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Hepatoprotective activity of Andrographis Paniculata and Swertia Chirayita
Food and Chemical Toxicology 49 (2011) 3367–3373
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Hepatoprotective activity of Andrographis Paniculata and Swertia Chirayita R. Nagalekshmi a, Aditya Menon b, Dhanya K. Chandrasekharan b, Cherupally Krishnan Krishnan Nair c,⇑ a
Amrita School of Pharmacy, Kochi 682041, Kerala, India Amala Cancer Research Centre, Thrissur 680555, Kerala, India c Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla 689101, Kerala, India b
a r t i c l e
i n f o
Article history: Received 18 July 2011 Accepted 20 September 2011 Available online 29 September 2011 Keywords: Andrographis paniculata Swertia chirayita Paracetamol Liver marker enzymes Hepatoprotection
a b s t r a c t Andrographis paniculata (Family: Acanthaceae) and Swertia chirayita (Family: Gentianaceae) are two controversial medicinal plants used as Kiriyattu, having similar therapeutic action and are used as a hepatoprotective and hepatostimulative agent. A. paniculata grows in southern parts of India and S. chirayita in the Himalayan region. The present work concerns on the ability of the extracts of these plants to offer protection against acute hepatotoxicity induced by paracetamol (150 mg/kg) in Swiss albino mice. Oral administration of A. paniculata or S. chirayita extract (100–200 mg/kg) offered a significant dose dependent protection against paracetamol induced hepatotoxicity as assessed in terms of biochemical and histopathological parameters. The paracetamol induced elevated levels of serum marker enzymes such as serum glutamate pyruvate transaminase (GPT), serum glutamate oxaloacetate transaminase (GOT), alkaline phosphatase (ALP), and bilirubin in peripheral blood serum and distorted hepatic tissue architecture along with increased levels of lipid peroxides (LPO) and reduction of superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx) in liver tissue. Administration of the plant extracts after paracetamol insult restored the levels of these parameters to control (untreated) levels. Thus the present study revealed that the extracts of A. paniculata or S. chirayita offered protection against hepatotoxicity induced by paracetamol. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The use of many herbs or their extracts for treatment of various ailments including cancer has been documented in Ayurveda medical system practiced primarily in the Indian subcontinent for 5000 years (Dahanukar et al., 2000). The immense potential of medicinal plants used in traditional systems has been well recognized in recent years (Hoareau and DaSilva, 1999). Andrographis paniculata (Family: Acanthaceae) and Swertia chirayita (Family: Gentianaceae) are controversial medicinal plants (Nair, 2004). S. chirayita is also known as S. chirata. Both are termed as Kiriyattu in Malayalam, and both have similar therapeutic action. The Sanskrit name Kiratatikta is used for both the plants, meaning black coloured with bitter taste which is also common to both. The extracts of these plants contain diterpenes, flavonoids and stigma sterols (Siripong et al., 1992). The bitterness, antihelmintic, hypoglycemic and antipyretic properties are attributed to amarogentin (most bitter compound isolated till date), swerchirin, swertiamarin, etc. A. paniculata and S. chirayita has many pharmacological activity such as anti-inflammatory, hepatoprotective, antidiarrho⇑ Corresponding author. Tel./fax: +91 04692600020. E-mail addresses: [email protected] (R. Nagalekshmi), 85.aditya@g mail.com (A. Menon), [email protected] (D.K. Chandrasekharan), [email protected] (C.K.K. Nair). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.09.026
eal, antiviral and antimalarial activity (Joshi and Dhawan, 2005; Mishra et al., 2009; Reena et al., 2001; Gupta et al., 1990; Wiart et al., 2005). The medicinal plants find application in pharmaceutical, cosmetic, agricultural and food industry. A. paniculata is commonly seen in south-Indian region and S. chirayita in Himalayan parts of India, Nepal and Bhutan. So commonly A. paniculata is a substitute of S. chirayita in southeastern region. A. paniculata is an annual shrub that grows abundantly in India and cultivated extensively in China and Thailand and S. chirayita grows luxuriously in temperate Himalayas at an altitude of 1220–3050 m. A. paniculata and S. chirayita has many similar pharmacological activity such as anti-inflammatory, anticarcinogenic, hepatoprotective, antidiarrhoeal, antiviral and antimalarial activity (Joshi and Dhawan, 2005; Mishra et al., 2009; Reena et al., 2001; Gupta et al., 1990, 2005; Kumar et al., 2004). There are no reports available on the comparative study of these two controversial medicinal plants. This paper reports on the comparative hepatoprotective activity of ethanolic extracts of these plants. Liver diseases remain as one of the serious health problems since we do not have satisfactory liver protective drugs in modern medicine for serious liver disorders. Herbal drugs play a role in the management of various liver disorders most of which enhance the natural healing processes of the liver. A number of plants have been shown to possess hepatoprotective properties by improving the antioxidant status (Gupta et al., 2005; Ajith et al., 2007;
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Bhattacharjee and Sil, 2007; Celik et al., 2009). The present study concerns the hepatoprotective activity of ethanol extracts of A. paniculata and S. chirayita.
biological activities. Qualitative tests were conducted for the detection of Alkaloids, Saponins, Flavanoids, tannins and phenolic compounds and steroids. Phytochemical components of both plant extracts are listed in Table 1.
2. Materials and methods
2.6. Free radical scavenging activity
2.1. Chemicals
In order to determine the free radical scavenging activity of the extracts the following parameters were assayed. DPPH Radical scavenging was determined by the method of Gadow et al. (1997) with minor modifications. Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe3+/ascorbate/EDTA/H2O2 system. The hydroxyl radicals attack deoxyribose, which result in thio barbituric acid reacting substance (TBARS) formation (Elizabeth and Rao, 1990).
