Hepatoprotective and antioxidant activity of Karisalai Karpam, a polyherbal Siddha formulation against acetaminophen-induced hepatic damage in rats

Abstract

Background:

The usage of Siddha medicine in Tamil Nadu and several parts of Southern India has considerably increased over the past two decades and it is steadily crossing the various geographies owing to its inexpensiveness compared to conventional medicines and has fairly high acceptance rates because of its herbal origin and therefore its nontoxic nature.

Aim:

This study aims to investigate the anti-hepatotoxic and antioxidant potential of the Karisalai Karpam formulation.

Materials and Methods:

Karisalai Karpam tablet at 50, 100, and 200 mg/kg/day, p.o. doses were administered orally to rats for three consecutive days. Single dose of acetaminophen (3 g/kg, p.o.) was administered on the 3^rd day. Animals were sacrificed 48 h after the administration of acetaminophen, and their serum bilirubin, different hepatic enzymes and in vivo antioxidant activity were estimated.

Statistical Analysis:

Data were evaluated using analysis of variance, followed by Tukey tests. A level of P < 0.05 was considered statistically significant.

Results:

Pretreatment with Karisalai Karpam tablet showed dose-dependent hepatoprotective activity. Karisalai Karpam tablet (200 mg/kg) reduces serum glutamic oxaloacetate transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase and total bilirubin, direct bilirubin by 67.8%, 72.3%, 47.6%, 61.3% and 62.9% respectively compared to disease control group. A significant increase (P < 0.001) in antioxidant enzyme level was observed in Karisalai Karpam treated animals. At higher doses, Karisalai Karpam prevented the depletion of glutathione in liver tissue.

Conclusion:

Results confirmed that Karisalai Karpam tablet could protect the liver against acetaminophen-induced oxidative damage possibly by increasing the antioxidant defence mechanism in rats.

INTRODUCTION

Synthetic drugs such as nonsteroidal anti-inflammatory drugs are used commonly as the main course or as adjuvant therapy for several disease conditions. Acetaminophen (paracetamol, n-acetyl-p-aminophenol) is a common and most widely used analgesic and antipyretic drug. This drug is associated with both intentional and accidental poisoning. Toxic effects on the liver due to the excess use of paracetamol may lead to death.[1] In view of this, use of the herb, herbal product, or phyto-constituents is becoming attractive as the first choice to avert the toxic effect of drugs on the liver.

Easy accessibility, low levels of technological input, low cost, broad continuing acceptance and relative low side-effects have increased the utilization of traditional medicines including Siddha medicine in many parts of India.[2] Karisalai Karpam tablet is a Siddha formulation containing seven plants. It is a proprietary medicine used to cure liver disorders such as jaundice, enlargement of liver and spleen, hepatospleenomegaly, anemia and is also beneficial in renopathic conditions and skin diseases. Until now, no research has been reported on the hepatoprotective activity of Karisalai Karpam. In view of above, the present study was designed to evaluate the hepatoprotective effect of Karisalai Karpam tablet against acetaminophen induced liver injury.

MATERIALS AND METHODS

Polyherbal formulation

Karisalai Karpam tablet is manufactured by SKM Siddha and Ayurveda Company (India) Limited, Erode, Tamil Nadu. The product is obtained from the SKM Siddha and Ayurveda Company (India) Limited, Erode (Batch No.: MHD 13002, Mfg date: April, 2013), and it is formulated using Karisalankanni (Eclipta prostrata L. 15%), Mañjal karisalai (Wedelia calendulaceae L. 15%), Avuri (Indigofera tinctoria L. 15%), Kottakkarandai (Sphaeranthus indicus L. 15%), Vallarai (Centella asiatica L. 15%), Kuppaimeni (Acalypha indica L. 15%), Siruseruppadai (Coldenia procumbens L. 5%), juice of E. prostrata L. and W. calendulaceae L. (q.s.).

Animals

Healthy Wistar rats (150–200 g) were used in the study. Animals were housed under standard environmental conditions. The Institutional Animal Ethical Committee (Reg. No. 1305/ac/09/CPCSEA) had approved the study.

Hepatoprotective activity

Rats were randomly divided into six groups (n = 6) in the following manner:

• Group I (healthy control): 0.5% sodium carboxymethyl cellulose (CMC) as vehicle

• Group II (disease control): 0.5% sodium CMC + paracetamol (3 g/kg)

• Group III (standard group): Silymarin (100 mg/kg, p.o. for 3 days) + paracetamol (3 g/kg)

• Groups IV-VI (test drug-treated group): Karisalai Karpam tablet (50, 100 and 200 mg/kg, p.o., for 3 days) + paracetamol (3 g/kg).

