|Year : 2015 | Volume
| Issue : 4 | Page : 198-202
Hepatoprotective and antioxidant activity of Karisalai Karpam, a polyherbal Siddha formulation against acetaminophen-induced hepatic damage in rats
Saikat Sen1, Raja Chakraborty2, Ganesh Thangavel3, Sivakumar Logaiyan3
1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Assam Down Town University, Guwahati, Assam, India
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Assam Down Town University, Guwahati, Assam, India
3 R&D Division, SKM Siddha and Ayurveda Company (India) Limited, Modakurichi, Erode, Tamilnadu, India
|Date of Web Publication||15-Jul-2015|
R and D Division, SKM Siddha and Ayurveda Company (India) Limited, Modakurichi, Erode, Tamil Nadu
Source of Support: None, Conflict of Interest: None
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.
Keywords: Antioxidant activity, hepatoprotective, Indian traditional medicine, Karisalai Karpam, Siddha formulation
|How to cite this article:|
Sen S, Chakraborty R, Thangavel G, Logaiyan S. Hepatoprotective and antioxidant activity of Karisalai Karpam, a polyherbal Siddha formulation against acetaminophen-induced hepatic damage in rats. Ancient Sci Life 2015;34:198-202
|How to cite this URL:|
Sen S, Chakraborty R, Thangavel G, Logaiyan S. Hepatoprotective and antioxidant activity of Karisalai Karpam, a polyherbal Siddha formulation against acetaminophen-induced hepatic damage in rats. Ancient Sci Life [serial online] 2015 [cited 2019 Nov 20];34:198-202. Available from: http://www.ancientscienceoflife.org/text.asp?2015/34/4/198/160863
| 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.  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.  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|| |
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 Karisalankanni0 (Eclipta prostrata L. 15%), Mañjal karisalai (Wedelia calendulaceae L. 15%), Avuri0 (Indigofera tinctoria L. 15%), Kottakkarandai (Sphaeranthus indicus L. 15%), Vallarai0 (Centella asiatica L. 15%), Kuppaimeni (Acalypha indica L. 15%), Siruseruppadai0 (Coldenia procumbens L. 5%), juice of E. prostrata L. and W. calendulaceae L. (q.s.).
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.
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.  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. 
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.  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.  For CAT, the rate of H 2 O 2 decomposition was determined spectrophotometrically at 240 nm,  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,  while GSH was measured by 5,5-dithiobis (2-nitrobenzoic acid) method.  All samples were assayed in triplicates.
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.
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.
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].
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].
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|
Click here to view
| 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.  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. ,, 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. ,, 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).  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 H 2 O 2 and O 2 , while CAT catalyzes H 2 O 2 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 H 2 O 2. 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.  GSH also scavenges toxic metabolites, including NAPQI, thus preventing damage of hepatocytes.  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.  The potent hepatoprotective effect of C. asiatica and S. indicus on CCl 4 induced liver injury in rats has been investigated. , Methanol extract and methanolic fraction of methanol extract of A. indica have shown significant hepatoprotective activity against thioacetamide-induced liver damage in albino rats.  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.  A study has shown that E. prostrata exerts significant hepatoprotective effect against CCl 4 and β-D-galactosamine induced toxicity but not against acetaminophen-induced toxicity in rats.  Hepatoprotective activity of aqueous extract of I. tinctoria (250, 500 mg/kg body weight) was investigated against paracetamol induced liver damage.  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
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ray SD, Mumaw VR, Raje RR, Fariss MW. Protection of acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl hemisuccinate pretreatment. J Pharmacol Exp Ther 1996;279:1470-83.
Subbarayappa BV. Siddha medicine: An overview. Lancet 1997;350:1841-4.
Devanna N, Chakraborty R, Sen S, De B. Tribal Medicinal Plants of Tripura, India: A Scientific Search. 1 st
ed. New Delhi, India: Serial Publishers; 2014. p. 143-8.
Kesiova M, Alexandrova A, Yordanova N, Kirkova M, Todorov S. Effects of diphenhydramine and famotidine on lipid peroxidation and activities of antioxidant enzymes in different rat tissues. Pharmacol Rep 2006;58:221-8.