Paracetamol was obtained from Variety Pharmaceuticals (P) Ltd., Shornur, Kerala, India. DPPH (1,1-diphenyl-2-picryl-hydrazyl), TBA (thio barbituric acid) and deoxyribose were from Sigma Chemical Company Inc., St. Louis, MO, USA. EDTA (ethylene diamine tetra acetic acid), NBT (nitroblue tetrazolium) and riboflavin were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. TCA (trichloro acetic acid) and ascorbic acid were from Merck Specialties Pvt. Ltd., Mumbai, India. All other chemicals were of analytical grade procured from reputed Indian manufacturers. 2.2. Animals Female Swiss albino mice of 8–10 week old weighing 20–25 g, were selected from inbred group maintained under standard condition of temperature (25 ± 5 °C) and humidity. Animals were provided with food and water ad libitum. All experiments in this study were carried out with the prior approval of the Institutional Animal Ethics Committee (IAEC) and were conducted strictly adhering to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of Government of India. 2.3. Plant materials A. paniculata (Batch No: B2NIN of KAPL, Athani) and S. chirayita (Batch No: B2N2N of KAPL, Athani) were obtained from Kerala Ayurveda Pvt. Ltd., Athani, Kerala, India. The plants were taxonomically identified as ‘A. paniculata (Burm.f) Wallich ex Nees and S. chirayita (Roxb.ex Flem) H.Karst’ by Dr. Ganesh, Associate Professor of Botany, Sree Krishna College, Guruvayur, Kerala, India. 2.4. Preparation of extracts Shade dried aerial parts of both plants were subjected for size reduction to coarse powder. The powders were extracted with 70% ethanol at 70 °C using soxhlet apparatus for 48 h. The ethanolic extracts of both plants were concentrated in water bath and lyophilisation was carried out at 55 °C to get solid crude extracts. The yield for A. paniculata was 5.8% w/w and that for S. chirayita was 1.4% w/w. The HPLC and HPTLC fingerprint profiles of the extracts are presented in Figs. 1 and 2. 2.5. Phytochemical screening The alcoholic extracts of A. paniculata (APE) and S. chirayita (SCE) were subjected to various phytochemical screening (Khandelwal, 2004; Ferguson, 1956) to test for the presence of secondary metabolites, which are responsible for various
2.7. Hepatoprotective activity A total of 35 animals were equally divided into 7 groups of five each. Group I served as normal control without any treatment. Animals of groups II, III, IV, V, VI and VII were administered with paracetamol (150 mg/kg orally after 18 h starvation) as single dose to induce hepatotoxicity, Group II served as paracetamol control. One hour after the paracetamol treatment, Group III served as positive control and received silymarin (75 mg/kg body weight) orally, Group IV and Group V were orally administered with APE 100 and 200 mg/kg and group VI and VII with SCE 100 and 200 mg/kg, respectively. Paracetamol was made into a suspension in 0.1% carboxy methyl cellulose. The plant extracts and silymarin were dissolved in sterile distilled water for oral administration to animals. A group exclusively treated with carboxy methyl cellulose was not kept, since it was reported to be non toxic (Eberhardt et al., 2006). It may be noted that higher doses (300–1000 mg/kg) of paracetamol were used to induce hepatotoxicity in mice in several reports (Chengelis et al., 1986; Xia et al., 2009). In our laboratory, our earlier studies showed induction of mortality in mice following paracetamol administration at doses 200 mg/kg or above. The differences in the responses of mice observed in our study from that of the previously reported ones could be due to the 18 h starvation prior to administration of paracetamol or due to the strain difference. After 24 h of the paracetamol treatment, blood was collected and serum was separated for the biochemical investigations. The liver was removed for investigations on oxidative stress (antioxidant profiles) and histopathological alterations.
2.7.1. Assessment of liver marker enzymes and antioxidants Serum biochemical parameters such as glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) (Reitman and Frankal, 1975), alkaline phosphatase (ALP) (Kind and King, 1954) and serum bilirubin (Malloy and Evelyn, 1937) were analyzed according to the reported methods. The liver homogenates (10% w/v) prepared in phosphate-buffered saline (PBS containing 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4 and 1.76 mM KH2PO4 in 1000 ml distilled water) were used for antioxidant studies such as lipid peroxidation (LPO) (Buege and Aust, 1978), superoxide dismutase (SOD) (McCord and Fridovich, 1969), glutathione peroxidase (GPx) (Hafemann et al., 1974) and reduced glutathione (GSH) (Moron et al., 1979).
Fig. 1. HPLC of Andrographis paniculata and Swertia chirayita extract.
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Fig. 2. HPTLC of Andrographis paniculata extract showing 14 peaks (left) and Swertia chirayita extract showing 10 peaks (right).
Table 1 Preliminary phyto-chemical studies of the plant extracts. Tests
Observation
Inference
Test for alkaloids (Hager’s test) Test for saponins (Foaming Test) Test for Flavanoids (sodium hydroxide test) Test for Tannins and Phenolic Compounds (a) Gelatin test (b) Ferric Chloride test Test for steroids (Salkowski Reaction)
Positive Positive Positive
Alkaloids Present in both plants Saponins present in both plants Flavanoids Present in both plants
Negative Positive for Andrographis paniculata extract
Tannins absent in both plants Phenolic compound present in Andrographis paniculata but not in Swertia chirayita
Positive
Steroids present in both plants
2.7.2. Histopathological studies Liver slices were fixed in 10% formalin and embedded in paraffin wax. Sections of 5 micron thickness were made using a microtome and stained with haematoxylin-eosin and observed under microscope. Photographs of each of the slides were taken at 40 magnification. 2.8. Statistical analysis The results are presented as mean ± SD of the studied group. Statistical analyses of the results were performed using ANOVA with Tukey–Kramer multiple comparisons test.
3. Results The yield for A. paniculata was 5.8% w/w and that for S. chirayita was 1.4% w/w after ethanolic extraction. The HPLC and HPTLC fingerprint profiles of the extracts are presented in Figs. 1 and 2. Various phytochemicals were found in both plants extracts. A. paniculata had 14 and S. chirayita had 10, compounds, respectively, on HPTLC. Two compounds were found identical in both extracts as confirmed by HPLC. The stable free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) with characteristic absorption at 515 nm was reduced by the extracts resulting in decrease in the absorption, which is directly related to the electron scavenging capacity of the extract. The ethanolic extract of S. chirayita and A. paniculata extract reduced DPPH radical in a concentration dependent manner (Fig. 3). Hydroxyl radicals produced by Fenton reaction can damage deoxyribose to produce malonaldehyde like substances, which can be detected by TBA reaction (Buege and Aust, 1978). Fig. 4 shows that
the plant extracts inhibited TBARS formation in this reaction and this suggest that the extracts prevented hydroxyl radical induced damage to deoxyribose. Administration of paracetamol (150 mg/kg) to Swiss albino mice resulted in acute hepatotoxicity as can be revealed from the liver antioxidant levels and serum marker enzyme levels (Table 2 and Table 3). The extent of membrane lipid peroxidation in liver tissues of animals administered with paracetamol was found to be significantly higher, while the animals treated with ethanolic extracts of A. paniculata or S. chirayita the paracetamol induced increase in lipid peroxidation was significantly reduced and the
Fig. 3. DPPH free radical scavenging activity by Andrographis paniculata extract and Swertia chirayita extract.
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Fig. 4. Hydroxyl radical scavenging activity by Andrographis paniculata extract and Swertia chirayita extract.
