxicants. Hepatic damage is associated with distortion of these metabolic functions (Wolf, 1999). Liver disease is still a worldwide health problem. Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects. In the absence of a reliable liver protective drug in modern medicine, there are a number of medicinal preparations in Ayurveda recommended for the treatment of liver disorders (Chatterjee 2000).

In the traditional system of Indian medicine, plant formulation and combined extracts of plants are used as the drug of choice rather than individual plant extracts and these herbal formulations are used for the treatment of a wide variety of diseases (Ansarullah et al., 2009). This therapeutic approach is often ignored by many and considered to be an alternative to conventional medicine by others due to lack of scientific validation of efficacy and safety (Yuan & Lin, 2000). Hence, there is a requirement for scientific proof (biological assays, animal models, clinical trials, and chemical standardization).

Punarnavashtak kwath (PNK) is an Ayurvedic polyherbal preparation mentioned in Ayurvedic literature Bhaishajya Ratnavali (Vidhyotiny 2004) for hepatic disorders and asthma. It consists of Boerhaavia diffusa Linn. (Nyctaginaceae), Picrorhiza kurroa Royle ex Benth. (Scrophulariaceae), Tinospora cordifolia (Willd.) Miers (Menispermaceae), Zingiber officinalis Rosc. (Zingiberaceae), Berberis aristata DC (Berberidaceae), Terminalia chebula Retz (Combretaceae), Azadirachta indica A. Juss. (Meliaceae), and Tricosanthes dioica Roxb. (Cucurbitaceae) plants. An aqueous extract of thinner roots of B. diffusa exhibited in vivo hepatoprotective activity against hepatic injury in rats (Rawat et al., 1997). P. kurroa and its active constituents were effective in preventing liver toxicity caused by numerous toxic agents (Saraswat et al., 1999). T. cordifolia showed significant in vivo hepatoprotective activity in CCl₄-induced hepatopathy in goats and in vitro inactivating property against Hepatitis B and E surface antigen (Singh et al., 2003). The aqueous ethanol extract of Z. officinalis showed hepatoprotective effect against acetaminophen-induced acute toxicity due to its direct radical scavenging capacity (Ajith et al., 2007). B. aristata and berberine (an alkaloid from B. aristata) were found to be protective against both paracetamol and CCl₄-induced liver damage (Janbaz, 1995). T. chebula extract was found to prevent the hepatotoxicity caused by the administration of rifampicin, isoniazid, and pyrazinamide (Tasduq et al., 2006). The aqueous extract of A. indica was found to offer protection against paracetamol-induced liver necrosis in rats (Biswas et al., 2002). T. dioica was reported as a hepatoprotective agent in ferrous sulfate (FeSO₄)-intoxicated rats (Ghaisas et al., 2008). Literature showed that scientific evidence is available for the individual plants but not for this formulation, so in this investigation, PNK has been evaluated for its hepatoprotective action against CCl₄-induced hepatotoxicity and its effect on HepG2 cell lines. The hepatotoxin used was CCl₄, as CCl₄-induced liver dysfunction in rats simulates liver cirrhosis in humans (Pérez-Tamayo, 1983; Wensing et al., 1990).

At present, one of the plant-derived medicines approved for use in liver cirrhosis and alcoholic liver diseases is silymarin. There are a number of studies that establish the efficacy of silymarin in these conditions (Saller et al., 2001). Therefore, in this study, silymarin was used as a positive control to compare the efficacy of PNK against CCl₄-induced hepatotoxicity.

Materials and methods

Materials

Carbon tetrachloride (extra pure) was obtained from Samir Tech-Chem (Baroda, India). Reduced glutathione and thiobarbituric acid were purchased from Kemphasol (Mumbai, India). Tris-HCl was obtained from Loba Chemie (Mumbai, India). All other chemicals were obtained from SD Fine Chemicals (Mumbai, India). HepG2 cell lines were obtained from the National Centre for Cell Sciences (Pune, India), minimum essential medium (MEM) was from Gibco (CA), and fetal calf serum was from Hyclone (UT). Dimethyl sulfoxide (DMSO), trypsin, and EDTA were obtained from Sigma Chemicals (St. Loius, MO). 3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Hi Media (Mumbai, India). Diagnostic kits purchased from Span Diagnostic (Baroda, India) were used for the measurement of biochemical parameters.