Qadrie ZL, Anandan R, Jacob B, Ashraf H. Liver protective activity of Indoneesiella echioides
against carbon tetrachloride (CCl 4
)-induced hepatotoxicity in rats. Pharmacologyonline 2011;2:416-29.
Huo HZ, Wang B, Liang YK, Bao YY, Gu Y. Hepatoprotective and antioxidant effects of licorice extract against CCl4-induced oxidative damage in rats. Int J Mol Sci 2011;12:6529-43.
Hsu CL, Yen GC. Effect of gallic acid on high fat diet-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. Br J Nutr 2007;98:727-35.
Raphael R. Pharmacological Activity of Plant Derived Drugs and the Mechanism of Action (with Special Reference to Phyllanthus amarus
Schum and Thonn), Ph.D thesis, Mahatma Gandhi University, Kottayam, India; 2004.
James LP, Mayeux PR, Hinson JA. Acetaminophen-induced hepatotoxicity. Drug Metab Dispos 2003;31:1499-506.
Ozer J, Ratner M, Shaw M, Bailey W, Schomaker S. The current state of serum biomarkers of hepatotoxicity. Toxicology 2008;245:194-205.
Hegde K, Joshi AB. Hepatoprotective effect of Carissa carandas
Linn root extract against CCl4 and paracetamol induced hepatic oxidative stress. Indian J Exp Biol 2009;47:660-7.
Kuriakose GC, Kurup MG. Effects of Aulosira fertilisima
against cisplatin-induced nephrotoxicity and oxidative stress in rats. Ren Fail 2010;32:224-33.
Ramachandra Setty S, Quereshi AA, Viswanath Swamy AH, Patil T, Prakash T, Prabhu K, et al.
Hepatoprotective activity of Calotropis procera
flowers against paracetamol-induced hepatic injury in rats. Fitoterapia 2007;78:451-4.
Olaleye MT, Akinmoladun AC, Ogunboye AA, Akindahunsi AA. Antioxidant activity and hepatoprotective property of leaf extracts of Boerhaavia diffusa
Linn against acetaminophen-induced liver damage in rats. Food Chem Toxicol 2010;48:2200-5.
Sen S, Chakraborty R. The role of antioxidants in human health. In: Silvana A, Hepel M, editors. Oxidative Stress: Diagnostics, Prevention, and Therapy. Washington, DC: American Chemical Society; 2011. p. 1-37.
Haldar PK, Gupta M, Mazumder UK, Kandar CC, Manikandan L. Hepatoprotective effect of Wedelia calendulaceae
against thioacetamide induced liver damage in rats. Pharmacologyonline 2007;3:414-21.
Pingale SS. Evaluation of effect of Centella asiatica
on CCl 4
induced rat liver damage. Pharmacologyonline 2008;3:537-43.
Mathews LA, Dhanyaraj D, Prathibhakumari PV, Prasad G. Hepatoprotective and antioxidant potential of Sphaeranthus indicus
0 [Linn] on liver damage in Wistar rats. Int J Pharm Pharm Sci 2012;4:222-5.
Kumar SVS, Kumar CV, Vardhan AV. Hepatoprotective activity of Acalypha indica
linn against thioacetamide induced toxicity. Int J Pharm Pharm Sci 2013;5:356-9.
Ganesan R, Venkatanarasimhan M, Pawar S, Reddy GP, Anandan T, Masilamani G. Hepatoprotective effect of Coldenia procumbens
linn against D-galactosamine induced acute liver damage in rats. Int J Integr Sci Innov Technol Sect B 2013;2:9-11.
Lin S, Yao C, Lin C, Lin Y. Hepatoprotective activity of Taiwan folk Medicine: Eclipta prostrate
Linn. against various hepatotoxins induced acute hepatotoxicity. Phytother Res 1996;10:483-90.
Muthulingam M, Mohandoss P, Indra N, Sethupathy S. Antihepatotoxic efficacy of Indigofera tinctoria (Linn.) on paracetamol induced liver damage in rats. Int J Pharm Biomed Res 2010;1:13-8.
[Table 1], [Table 2]