S. chirayita extract was found to be more effective as can be evidenced from the data presented in Table 2. Paracetamol treatment also caused a significant decrease in the level of tissue antioxidant enzymes such as super oxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and the cellular antioxidant molecule, reduced glutathione (GSH) in liver tissue when compared with control group. Administration of the extracts of A. paniculata or S. chirayita extract following the paracetamol insult, significantly improved the levels of SOD, GPx and GSH in a dose dependent manner to almost that of the control untreated mice. It can be seen from the data presented in Table 3 that the levels of serum glutamate oxaloacetate tansaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), globulin and bilirubin were significantly elevated in the par-
acetamol treated animals when compared to the control untreated, indicating induction of hepatic damage due to the paracetamol administration. Administration of the extract of A. paniculata or S. chirayita protected against the paracetamol induced hepatotoxicity in a dose dependent manner as revealed by the levels of the serum marker enzymes. The extract treatments significantly reversed the elevated levels of GOT, GPT, ALP and bilirubin (P < 0.001). From the results it can be realized that A. paniculata or S. chirayita prevented the manifestation of paracetamol induced hepatotoxicity in dose dependent manner and upon comparison of the tissue-antioxidant parameters on the basis of kg-body-weight doses of the extracts administered, S. chirayita offered better protection than A. paniculata even though the difference is not much significant. The cellular architecture of the liver tissue of the different groups of mice as studied by the histo-pathological analysis is presented in Fig. 5. The liver tissues of the untreated control animals showed normal architecture of hepatic cells with clear cytoplasm and slightly dilated central veins, normal kupffer cells and all cells had normal large nuclei (Fig. 5 – Normal). In paracetamol treated animals the liver tissue showed distorted architecture with markedly congested central veins, extensive area of necrosis and hemorrhage, nuclei were distorted and some of the hepatocytes contain vacuolated cytoplasm (Fig. 5 – Paracetamol). In the animals treated with the plant extracts, the normal architecture of the liver tissue was discernible (Fig. 5 – APE (100 mg/kg), APE (200 mg/kg), SCE (100 mg/kg), and SCE (200 mg/kg)). The histological observations supported the results obtained on induction of hepatotoxicity by paracetamol and reversal of the toxicity by APE or SCE as discernible from the levels of tissue- antioxidants and lipid peroxidation and serum marker enzymes.
Table 2 Effect of administration of the extracts of Andrographis paniculata and Swertia chirayita on antioxidants levels and lipid peroxidation in liver tissue of Swiss albino mice treated with paracetamol. Values are expressed as mean ± SD (n = 4). Data were analyzed using ANOVA with Tukey–Kramer multiple comparisons test. Normal
Paracetamol (150 mg/kg)
GSH (n mol/mg 24.16 ± 2.33 8.39 ± 1.10 protein) GPx (Unit/mg 22.40 ± 1.39 9.56 ± 2.63 protein) SOD (Unit/mg 12.26 ± 1.80 5.75 ± 0.20 protein) MDA (n mol/mg 1.06 ± 0.89 4.79 ± 1.70 protein) a b c d e f g
Paracetamol (150 mg/ Paracetamol (150 mg/ Paracetamol (150 mg/kg) + SCE kg) + APE kg) + APE (100 mg/kg) (200 mg/kg) (100 mg/kg)
Paracetamol (150 mg/kg) + SCE (200 mg/kg)
17.68 ± 1.60a
20.27 ± 1.78a,f
18.64 ± 0.10a
20.95 ± 1.58a,d,e,g
25.38 ± 0.04a
20.43 ± 1.40a,b
22.30 ± 0.33a,c
20.60 ± 0.41a,b
24.07 ± 2.62a,e,g
8.73 ± 0.17a
11.70 ± 0.41a,b
12.44 ± 0.24a,b
11.84 ± 1.12a,b
12.30 ± 1.10a,b
1.1 ± 0.18a
2.50 ± 0.39a,d
1.90 ± 0.30a
2.26 ± 0.39a
2.04 ± 0.38a
Paracetamol (150 mg/ kg) + Silymarin (75 mg/kg) 18.6 ± 0.08a
p < 0.001 When compared to Paracetamol. p < 0.001 When compared to Silymarin (75 mg/kg). p 6 0.01 When compared to Silymarin (75 mg/kg). p 6 0.05 When compared to Silymarin (75 mg/kg). p 6 0.001 When compared to APE (100 mg/kg). p 6 0.05 When compared to APE (100 mg/kg). p 6 0.01 When compared to SCE (100 mg/kg).
Table 3 Effect of administration of the extracts of Andrographis paniculata and Swertia chirayita on serum markers for assessing hepatotoxicity in Swiss albino mice. Values are expressed as mean ± SD (n = 4). Data were analyzed using ANOVA with Tukey–Kramer multiple comparisons test. Normal
Paracetamol (150 mg/kg)
SGOT (IU/L) 40.15 ± 6.73 230.30 ± 26.13 SGPT (IU/L) 31.61 ± 3.38 160.50 ± 76.47 ALP (KA Unit) 110.26 ± 22.16 213.60 ± 22.35 BILIRUBIN (mg/dL) 0.72 ± 0.04 1.67 ± 0.88 a b c
p 6 0.001 When compared to Paracetamol. p < 0.001 When compared to Silymarin (75 mg/kg). p < 0.05 When compared to Silymarin (75 mg/kg).
Paracetamol (150 mg/ kg) + Silymarin (75 mg/kg)
Paracetamol Paracetamol (150 mg/kg) + APE (150 mg/ kg) + APE(100 mg/ (200 mg/kg) kg)
Paracetamol (150 mg/kg) + SCE (100 mg/kg)
Paracetamol (150 mg/ kg) + SCE (200 mg/kg)
63.81 ± 31.34a 38.14 ± 0.82 a 162 ± 0.49a 0.75 ± 0.10 a
54.47 ± 22.30a 65.40 ± 20.53a 124.17 ± 32.99a,c 0.73 ± 0.11a
47.76 ± 2.45a 34.88 ± 5.92a 110.29 ± 28.46a,b 0.63 ± 0.10a
43.71 ± 4.45a 22.69 ± 0.30a 94.69 ± 17.74a,b 0.55 ± 0.10a
48.25 ± 21.64a 42.21 ± 5.12a 110.59 ± 17.12a,b 0.63 ± 0.10a
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4. Discussion The present work explored the ability of ethanolic extracts of A. paniculata or S. chirayita to offer protection against hepatotoxicity induced by ‘over the counter drug’, paracetamol. Liver is a major metabolic organ affected by various chemicals and toxins and liver injuries induced by various hepatotoxins have been recognized as a major toxicological problem for years (Azer et al., 1997). In the absence of reliable liver protective drugs in modern medical practices, herbs play an important role in the management of various liver disorders. A number of plants show hepatoprotective activity (Hamza, 2010; Hwang et al., 2009; Olaleye et al., 2010; Malhotra et al., 2001). A. paniculata (Kalmegh) is used extensively in the Indian traditional system of medicine as a hepatoprotective and hepatostimulative agent. The aqueous extract of A. paniculata inhibited BHC induced liver toxicity in Swiss male mice (Trivedi et al., 2007; Trivedi and Rawal, 2000; Sutha et al., 2010) and ethanol induced hepatotoxicity in albino wistar rats (Vetriselvan et al., 2011) Andrographolide offered protection against galactosamine or paracetamol induced toxicity to hepatic tissue (Handa and Sharma, 1990).