Collection of plants and preparation of formulation

B. diffusa root, T. cordifolia stem, T. dioica leaves, and A. indica bark were collected from the medicinal garden of APMC College of Pharmaceutical Education and Research (January, 2008), while other plants, P. kurroa stem, B. aristata stem, T. chebula fruit, and Z. officinalis rhizome, were purchased from the market. All the plants were authenticated by Mukesh Prajapati, botanist, HNSB Ltd. Science College, Himatnagar, and voucher specimens of all plants were kept in the Department of Pharmacognosy, APMC College of Pharmaceutical Education and Research, Himatnagar (APMC 0801 to 0808). PNK (decoction) was prepared by boiling the powdered drugs (Table 1) in equal quantities in proportion with 16 times the amount of water reduced to one fourth and strained through cloth. The filtrate was evaporated and dried under reduced pressure (Vidhyotiny, 2004). Yield of the extract was 10% w/w with respect to air-dried drugs.

Table 1.  Composition of punarnavashtak kwath (PNK).

No	Botanical name	Family	Part used
1	Boerhaavia diffusa Linn.	Nyctaginaceae	Root
2	Picrorhiza kurroa Royle ex Benth.	Scrophulariaceae	Root
3	Berberis aristata DC	Berberidaceae	Stem
4	Tinospora cordifolia (Willd.) Miers	Menispermaceae	Stem
5	Terminalia chebula Retz	Combretaceae	Fruit
6	Azadirachta indica A. Juss.	Meliaceae	Bark
7	Zingiber officinalis Rosc.	Zingiberaceae	Rhizome
8	Tricosanthes dioica Roxb.	Cucurbitaceae	Leaf

Preliminary phytochemical screening

The dried extract of PNK was subjected to the preliminary phytochemical analysis for the presence of different phytoconstituents (Khandelwal, 2000). Thin layer chromatography (TLC) study of PNK was also done for the presence of different phytoconstituents (Wagner & Bladt, 2002).

Preparation of test sample

For the in vivo study, PNK was dissolved in distilled water and was given orally in different doses. Silymarin was suspended in sodium carboxymethyl cellulose (0.3%) in distilled water. The doses are expressed as milligrams of dried extract per kilogram of rat.

For investigations of the HepG2 cell line, dried extract of PNK was dissolved in MEM to obtain a stock solution of 1 mg/mL, which was stored at −20°C before use. Further dilutions were made to get concentrations ranging from 1 to 15 µg/mL with respective media. A suspension of standard powdered silymarin (50 µg/mL) was also prepared. Standard silymarin powder was first dissolved in DMSO and further dilution was done with MEM to get a concentration of 50 µg/mL, as it is not directly soluble in media. All the dilutions were done under aseptic conditions only.

Animals

Healthy untreated Wistar rats of either sex (equal ratio) weighing 180–250 g (16–18 weeks old) were used for hepatoprotective activity and Swiss albino female mice weighing between 25 and 30 g (10–12 weeks old) were used for acute toxicity study. All animals (mice and rats) were collected from the animal house, Zydus Cadila Pharmaceuticals, Ahmedabad. The animals were grouped and housed in polyacrylic cages with not more than two animals per cage and maintained under well-controlled conditions of temperature (27 ± 2°C), humidity (55 ± 5%), and 12/12 h light–dark cycle. Conventional laboratory diet and tap water were provided ad libitum. The protocol of the experiments was approved by the Institutional Animal Ethical Committee as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India (Proposal no. 08/02, APMC, Himatnagar).

Acute toxicity study

The animals (mice) were divided into seven groups of six animals each. The control group received normal saline (2 mL/kg body weight p.o.) while other groups received 300, 600, 800, 1000, 2000, and 3000 mg/kg of the test extract. Immediately after dosing, the animals were observed continuously for the first 4 h for any behavioral changes. They were then kept under observation for 14 days after drug administration to find out the mortality if any. Observations were made twice daily, one at 7 a.m. and again at 7 p.m. (OECD 2001).