The animals in above stated groups were treated with respective drug or vehicle once daily for 3 consecutive days followed by oral administration of 3 g/kg of acetaminophen on the 3^rd day to all animals except those in Group I.[3] All the standard and test drugs were suspended in 0.5% sodium CMC and administered orally. Animals were anaesthetized and sacrificed 48 h after administration of acetaminophen. Blood samples were collected and centrifuged at 4000 ×g for 30 min to obtain the serum used for the analyses of serum glutamic oxaloacetate transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin (TB), direct bilirubin (DB), using commercial biochemical kit obtained from Agapee Diagnostic Ltd., Kerala.[3]

In vivo antioxidant activity

Liver samples were homogenized in 0.15 M KCl-10 mM potassium phosphate buffer (pH 7.4) to give a 10% (w/v) liver homogenate.[4] The liver homogenates were then centrifuged at 3000 ×g for 10 min to get clear supernatant, which was used for the determination of antioxidant status. The content of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and nonenzymatic antioxidant glutathione (GSH) in liver tissue was determined. In brief, SOD activity was estimated by the inhibition of autocatalyzed adrenochrome formation at 480 nm in the presence of tissue homogenate.[5] For CAT, the rate of H₂O₂ decomposition was determined spectrophotometrically at 240 nm,[6] GPx activity evaluated using the molar extinction coefficient of nicotinamide adenine dinucleotide phosphate (NADPH) 6220/M/cm and expressed as μM reduced NADPH oxidized/min/mg protein at 37°C,[7] while GSH was measured by 5,5-dithiobis (2-nitrobenzoic acid) method.[8] All samples were assayed in triplicates.

Histopathological examination

After the animals were sacrificed, sections of livers from each group were fixed immediately in 10% neutral formalin for a period of at least 24 h, dehydrated in graded (50–100%) alcohol and embedded in paraffin. Cross-sections of the liver tissue (5–6 μM thickness) were prepared and stained with hematoxylin-eosin dye. These sections were subjected to microscopical examination.

Statistical analysis

The data were subjected to analysis of variance (ANOVA) and expressed as mean ± standard error of mean (n = 6). Statistical analysis was carried out by ANOVA followed by Tukey tests. A level of P < 0.05 was used as the criterion for statistical significance.

RESULTS

This study was planned with a view to evaluate the efficacy of Karisalai Karpam tablet against acetaminophen-induced liver disorder and to investigate its possible underlying mechanisms.

Hepatoprotective activity

In our study, we observed that the treatment with the toxic dose of paracetamol results in significant (P < 0.001) increase in SGPT, SGOT, ALP activity and TB, DB level in serum in negative control group thereby confirming drug induced hepatocellular toxicity. Treatment with Karisalai Karpam tablet was found to normalize the level of these enzymes. Karisalai Karpam showed dose-dependent activity and in higher dose it prevented the increase in SGPT, SGOT, ALP, TB, and DB by 67.8%, 72.3%, 47.6%, 61.3% and 62.9% respectively as compared to the disease control group. The hepatoprotective activity was comparable to the standard drug silymarin [Table 1].

Table 1.

Hepatoprotective activity of KKT

In vivo antioxidant activity

Marked decrease in concentration of GSH, SOD, CAT and GPx was observed in paracetamol treated animals. Karisalai Karpam at 100 and 200 mg/kg significantly produced in vivo antioxidant activity by restoring liver GSH, blood GSH, SOD, CAT, GPx level. Karisalai Karpam (200 mg/kg) was found effective in increasing in liver and blood GSH, SOD and CAT [Table 2].

Table 2.

In vivo antioxidant activity of KKT

Histopathological examination

Histopathological studies also have confirmed that Karisalai Karpam showed a protective effect. At 100 and 200 mg/kg dose, the Karisalai Karpam treated rat livers showed only mild congestion which is comparable with that of standard [Figure 1].

Figure 1.