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Earlier works showed that A. paniculata at a dose of 500 mg/kg of is nontoxic to mice (Guo et al., 1988) and S. chirayita is safe for animals up to a dose of 2000 mg/kg (Bhargava et al., 2009). In our study, paracetamol is used as a model system to induce acute hepatotoxicity in Swiss albino mice. Assessment of liver function is made by estimating the activities of serum GPT, GOT, ALP and bilirubin which are present higher concentration in cytoplasm. When there is hepatopathy, these molecules leak into the blood stream in compliance with the extent of liver damage (Nkosi et al., 2005). Bilirubin is one of the most useful clinical clues to the severity of necrosis and its accumulation is a measure of binding, conjugation and excretory capacity of hepatocyte. After normal therapeutic doses, paracetamol is metabolized, by phase II drug-metabolizing enzymes such as the glucuronyl and sulfotransferases (Anker and Smilkstein, 1994). When high doses are in taken, the major portion of paracetamol dose is available to undergo bioactivation by the cytochrome P450 system (CYP2E1, CYP3A4, CYP1A2) to create a highly reactive intermediate, N-acetyl-p-benzoquinoneimine (NAPQI) (Dahlin et al., 1984; Nelson 1995; Patten et al., 1993; Raucy et al., 1989). This intermediate can covalently binds to cellular macromolecules, causing damage and cell death (Rogers
Fig. 5. Histopathological analysis of liver tissue of animals treated with paracetamol and administrated with the extracts of Andrographis paniculata or Swertia chirayita. Normal – liver histopathology of untreated animal, Paracetamol – liver histopathology of paracetamol treated animal, Silymarin – liver histopathology of paracetamol treated animals administered with Silymarin (75 mg/ kg), APE (100 mg/kg) – liver histopathology of paracetamol treated animal administered with 100 mg/kg of Andrographis paniculata extract, APE (200 mg/kg) – liver histopathology of paracetamol treated animal administered with 200 mg/kg of Andrographis paniculata extract, SCE (100 mg/kg) – liver histopathology of paracetamol treated animal administered with 100 mg/kg of Swertia chirayita extract, SCE (200 mg/kg) – liver histopathology of paracetamol treated animal administered with 200 mg/kg of Swertia chirayita extract.
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et al., 1997). Accidental or incidental paracetamol overdose may be associated with toxic liver damage leading to potentially fatal, hepatic centrilobular necrosis and liver failure (James et al., 2003; Bergman et al., 1996). This toxic reaction is associated with metabolic activation that results in the formation of NAPQI, which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione (Henderson et al., 2000). At higher doses glutathione becomes depleted and the excess of NAPQI arylates and oxidizes hepatic proteins (Fountoulakis et al., 2000). Oxidative stress is another mechanism that has been postulated to be important in the development of paracetamol toxicity. Toxicity begins with the change in endoplasmic reticulum, which results in the loss of metabolic enzymes located in the intracellular structures (Recknagel, 1983). It was observed that the levels of cellular antioxidant enzymes and molecules are decreased significantly in paracetamol treated animals. The declined antioxidant enzyme activity is responsible for the increased lipid peroxidation measured as thiobarbituric acid reacting substance malondialdehyde (MDA), which causing loss of membrane fluidity, membrane integrity, and finally loss of cell functions of liver (Smith et al., 1987; Halliwell and Gutteridge, 1989). This peroxidative damage to membranes results in the leakage of enzymes, and metabolites to circulation. In the present study, it was observed that, the animals treated with paracetamol showed elevated levels of serum markers such as GPT, GOT, ALP and bilirubin. Normally, GOT and ALP are present in high concentration in liver. Due to hepatocyte necrosis or abnormal membrane permeability, these enzymes are released from the cells and their levels in the blood increases. GPT is a sensitive indicator of acute liver damage and elevation of this enzyme in non hepatic diseases is unusual. GPT is more selectively a liver paranchymal enzyme than GOT (Shah et al., 2002). The rise in the SGOT is usually accompanied by an elevation in the levels of SGPT, which play a vital role in the conversion of amino acids to keto acids (Sallie et al., 1999). Oral administration of ethanolic extract of A. paniculata or S. chirayita at the doses of 100 and 200 mg/kg significantly prevented the elevation of the serum markers of hepatotoxicity and restored the tissue antioxidant levels and decreased the lipid peroxidation levels in liver. The results would suggest that the extracts protected the membrane integrity of the liver cells against paracetamol induced leakage of marker enzymes into the circulation. The elevated levels of alkaline phosphatase reflected the pathological alteration in biliary flow (Ploa and Hewitt, 1989). The paracetamol induced elevation of this enzyme in the serum is lined up with high level of serum bilirubin. Administration of the ethanol extract of A. paniculata or S. chirayita decreased the ALP activity and serum bilirubin levels and stabilized biliary dysfunction and normal functional status of the liver (Suky et al., 2011). The increase in lipid peroxidation in liver due to paracetamol administration leading to tissue damage (Muriel et al., 1992) is due to the failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with ethanol extract of A. paniculata or S. chirayita significantly reversed these changes. Administration of ethanolic extract of A. paniculata or S. chirayita effectively prevented the decrease in SOD and GPX activities which may be due to the scavenging of radicals by the extract. Thus the present study indicates that A. paniculata or S. chirayita maintain the cellular integrity of hepatic tissues and helped its regeneration. Slightly better protective effect is noticed in case of GSH and GPx activities for the extract of Swertia while the extracts of Andrographis and Swertia showed almost similar activity in the case of SOD and MDA levels. The protective ability of these plant extracts towards paracetamol induced hepatotoxicity might be attributed to the free radical
scavenging activity of the extracts. The findings suggest the therapeutic use of these plants for liver ailments for ameliorating hepatotoxicity.
5. Conclusions The present work analyzed the hepatoprotective potential of ethanolic extracts of two plants, A. paniculata and S. chirayita. The results indicated the potential of these two plant extracts to offer protection against the acute hepatotoxicity induced by paracetamol. The S. chirayita extract has been found to provide higher hepatoprotection than A. paniculata.