Hepatoprotective effect of PNK in CCl₄-induced liver damage

The animals (rats) were divided into six groups each consisting of six animals. Carbon tetrachloride diluted with liquid paraffin (1:1) was administered in a dose of 1 mL/kg body weight intraperitoneally for 2 days to all animal groups except the control group to induce hepatotoxicity. Groups III, IV, V, and VI received CCl₄, and after 30 min of first dose of CCl₄, they were treated with the listed dose of PNK or silymarin (Rao and Mishra 1998; Porchezhian and Ansari 2005).

Group I: Normal control animals (received vehicle).

Group II: Carbon tetrachloride-treated control animals.

Group III: PNK-treated animals (50 mg/kg bw, p.o. 3 times at 12 h interval).

Group IV: PNK-treated animals (100 mg/kg bw, p.o. 3 times at 12 h interval).

Group V: PNK-treated animals (150 mg/kg bw, p.o. 3 times at 12 h interval).

Group VI: Silymarin-treated animals (50 mg/kg bw, p.o. 3 times at 12 h interval).

After the last dose of PNK, thiopentone sodium was injected (40 mg/kg bw i.p.; Gujrati et al. 2007) to all groups to record sleeping time. After 36 h of carbon tetrachloride treatment, blood was collected and serum was separated and analyzed for biochemical parameters. After killing, the liver was isolated and used for biochemical parameters and histopathological analysis.

Biochemical studies

Blood was obtained from the retro-orbital plexus of all animals. Serum was separated by centrifugation at 800g at 30°C for 15 min and used for the estimation of various biochemical parameters, namely serum aspartate transaminase (AST), serum alanine transaminase (ALT) (Reitman & Frankel 1957), serum alkaline phosphatase (ALP) (Kind & King 1954), serum bilirubin (SBRN) (Malloy & Evelyn 1937), and total protein content (Lowry et al., 1951).

Antioxidant activity

For estimating antioxidant activity, the animals were killed by light ether anesthesia and their livers were excised, rinsed in ice-cold normal saline, followed by 0.15 M Tris-HCl (pH 7.4) blotted dry and weighed. A 10% w/v of homogenate was prepared in 0.15 M Tris-HCl buffer and processed for the estimation of thiobarbituric acid reactive substance (TBARS) (Ohkawa et al., 1979). After precipitating proteins with trichloroacetic acid, part of the homogenate was used for estimation of glutathione (GSH) (Ellman, 1959). The remaining homogenate was centrifuged at 10,500g for 20 min at 4°C. The supernatant thus obtained was used for the estimation of superoxide dismutase (SOD) (Misra & Fridovich, 1972) and catalase (CAT) (Aebi, 1984).

Histopathological studies

Paraffin sections (7-µm thick) of buffered formalin-fixed liver samples were stained with hematoxylin-eosin to study the histological structure of control and treated rat liver.

Hepatoprotective activity of PNK on HepG2 cell line

The screening of hepatoprotective activity was based on protection of human liver-derived HepG2 cells against CCl₄-induced damage (Ira et al., 1997; Vijayan et al., 2003) determined by estimating mitochondrial synthesis using tetrazolium assay (Ke et al., 1999; Brugisser et al., 2002). HepG2 cells were routinely grown and subcultured as monolayers in MEM supplemented with 10% fetal calf serum. The experiment in this investigation was conducted with the cells that had been initially batch cultured for 10 days. At this stage, the cells were harvested and plated at approximately 2 × 10⁵ cells/well in 96-well microtiter plates (Nunclon, Denmark) and placed in a humidified atmosphere of 5% CO₂ at 37°C for a 24 h. After a 24-h period, the cells were incubated for 1 h with 100 µL medium containing 1% CCl₄ (toxicant), with or without various concentration of extract (1–15 µg/mL) or the standard (50 µg/mL). Some cells were incubated with fresh medium alone (as normal/control). At the end of the period, the medium was removed and replaced with 100-µL fresh medium (washing step), so that there was no direct interaction between the test extract and MTT (Brugisser et al., 2002). The cytotoxicity was assessed by estimating viability of HepG2 cells by MTT reduction assay (Ke et al., 1999; Brugisser et al., 2002). After the washing step, 10 µL MTT (5 mg/mL MTT prepared in MEM without phenol red) was added to each well. The plate was gently shaken and incubated for 3 h at 37°C in a humidified 5% CO₂ atmosphere. At the end of the period, 100-µL MTT solubilizing buffer (propanol) was added to each well and the plate was gently shaken to solubilize the blue-colored formazan. The plates were directly read on a microtiter plate reader SPECTRAmax 190 (Molecular Devices, USA) at 540 nm.