(a) H and E-stained sections of normal rat liver, no sign of inflammation or necrosis. (b) Liver section of negative control group showing extensive hemorrhagic necrosis particularly around the central vein. (c) Liver section of silymarin (100 mg/kg) treated rats exhibit almost normal hepatic parenchyma with lesser degree of congestion. (d) Section of Karisalai Karpam tablet (50 mg/kg) treated rat liver showing congestion, microvesicular steatosis and congestion of sinusoids but to a lesser extent. (e) Section of Karisalai Karpam tablet (100 mg/kg) treated rat liver showing minimal degree of congestion, no sign of necrosis and portal triads with few inflammatory cells. (f) Liver section of Karisalai Karpam tablet (200 mg/kg) treated rat showing normal hepatic parenchyma with mild congestion of sinusoids

DISCUSSION

Paracetamol, soon after administration is rapidly absorbed in the stomach and small intestine and is metabolized in the liver. A small portion (5–10%) of paracetamol is converted to its reactive metabolite n-acetyl-p-benzo-quinoneimine (NAPQI) by hepatic cytochrome P450, which is responsible for oxidative stress and liver injury.[9] SGPT and SGOT are released from the liver cells during liver injury, which results increased serum levels of these enzymes. Thus, the estimation of SGOT and SGPT is considered as a specific test for detecting liver abnormalities.[10,11,12] ALP is considered as a leading marker of hepatobiliary effects and cholestasis (moderate to marked elevations). Inflammation or damage of hepatocytes results in the increase of ALP level in bloodstream. Several experimental investigations have reported that the hepatocellular damage caused by paracetamol increases the level of ALP in animal models.[10,13,14] Injury of hepatocyte and/or obstruction in excretory ducts of the liver interferes with the capability of the liver to excrete normal levels of bilirubin. Thus, serum bilirubin is generally used to identify proper liver functioning. TB is a combination of indirect bilirubin (nonhepatic) and DB (hepatic).[14] Results indicate that Karisalai Karpam tablet possesses hepatoprotective effects possibly by maintaining the structural integrity of hepatocellular membrane and liver cell architecture against paracetamol-induced damage.

SOD is involved in catalyzing the dismutation of superoxide to H₂O₂ and O₂, while CAT catalyzes H₂O₂ to water and molecular oxygen to avert the oxidative damage. GPx is present in the cytoplasm and prevents the formation of hydroxyl radicals and oxidative cell injury caused by H₂O₂.[15] GSH is the most important non-enzymatic antioxidant defense system against oxidative stress. GSH plays an important role in detoxifying foreign compounds, free radicals, and regulating thiol-disulfide status of the cell.[15] GSH also scavenges toxic metabolites, including NAPQI, thus preventing damage of hepatocytes.[13] In this study, Karisalai Karpam, specially at 200 mg/kg dose showed significant protective effect as is evident by the restored GSH levels near to normal, and inhibition of the depletion of antioxidant enzymes like SOD, CAT, GPx level. In vivo antioxidant effect of Karisalai Karpam tablet might have contributed toward its hepatoprotective effect. Several researchers based on the traditional application of these plants have investigated their hepatoprotective activity. Methanol extract of leaves of W. calendulaceae was investigated for its hepatoprotective activity, and it was found that the extract exerts hepatoprotective effect against thioacetamide-induced toxicity in rats.[16] The potent hepatoprotective effect of C. asiatica and S. indicus on CCl₄ induced liver injury in rats has been investigated.[17,18] Methanol extract and methanolic fraction of methanol extract of A. indica have shown significant hepatoprotective activity against thioacetamide-induced liver damage in albino rats.[19] Chloroform extract of whole plant of C. procumbens Linn. was tested for anti-hepatotoxic activity and the results confirmed that the extract showed a significant effect at 200 mg/kg against D-galactosamine (D-GalN) induced hepatotoxicity.[20] A study has shown that E. prostrata exerts significant hepatoprotective effect against CCl₄ and β-D-galactosamine induced toxicity but not against acetaminophen-induced toxicity in rats.[21] Hepatoprotective activity of aqueous extract of I. tinctoria (250, 500 mg/kg body weight) was investigated against paracetamol induced liver damage.[22] Histopathological studies also have confirmed the protective effects of Karisalai Karpam in this study. Oxidative stress is one of the underlying mechanisms responsible for paracetamol induced hepatotoxicity. Both paracetamol and its reactive metabolite increase the generation of free radicals and causes depletion of natural antioxidant defense mechanism. Karisalai Karpam tablet has shown prominent antioxidant activity by inhibiting the depletion of antioxidant enzymes and GSH levels in consequence of paracetamol administration, which may have played a key role in preventing paracetamol induced hepatotoxicity in Karisalai Karpam treated animals.

CONCLUSION

This study shows that Karisalai Karpam tablet possesses the significant hepatoprotective property, and this may in part be explained by the antioxidant activity of Karisalai Karpam tablet. Currently, the incidence of drug-related liver damage is increasing in an alarming rate on one hand while on the other hand, the use of paracetamol is extensive and often abused. This study establishes the protective effect of Karisalai Karpam tablet against paracetamol-induced liver damage, which provides a support for its use in treatment of liver disorders.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.