Conflict of Interest The authors declare that there are no conflict of interest. Acknowledgment The authors thank Kerala State Council for Science, Technology and Engineering, Govt. Kerala, India for the financial support through a research grant awarded to CKKN. They are thankful to Mss. Variety Pharmaceuticals (P) Ltd., Shornur, Kerala, India for giving Paracetamol as a gift for the research studies. References Ajith, T.A., Hema, U., Aswathy, M.S., 2007. Zingiber officinale Roscoe prevents acetaminophen-induced acute hepatotoxicity by enhancing hepatic antioxidant status. Food Chem. Toxicol. 45, 2267–2272. Anker, A.L., Smilkstein, M.J., 1994. Acetaminophen concepts and controversies. Emerg. Med. Clin. NA 12, 335–349. Azer, S.A., Mc Caughan, G.M., Stacey, N.H., 1997. Liver and Environmental Xenobiotics. Narosa Publishing House, New Delhi, p. 178. Bergman, K., Muller, L., Teigen, S.W., 1996. Series: current issues in mutagenesis and carcinogenesis, No. 65. The genotoxicity and carcinogenicity of paracetamol: a regulatory (re)view. Mutat. Res. 349, 263–288. Bhargava, S., Rao, P.S., Bhargava, P., Shukla, S., 2009. Antipyretic potential of Swertia chirata buch ham. Root extract. Sci. Pharm. 77, 617–623. Bhattacharjee, R., Sil, P.C., 2007. Protein isolate from the herb, Phyllanthus niruri L. (Euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver damage via its antioxidant properties. Food Chem. Toxicol. 45, 817–826. Buege, A.J., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Celik, I., Temur, A., Isik, I., 2009. Hepatoprotective role and antioxidant capacity of pomegranate (Punica granatum) flowers infusion against trichloroacetic acidexposed in rats. Food Chem. Toxicol. 47, 145–149. Chengelis, C.P., Dodd, D.C., Means, J.R., Kotsonis, F.N., 1986. Protection by zinc against paracetamol induced hepatotoxicity in mice. Fundam. Appl. Toxicol. 6, 278–284. Dahanukar, S.A., Kulkarni, R.A., Rege, N.N., 2000. Pharmacology of medicinal plants and natural products. Ind. J. Pharm. 32, S81-S11. Dahlin, D.C., Miwa, G.T., Lu, A.Y., Nelson, S.D., 1984. N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen. Proc. Natl. Acad. Sci. USA 81, 1327–1331. Eberhardt, T.L., Min, S., Han, J.S., 2006. Phosphate removal by refined aspen wood fiber treated with carboxymethyl cellulose and ferrous chloride. Bioresour. Technol. 97, 2371–2377. Elizabeth, K., Rao, M.N.A., 1990. Oxygen radical scavenging activity of curcumin. Int. J. Pharmacol. 58, 379–382. Ferguson, N.M., 1956. A Text Book of Pharmacognosy, New York, The Macmillan Company, pp. 189–190. Fountoulakis, M., Berndt, P., Boelsterli, U.A., Winter, M., Suter, L.A., 2000. Two diamensional database of mouse liver proteins: changes in hepatic protein levels following treatment with acetaminophen or its nontoxic regioisimer 3acetamidophenol. Electrophoresis 21, 2148–2161. Gadow, A., Joubert, E., Hansmann, C.F., 1997. Comparision of the antioxidant activity of asplathin with that of other plant phenols of roobios tea (Asplathus linearis) alpha-tocopherol, BHT and BHA. J. Agric. Food Chem. 45, 632–638. Guo, S.Y., Li, D.Z., Li, W.S., FU, A.H., Zhang, L.F., 1988. Study of the toxicity of androgrographolide in rabbits. J. Beijing Med. Univ. 5, 422–428. Gupta, M., Mazumder, U.K., Kumar, R.S., Sivakumar, T., Gomathi, P., Rajeshwar, Y., 2005. Antioxidant defense system induced by a methanol extract of Caesalpinia bonducella in rat liver. Pharm. Biol. 43, 411–419.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Hepatoprotective activity of Andrographis Paniculata and Swertia Chirayita R. Nagalekshmi a, Aditya Menon b, Dhanya K. Chandrasekharan b, Cherupally Krishnan Krishnan Nair c,⇑ a
Amrita School of Pharmacy, Kochi 682041, Kerala, India Amala Cancer Research Centre, Thrissur 680555, Kerala, India c Pushpagiri Institute of Medical Sciences and Research Centre, Thiruvalla 689101, Kerala, India b
a r t i c l e
i n f o
Article history: Received 18 July 2011 Accepted 20 September 2011 Available online 29 September 2011 Keywords: Andrographis paniculata Swertia chirayita Paracetamol Liver marker enzymes Hepatoprotection
a b s t r a c t Andrographis paniculata (Family: Acanthaceae) and Swertia chirayita (Family: Gentianaceae) are two controversial medicinal plants used as Kiriyattu, having similar therapeutic action and are used as a hepatoprotective and hepatostimulative agent. A. paniculata grows in southern parts of India and S. chirayita in the Himalayan region. The present work concerns on the ability of the extracts of these plants to offer protection against acute hepatotoxicity induced by paracetamol (150 mg/kg) in Swiss albino mice. Oral administration of A. paniculata or S. chirayita extract (100–200 mg/kg) offered a significant dose dependent protection against paracetamol induced hepatotoxicity as assessed in terms of biochemical and histopathological parameters. The paracetamol induced elevated levels of serum marker enzymes such as serum glutamate pyruvate transaminase (GPT), serum glutamate oxaloacetate transaminase (GOT), alkaline phosphatase (ALP), and bilirubin in peripheral blood serum and distorted hepatic tissue architecture along with increased levels of lipid peroxides (LPO) and reduction of superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx) in liver tissue. Administration of the plant extracts after paracetamol insult restored the levels of these parameters to control (untreated) levels. Thus the present study revealed that the extracts of A. paniculata or S. chirayita offered protection against hepatotoxicity induced by paracetamol. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction The use of many herbs or their extracts for treatment of various ailments including cancer has been documented in Ayurveda medical system practiced primarily in the Indian subcontinent for 5000 years (Dahanukar et al., 2000). The immense potential of medicinal plants used in traditional systems has been well recognized in recent years (Hoareau and DaSilva, 1999). Andrographis paniculata (Family: Acanthaceae) and Swertia chirayita (Family: Gentianaceae) are controversial medicinal plants (Nair, 2004). S. chirayita is also known as S. chirata. Both are termed as Kiriyattu in Malayalam, and both have similar therapeutic action. The Sanskrit name Kiratatikta is used for both the plants, meaning black coloured with bitter taste which is also common to both. The extracts of these plants contain diterpenes, flavonoids and stigma sterols (Siripong et al., 1992). The bitterness, antihelmintic, hypoglycemic and antipyretic properties are attributed to amarogentin (most bitter compound isolated till date), swerchirin, swertiamarin, etc. A. paniculata and S. chirayita has many pharmacological activity such as anti-inflammatory, hepatoprotective, antidiarrho⇑ Corresponding author. Tel./fax: +91 04692600020. E-mail addresses: [email protected] (R. Nagalekshmi), 85.aditya@g mail.com (A. Menon), [email protected] (D.K. Chandrasekharan), [email protected] (C.K.K. Nair). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.09.026
eal, antiviral and antimalarial activity (Joshi and Dhawan, 2005; Mishra et al., 2009; Reena et al., 2001; Gupta et al., 1990; Wiart et al., 2005). The medicinal plants find application in pharmaceutical, cosmetic, agricultural and food industry. A. paniculata is commonly seen in south-Indian region and S. chirayita in Himalayan parts of India, Nepal and Bhutan. So commonly A. paniculata is a substitute of S. chirayita in southeastern region. A. paniculata is an annual shrub that grows abundantly in India and cultivated extensively in China and Thailand and S. chirayita grows luxuriously in temperate Himalayas at an altitude of 1220–3050 m. A. paniculata and S. chirayita has many similar pharmacological activity such as anti-inflammatory, anticarcinogenic, hepatoprotective, antidiarrhoeal, antiviral and antimalarial activity (Joshi and Dhawan, 2005; Mishra et al., 2009; Reena et al., 2001; Gupta et al., 1990, 2005; Kumar et al., 2004). There are no reports available on the comparative study of these two controversial medicinal plants. This paper reports on the comparative hepatoprotective activity of ethanolic extracts of these plants. Liver diseases remain as one of the serious health problems since we do not have satisfactory liver protective drugs in modern medicine for serious liver disorders. Herbal drugs play a role in the management of various liver disorders most of which enhance the natural healing processes of the liver. A number of plants have been shown to possess hepatoprotective properties by improving the antioxidant status (Gupta et al., 2005; Ajith et al., 2007;
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Bhattacharjee and Sil, 2007; Celik et al., 2009). The present study concerns the hepatoprotective activity of ethanol extracts of A. paniculata and S. chirayita.