Statistical analysis

Results are expressed as mean ± SEM. The statistical difference was analyzed by one-way analysis of variance followed by the Tukey–Kramer multiple comparison test. Significance was calculated as the P value, and P values of less than 0.05 were regarded as statistically significant.

Results

Preliminary phytochemical screening

Preliminary phytochemical screening showed the presence of alkaloids, tannins, carbohydrates flavonoids, and saponins in PNK (Table 2). The TLC pattern of PNK showed the presence of tannins, alkaloids, phenolic compounds, saponins, and bitter principle (Table 3).

Table 2.  Preliminary phytochemical screening of PNK (chemical test).

Constituents	Test	Results
Alkaloid	Dragendorff test Hager’s test Wagner’s test	Positive Positive Positive
Tannin	Gelatin test Lead acetate test	Positive
Phenolic	Ferric chloride test Folin-Ciocalteu test	Positive
Saponin	Foam test	Positive
Carbohydrate	Molisch test	Positive
Flavonoid	Shinoda test	Positive

Table 3.  TLC pattern of PNK solvent system: toluene:ethylacetate:methanol:water (3:3:0.2:0.8).

Detection	Rf value	Inference
UV light at 254 nm	0.12, 0.29, 0.39, 0.49, 0.55	–
UV light at 366 nm	0.13 (blue) 0.2 (yellow) 0.24 (blue) 0.38 (blue) 0.39 (blue) 0.54 (blue) 0.69 (blue) 0.73 (blue) 0.77 (blue)	–
Dragendorff reagent	0.2	Alkaloid
Anisaldehyde sulfuric acid	0.19 (brown) 0.49 (brown) 0.54 (violet) 0.65 (brown) 0.78 (blue) 0.9 (brown)	Bitter principle
Vanillin sulfuric acid	0.19 (blue) 0.29 (brown), 0.42 (brown), 0.53 (violet), 0.6 (violet), 0.64 (brown), 0.72 (violet)	Bitter principle and saponin
Ferric chloride reagent	0.39 (blue) 0.56 (blue)	Phenolic and tannin

Acute toxicity study

In the acute toxicity study, it was observed that there was no mortality at any of the tested doses (up to 3000 mg/kg) at the end of 14 days of observation, so the LD₅₀ was more than 3 g/kg.

Hepatoprotective effect of PNK in CCl₄-induced liver damage

Intoxication of rats treated with CCl₄ significantly (P < 0.001) altered the biochemical parameters (ALT, AST, ALP, SBRN, and total protein) when compared with normal control rats, coupled with marked hepatic oxidative stress (Table 4). Thiopentone-induced sleeping time was also significantly (P < 0.001) increased in CCl₄-treated group (Table 5). CCl₄ challenge significantly decreased (P < 0.001) the level of SOD and CAT in liver. Intracellular antioxidant GSH level was also significantly depleted (P < 0.001). The level of TBARS, which is produced as a result of lipid peroxidation, was significantly increased (P < 0.001) (Table 6).

Table 4.  Effect of PNK on different biochemical parameters in CCl₄-induced hepatotoxicity in rats.

Group	AST (U/L)	ALT (U/L)	ALP (U/L)	SBRN (mg%)	Protein (mg/dL)
Control	24 ± 1.26	24.66 ± 2.06	60.16 ± 2.15	0.5 ± 0.03	6.24 ± 0.18
Toxicant	166.0 ± 4.47^†	160.83 ± 7.2^†	154.0 ± 9.8^†	0.96 ± 0.06^†	4.43 ± 0.24^†
Test (50 mg/kg)	95.33 ± 9.0***	77.33 ± 8.19***	115.67 ± 6.71*	0.81 ± 0.06^NS	4.98 ± 0.18***
Test (100 mg/kg)	80.5 ± 6.34***	60.83 ± 8.52***	84.0 ± 6.22***	0.63 ± 0.06**	5.43 ± 0.24***
Test (150 mg/kg)	67.17 ± 4.25***	55.17 ± 6.1***	73.5 ± 4.22***	0.55 ± 0.08***	5.48 ± 0.25***
Silymarin(50 mg/kg)	57.0 ± 3.97***	52.5 ± 4.68***	66.83 ± 1.54***	0.51 ± 0.02***	5.6 ± 0.17***

Values are expressed as mean ± SEM of 6 animals in each group. †P < 0.001 relative to control group; ***P < 0.001 relative to toxicant group; **P < 0.01 relative to toxicant group; *P < 0.05 relative to toxicant group; NS, not significant.

Table 5.  Effect of PNK on thiopentone sodium-induced sleeping time in CCl₄-induced hepatotoxicity in rats.

Group	Thiopentone-induced sleeping time
	Onset(s)	Duration (min)
Control	168.33 ± 8.23	77.5 ± 3.82
Toxicant	62 ± 5.13^†	186.00 ± 12.74^†
Test (50 mg/kg)	103.67 ± 7.04**	129.0 ± 9.61***
Test (100 mg/kg)	134.17 ± 8.51***	113.67 ± 7.13***
Test (150 mg/kg)	139.83 ± 7.4***	105.33 ± 6.89***
Silymarin (50 mg/kg)	144.5 ± 10.3***	96.67 ± 9.19***

Values are expressed as mean ± SEM of 6 animals in each group. †P < 0.001 relative to control group; ***P < 0.001 relative to toxicant group; **P < 0.01 relative to toxicant group.

Table 6.  Effect of PNK on lipid peroxidation (TBARS), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) level in CCl₄-induced hepatotoxicity in rats.

Group	TBARS (μmol/mg protein)	SOD (U/mg protein)	Catalase (U/min/mg protein)	Glutathione (nmol/mg protein)
Control	0.53 ± 0.05	11.11 ± 1.1	12.94 ± 0.73	101.77 ± 3.97
Toxicant	3.26 ± 0.45†	3.66 ± 0.24 †	1.65 ± 0.47 †	48.37 ± 2.79 †
Test (50 mg/kg)	1.55 ± 0.2***	5.94 ± 0.09^NS	5.69 ± 1.19^NS	63.16 ± 7.03^NS
Test (100 mg/kg)	1.01 ± 0.08***	6.84 ± 0.31**	7.07 ± 1.198*	72.18 ± 2.36*
Test (150 mg/kg)	0.96 ± 0.06***	7.25 ± 0.1**	7.19 ± O.19*	78.26 ± 6.65**
Silymarin (50 mg/kg)	0.95 ± 0.05***	7.61 ± 0.42***	8.38 ± 1.47**	78.96 ± 2.36**

Values are expressed as mean ± SEM of 6 animals in each group. †P < 0.001 relative to control group; ***P < 0.001 relative to toxicant group; **P < 0.01 relative to toxicant group; *P < 0.05 relative to toxicant group. NS, not significant.

Treatment with PNK showed a significant decrease in the levels of marker enzymes, namely ALT, AST, ALP, and SBRN compared with CCl₄-intoxicated rats. A dose-dependent significant increase in total protein level was also observed in PNK-treated animals as compared with CCl₄-treated groups (Table 4). At 100 and 150 mg/kg, PNK showed significant protective effect (P < 0.01). A significant reduction (P < 0.001) in thiopentone-induced sleeping time was observed in PNK (100 and 150 mg/kg)-treated animals compared with the CCl₄-treated group (Table 5).