biological activities. Qualitative tests were conducted for the detection of Alkaloids, Saponins, Flavanoids, tannins and phenolic compounds and steroids. Phytochemical components of both plant extracts are listed in Table 1.
2. Materials and methods
2.6. Free radical scavenging activity
2.1. Chemicals
In order to determine the free radical scavenging activity of the extracts the following parameters were assayed. DPPH Radical scavenging was determined by the method of Gadow et al. (1997) with minor modifications. Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe3+/ascorbate/EDTA/H2O2 system. The hydroxyl radicals attack deoxyribose, which result in thio barbituric acid reacting substance (TBARS) formation (Elizabeth and Rao, 1990).
Paracetamol was obtained from Variety Pharmaceuticals (P) Ltd., Shornur, Kerala, India. DPPH (1,1-diphenyl-2-picryl-hydrazyl), TBA (thio barbituric acid) and deoxyribose were from Sigma Chemical Company Inc., St. Louis, MO, USA. EDTA (ethylene diamine tetra acetic acid), NBT (nitroblue tetrazolium) and riboflavin were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. TCA (trichloro acetic acid) and ascorbic acid were from Merck Specialties Pvt. Ltd., Mumbai, India. All other chemicals were of analytical grade procured from reputed Indian manufacturers. 2.2. Animals Female Swiss albino mice of 8–10 week old weighing 20–25 g, were selected from inbred group maintained under standard condition of temperature (25 ± 5 °C) and humidity. Animals were provided with food and water ad libitum. All experiments in this study were carried out with the prior approval of the Institutional Animal Ethics Committee (IAEC) and were conducted strictly adhering to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of Government of India. 2.3. Plant materials A. paniculata (Batch No: B2NIN of KAPL, Athani) and S. chirayita (Batch No: B2N2N of KAPL, Athani) were obtained from Kerala Ayurveda Pvt. Ltd., Athani, Kerala, India. The plants were taxonomically identified as ‘A. paniculata (Burm.f) Wallich ex Nees and S. chirayita (Roxb.ex Flem) H.Karst’ by Dr. Ganesh, Associate Professor of Botany, Sree Krishna College, Guruvayur, Kerala, India. 2.4. Preparation of extracts Shade dried aerial parts of both plants were subjected for size reduction to coarse powder. The powders were extracted with 70% ethanol at 70 °C using soxhlet apparatus for 48 h. The ethanolic extracts of both plants were concentrated in water bath and lyophilisation was carried out at 55 °C to get solid crude extracts. The yield for A. paniculata was 5.8% w/w and that for S. chirayita was 1.4% w/w. The HPLC and HPTLC fingerprint profiles of the extracts are presented in Figs. 1 and 2. 2.5. Phytochemical screening The alcoholic extracts of A. paniculata (APE) and S. chirayita (SCE) were subjected to various phytochemical screening (Khandelwal, 2004; Ferguson, 1956) to test for the presence of secondary metabolites, which are responsible for various
2.7. Hepatoprotective activity A total of 35 animals were equally divided into 7 groups of five each. Group I served as normal control without any treatment. Animals of groups II, III, IV, V, VI and VII were administered with paracetamol (150 mg/kg orally after 18 h starvation) as single dose to induce hepatotoxicity, Group II served as paracetamol control. One hour after the paracetamol treatment, Group III served as positive control and received silymarin (75 mg/kg body weight) orally, Group IV and Group V were orally administered with APE 100 and 200 mg/kg and group VI and VII with SCE 100 and 200 mg/kg, respectively. Paracetamol was made into a suspension in 0.1% carboxy methyl cellulose. The plant extracts and silymarin were dissolved in sterile distilled water for oral administration to animals. A group exclusively treated with carboxy methyl cellulose was not kept, since it was reported to be non toxic (Eberhardt et al., 2006). It may be noted that higher doses (300–1000 mg/kg) of paracetamol were used to induce hepatotoxicity in mice in several reports (Chengelis et al., 1986; Xia et al., 2009). In our laboratory, our earlier studies showed induction of mortality in mice following paracetamol administration at doses 200 mg/kg or above. The differences in the responses of mice observed in our study from that of the previously reported ones could be due to the 18 h starvation prior to administration of paracetamol or due to the strain difference. After 24 h of the paracetamol treatment, blood was collected and serum was separated for the biochemical investigations. The liver was removed for investigations on oxidative stress (antioxidant profiles) and histopathological alterations.
2.7.1. Assessment of liver marker enzymes and antioxidants Serum biochemical parameters such as glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) (Reitman and Frankal, 1975), alkaline phosphatase (ALP) (Kind and King, 1954) and serum bilirubin (Malloy and Evelyn, 1937) were analyzed according to the reported methods. The liver homogenates (10% w/v) prepared in phosphate-buffered saline (PBS containing 137 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4 and 1.76 mM KH2PO4 in 1000 ml distilled water) were used for antioxidant studies such as lipid peroxidation (LPO) (Buege and Aust, 1978), superoxide dismutase (SOD) (McCord and Fridovich, 1969), glutathione peroxidase (GPx) (Hafemann et al., 1974) and reduced glutathione (GSH) (Moron et al., 1979).