Treatment with PNK at doses of 100 and 150 mg/kg significantly prevented the increase in TBARS level (P < 0.001) and brought them near a normal level, and GSH, SOD and CAT activities were significantly increased (P < 0.05) in PNK-treated groups (Table 6). The effects of PNK were compared with the standard reference drug silymarin.

In the histopathological studies, normal animals showed central vein normal liver parenchymal cells (Figure 1a). CCl₄-intoxicated animals showed extensive necrosis, inflammation, and infiltration by lymphocytes (Figure 1b). In the treated groups, at the lowest dose, necrosis, sparse lymphocyte infiltration, and occasional regenerating parenchymal cells were seen (Figure 1c). With 100 and 150 mg/kg, there was a greater amount of regeneration with mild inflammation and some lymphocytic infiltration in the necrotic area (Figure 1d and 1e). Standard silymarin showed a greater number of regenerating liver cells around the necrotic area (Figure 1f). Thus, the histopathology studies also support the protective effect of PNK.

Figure 1.  Photomicrograph of liver section taken from rats against CCl₄-induced hepatotoxicity. (A) Normal control rat. Section of liver showing hepatic cells with nuclei, cytoplasm, central vein, and portal triad. (B) Liquid paraffin: CCl₄-treated rats. Section of liver showing marked necrosis, inflammation, lymphocytic infiltration severe. (C) PNK (50 mg/kg)-treated rats. Section of liver showing necrosis, lymphocytic infiltration and occasional regenerating parenchymal cells. (D–F) PNK (100 and 150 mg/kg) and silymarin-treated rats. Section of liver showing mild inflammatory changes and greater area of regeneration.

Hepatoprotective effects on HepG2 cell line

The CCl₄-exposed HepG2 cells showed 25.37% viability. These exposed cells when treated with different concentrations of PNK showed a significant dose-dependent increase in the percentage viability when compared with the CCl₄-exposed cells. The percentage viability ranged between 30 and 88% at 1–15 µg/mL concentration of PNK (Table 7). The increase in the percentage cell viability of HepG2 cells treated with PNK at 15 µg/mL concentration was the same as that produced by standard silymarin at 50 µg/mL, from which we can infer that PNK required a lower dose than silymarin in the HepG2 cell line study.

Table 7.  Effect of PNK on CCl₄ intoxicated HepG2 cell lines.

Treatments	Conc (µg/mL)	%Viability
Control	—	100
CCl4	—	25.37 ± 2.14†
CCl4 + STD	50	84.48 ± 2.03**
CCl4+ Test	15	87.84 ± 2.03**
	10	70.94 ± 2.26**
	7	59.25 ± 1.19**
	5	48.32 ± 4.37**
	3	37.58 ± 1.06**
	1	30.43 ± 1.98*

Average of five determinations, four replicates. CCl₄, carbon tetrachloride (1%)-treated cells; STD, standard silymarin 50 µg/mL; Test, different concentrations of PNK in µg/mL.

†P < 0.001 when compared with normal cells; **P < 0.001 when compared with CCl₄-intoxicated cells; *P < 0.01 when compared to CCl₄ intoxicated cells.

Discussion

It is well established that the cleavage of a carbon–chloride bond (C–Cl bond) of carbon tetrachloride leads the formation of trichloromethyl free radical, which further reacts with O₂ and is converted into a peroxy radical, which is involved in the pathogenesis of liver injury (Cheeseman et al., 1985). The abnormal higher level of serum AST, ALT, ALP, and SBRN observed in our study (Table 4) is the consequence of carbon tetrachloride-induced liver dysfunction and denotes the damage to the hepatic cells (Singh et al., 1999). Feeding of PNK (100 and 150 mg/kg) to CCl₄-treated rats significantly reduced the increased level of serum ALT, AST, ALP, and SBRN, and it seemed to offer protection and maintain the functional integrity of hepatic cells. In hepatotoxicity, a depression in total protein is also observed due to the defect in protein biosynthesis (Clauson, 1989; Dubey et al., 1999). This is due to the disruptions and disassociation of polyribosome from endoplasmic reticulum after CCl₄ administration. Treatment with PNK exhibited significant restoration of the altered biochemical parameters toward normal in CCl₄-intoxicated rats. This may be due to the promotion of the assembly of ribosomes in the endoplasmic reticulum to facilitate uninterrupted protein biosynthesis.

Barbiturates are a class of xenobiotics that are extensively metabolized in the liver. Deranged liver function leads to delay in the clearance of barbiturates, resulting in a longer duration of hypnotic effect (Kulkarni, 1999). In this study, administration of thiopentone sodium to rats treated with an acute dose of CCl₄ resulted in an increased duration of thiopentone sleep time. Treatment with PNK decreased the thiopentone-induced sleep time, which is the indirect evidence of hepatoprotective activity (Table 5). These findings can be further corroborated by histopathological studies.

Enhanced lipid peroxidation expressed in terms of TBARS and reduced activities of SOD and CAT observed in CCl₄-treated rats in our study (Table 6) confirms the hepatic damage to the rats (Kaplowitz et al., 1986). Oral administration of PNK caused a significant decrease in TBARS level indicating the anti-lipid peroxidative and/or adaptive nature of the system brought by PNK against the damaging effects of free radicals produced by the carbon tetrachloride.

Glutathione is one of the most abundant tripeptide, nonenzymatic biological antioxidants present in the liver. Its functions are concerned with the removal of free-radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals and maintenance of membrane protein thiols, and as a substrate for glutathione peroxidase and glutathione transferase (Prakash et al., 2001). In this study, the decreased level of GSH has been associated with an enhanced lipid peroxidation in CCl₄-treated rats. Administration of PNK significantly increased the level of glutathione.

SOD has been reported as one of the most important enzymes in the enzymatic antioxidant defense system (Curtis et al., 1972). It scavenges the superoxide anion to form hydrogen peroxide, hence diminishing the toxic effect caused by this radical. In this study, it was observed that PNK significantly (P < 0.05) increased hepatic SOD activity in CCl₄-induced liver damage in rats. This showed that PNK can reduce reactive free radicals, thereby reducing oxidative damage to the tissues besides improving activity of hepatic antioxidant enzymes.

CAT is an enzymatic antioxidant widely distributed in all animal tissues, and the highest activity is found in the red blood cells and liver. CAT decomposes hydrogen peroxide and protects the tissue from highly reactive hydroxyl radicals (Chance et al., 1952). Therefore, reduction in the activity of these enzymes may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide. Administration of PNK increases the activity of CAT in CCl₄-induced liver damage in rats to prevent the accumulation of excessive free radicals, protecting the liver from CCl₄ intoxication.

HepG2 cells, a human hepatoma cell line, are considered a good model to study in vitro xenobiotic metabolism and toxicity to the liver, because they retain many of the specialized functions that characterize normal human hepatocytes (Knasmüller et al., 1998). The investigation carried out on human liver-derived HepG2 cells against CCl₄-induced damage also confirmed the hepatoprotective nature of the PNK.

The results from this study indicate a good correlation between in vivo and in vitro studies. The effects were compared with those of the standard drug silymarin. Our findings support the reported therapeutic use of this formulation in Ayurvedic literature for liver ailments. This may be due to synergistic effects between different herbs in a formulation (Williamson, 2001). Significant effect of PNK on antioxidant enzyme levels showed hepatoprotective activity by its antioxidant effect. Further study is in progress for the effect of PNK in other toxicant (paracetamol, ethanol)-induced hepatotoxicity for mechanism of action. Clinical study is also in progress to support traditional use of this formulation in jaundice and other hepatic disorders. The study has been registered in the clinical trial registry of India (CTRI/2009/091/000719, 22-09-2009).

Acknowledgments

The authors thank Mukul Jain (Vice President and Head), Prabodha Swain (Principal Scientist), and Gaurav Pandya (Research Associate) of the Pharmacology Department, Zydus Research Centre, Ahmedabad, for providing facilities to work on HepG2 cell lines.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.