Fig. 1. HPLC of Andrographis paniculata and Swertia chirayita extract.
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Fig. 2. HPTLC of Andrographis paniculata extract showing 14 peaks (left) and Swertia chirayita extract showing 10 peaks (right).
Table 1 Preliminary phyto-chemical studies of the plant extracts. Tests
Observation
Inference
Test for alkaloids (Hager’s test) Test for saponins (Foaming Test) Test for Flavanoids (sodium hydroxide test) Test for Tannins and Phenolic Compounds (a) Gelatin test (b) Ferric Chloride test Test for steroids (Salkowski Reaction)
Positive Positive Positive
Alkaloids Present in both plants Saponins present in both plants Flavanoids Present in both plants
Negative Positive for Andrographis paniculata extract
Tannins absent in both plants Phenolic compound present in Andrographis paniculata but not in Swertia chirayita
Positive
Steroids present in both plants
2.7.2. Histopathological studies Liver slices were fixed in 10% formalin and embedded in paraffin wax. Sections of 5 micron thickness were made using a microtome and stained with haematoxylin-eosin and observed under microscope. Photographs of each of the slides were taken at 40 magnification. 2.8. Statistical analysis The results are presented as mean ± SD of the studied group. Statistical analyses of the results were performed using ANOVA with Tukey–Kramer multiple comparisons test.
3. Results The yield for A. paniculata was 5.8% w/w and that for S. chirayita was 1.4% w/w after ethanolic extraction. The HPLC and HPTLC fingerprint profiles of the extracts are presented in Figs. 1 and 2. Various phytochemicals were found in both plants extracts. A. paniculata had 14 and S. chirayita had 10, compounds, respectively, on HPTLC. Two compounds were found identical in both extracts as confirmed by HPLC. The stable free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) with characteristic absorption at 515 nm was reduced by the extracts resulting in decrease in the absorption, which is directly related to the electron scavenging capacity of the extract. The ethanolic extract of S. chirayita and A. paniculata extract reduced DPPH radical in a concentration dependent manner (Fig. 3). Hydroxyl radicals produced by Fenton reaction can damage deoxyribose to produce malonaldehyde like substances, which can be detected by TBA reaction (Buege and Aust, 1978). Fig. 4 shows that
the plant extracts inhibited TBARS formation in this reaction and this suggest that the extracts prevented hydroxyl radical induced damage to deoxyribose. Administration of paracetamol (150 mg/kg) to Swiss albino mice resulted in acute hepatotoxicity as can be revealed from the liver antioxidant levels and serum marker enzyme levels (Table 2 and Table 3). The extent of membrane lipid peroxidation in liver tissues of animals administered with paracetamol was found to be significantly higher, while the animals treated with ethanolic extracts of A. paniculata or S. chirayita the paracetamol induced increase in lipid peroxidation was significantly reduced and the
Fig. 3. DPPH free radical scavenging activity by Andrographis paniculata extract and Swertia chirayita extract.
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Fig. 4. Hydroxyl radical scavenging activity by Andrographis paniculata extract and Swertia chirayita extract.
S. chirayita extract was found to be more effective as can be evidenced from the data presented in Table 2. Paracetamol treatment also caused a significant decrease in the level of tissue antioxidant enzymes such as super oxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and the cellular antioxidant molecule, reduced glutathione (GSH) in liver tissue when compared with control group. Administration of the extracts of A. paniculata or S. chirayita extract following the paracetamol insult, significantly improved the levels of SOD, GPx and GSH in a dose dependent manner to almost that of the control untreated mice. It can be seen from the data presented in Table 3 that the levels of serum glutamate oxaloacetate tansaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), globulin and bilirubin were significantly elevated in the par-
acetamol treated animals when compared to the control untreated, indicating induction of hepatic damage due to the paracetamol administration. Administration of the extract of A. paniculata or S. chirayita protected against the paracetamol induced hepatotoxicity in a dose dependent manner as revealed by the levels of the serum marker enzymes. The extract treatments significantly reversed the elevated levels of GOT, GPT, ALP and bilirubin (P < 0.001). From the results it can be realized that A. paniculata or S. chirayita prevented the manifestation of paracetamol induced hepatotoxicity in dose dependent manner and upon comparison of the tissue-antioxidant parameters on the basis of kg-body-weight doses of the extracts administered, S. chirayita offered better protection than A. paniculata even though the difference is not much significant. The cellular architecture of the liver tissue of the different groups of mice as studied by the histo-pathological analysis is presented in Fig. 5. The liver tissues of the untreated control animals showed normal architecture of hepatic cells with clear cytoplasm and slightly dilated central veins, normal kupffer cells and all cells had normal large nuclei (Fig. 5 – Normal). In paracetamol treated animals the liver tissue showed distorted architecture with markedly congested central veins, extensive area of necrosis and hemorrhage, nuclei were distorted and some of the hepatocytes contain vacuolated cytoplasm (Fig. 5 – Paracetamol). In the animals treated with the plant extracts, the normal architecture of the liver tissue was discernible (Fig. 5 – APE (100 mg/kg), APE (200 mg/kg), SCE (100 mg/kg), and SCE (200 mg/kg)). The histological observations supported the results obtained on induction of hepatotoxicity by paracetamol and reversal of the toxicity by APE or SCE as discernible from the levels of tissue- antioxidants and lipid peroxidation and serum marker enzymes.
Table 2 Effect of administration of the extracts of Andrographis paniculata and Swertia chirayita on antioxidants levels and lipid peroxidation in liver tissue of Swiss albino mice treated with paracetamol. Values are expressed as mean ± SD (n = 4). Data were analyzed using ANOVA with Tukey–Kramer multiple comparisons test. Normal
Paracetamol (150 mg/kg)
GSH (n mol/mg 24.16 ± 2.33 8.39 ± 1.10 protein) GPx (Unit/mg 22.40 ± 1.39 9.56 ± 2.63 protein) SOD (Unit/mg 12.26 ± 1.80 5.75 ± 0.20 protein) MDA (n mol/mg 1.06 ± 0.89 4.79 ± 1.70 protein) a b c d e f g
Paracetamol (150 mg/ Paracetamol (150 mg/ Paracetamol (150 mg/kg) + SCE kg) + APE kg) + APE (100 mg/kg) (200 mg/kg) (100 mg/kg)
Paracetamol (150 mg/kg) + SCE (200 mg/kg)
17.68 ± 1.60a
20.27 ± 1.78a,f
18.64 ± 0.10a
20.95 ± 1.58a,d,e,g
25.38 ± 0.04a
20.43 ± 1.40a,b
22.30 ± 0.33a,c
20.60 ± 0.41a,b
24.07 ± 2.62a,e,g
8.73 ± 0.17a
11.70 ± 0.41a,b
12.44 ± 0.24a,b
11.84 ± 1.12a,b
12.30 ± 1.10a,b
1.1 ± 0.18a
2.50 ± 0.39a,d
1.90 ± 0.30a
2.26 ± 0.39a
2.04 ± 0.38a
Paracetamol (150 mg/ kg) + Silymarin (75 mg/kg) 18.6 ± 0.08a
p < 0.001 When compared to Paracetamol. p < 0.001 When compared to Silymarin (75 mg/kg). p 6 0.01 When compared to Silymarin (75 mg/kg). p 6 0.05 When compared to Silymarin (75 mg/kg). p 6 0.001 When compared to APE (100 mg/kg). p 6 0.05 When compared to APE (100 mg/kg). p 6 0.01 When compared to SCE (100 mg/kg).
Table 3 Effect of administration of the extracts of Andrographis paniculata and Swertia chirayita on serum markers for assessing hepatotoxicity in Swiss albino mice. Values are expressed as mean ± SD (n = 4). Data were analyzed using ANOVA with Tukey–Kramer multiple comparisons test. Normal
Paracetamol (150 mg/kg)
SGOT (IU/L) 40.15 ± 6.73 230.30 ± 26.13 SGPT (IU/L) 31.61 ± 3.38 160.50 ± 76.47 ALP (KA Unit) 110.26 ± 22.16 213.60 ± 22.35 BILIRUBIN (mg/dL) 0.72 ± 0.04 1.67 ± 0.88 a b c
p 6 0.001 When compared to Paracetamol. p < 0.001 When compared to Silymarin (75 mg/kg). p < 0.05 When compared to Silymarin (75 mg/kg).
Paracetamol (150 mg/ kg) + Silymarin (75 mg/kg)
Paracetamol Paracetamol (150 mg/kg) + APE (150 mg/ kg) + APE(100 mg/ (200 mg/kg) kg)
Paracetamol (150 mg/kg) + SCE (100 mg/kg)
Paracetamol (150 mg/ kg) + SCE (200 mg/kg)
63.81 ± 31.34a 38.14 ± 0.82 a 162 ± 0.49a 0.75 ± 0.10 a
54.47 ± 22.30a 65.40 ± 20.53a 124.17 ± 32.99a,c 0.73 ± 0.11a
47.76 ± 2.45a 34.88 ± 5.92a 110.29 ± 28.46a,b 0.63 ± 0.10a
43.71 ± 4.45a 22.69 ± 0.30a 94.69 ± 17.74a,b 0.55 ± 0.10a
48.25 ± 21.64a 42.21 ± 5.12a 110.59 ± 17.12a,b 0.63 ± 0.10a
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4. Discussion The present work explored the ability of ethanolic extracts of A. paniculata or S. chirayita to offer protection against hepatotoxicity induced by ‘over the counter drug’, paracetamol. Liver is a major metabolic organ affected by various chemicals and toxins and liver injuries induced by various hepatotoxins have been recognized as a major toxicological problem for years (Azer et al., 1997). In the absence of reliable liver protective drugs in modern medical practices, herbs play an important role in the management of various liver disorders. A number of plants show hepatoprotective activity (Hamza, 2010; Hwang et al., 2009; Olaleye et al., 2010; Malhotra et al., 2001). A. paniculata (Kalmegh) is used extensively in the Indian traditional system of medicine as a hepatoprotective and hepatostimulative agent. The aqueous extract of A. paniculata inhibited BHC induced liver toxicity in Swiss male mice (Trivedi et al., 2007; Trivedi and Rawal, 2000; Sutha et al., 2010) and ethanol induced hepatotoxicity in albino wistar rats (Vetriselvan et al., 2011) Andrographolide offered protection against galactosamine or paracetamol induced toxicity to hepatic tissue (Handa and Sharma, 1990).
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Earlier works showed that A. paniculata at a dose of 500 mg/kg of is nontoxic to mice (Guo et al., 1988) and S. chirayita is safe for animals up to a dose of 2000 mg/kg (Bhargava et al., 2009). In our study, paracetamol is used as a model system to induce acute hepatotoxicity in Swiss albino mice. Assessment of liver function is made by estimating the activities of serum GPT, GOT, ALP and bilirubin which are present higher concentration in cytoplasm. When there is hepatopathy, these molecules leak into the blood stream in compliance with the extent of liver damage (Nkosi et al., 2005). Bilirubin is one of the most useful clinical clues to the severity of necrosis and its accumulation is a measure of binding, conjugation and excretory capacity of hepatocyte. After normal therapeutic doses, paracetamol is metabolized, by phase II drug-metabolizing enzymes such as the glucuronyl and sulfotransferases (Anker and Smilkstein, 1994). When high doses are in taken, the major portion of paracetamol dose is available to undergo bioactivation by the cytochrome P450 system (CYP2E1, CYP3A4, CYP1A2) to create a highly reactive intermediate, N-acetyl-p-benzoquinoneimine (NAPQI) (Dahlin et al., 1984; Nelson 1995; Patten et al., 1993; Raucy et al., 1989). This intermediate can covalently binds to cellular macromolecules, causing damage and cell death (Rogers
Fig. 5. Histopathological analysis of liver tissue of animals treated with paracetamol and administrated with the extracts of Andrographis paniculata or Swertia chirayita. Normal – liver histopathology of untreated animal, Paracetamol – liver histopathology of paracetamol treated animal, Silymarin – liver histopathology of paracetamol treated animals administered with Silymarin (75 mg/ kg), APE (100 mg/kg) – liver histopathology of paracetamol treated animal administered with 100 mg/kg of Andrographis paniculata extract, APE (200 mg/kg) – liver histopathology of paracetamol treated animal administered with 200 mg/kg of Andrographis paniculata extract, SCE (100 mg/kg) – liver histopathology of paracetamol treated animal administered with 100 mg/kg of Swertia chirayita extract, SCE (200 mg/kg) – liver histopathology of paracetamol treated animal administered with 200 mg/kg of Swertia chirayita extract.
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et al., 1997). Accidental or incidental paracetamol overdose may be associated with toxic liver damage leading to potentially fatal, hepatic centrilobular necrosis and liver failure (James et al., 2003; Bergman et al., 1996). This toxic reaction is associated with metabolic activation that results in the formation of NAPQI, which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione (Henderson et al., 2000). At higher doses glutathione becomes depleted and the excess of NAPQI arylates and oxidizes hepatic proteins (Fountoulakis et al., 2000). Oxidative stress is another mechanism that has been postulated to be important in the development of paracetamol toxicity. Toxicity begins with the change in endoplasmic reticulum, which results in the loss of metabolic enzymes located in the intracellular structures (Recknagel, 1983). It was observed that the levels of cellular antioxidant enzymes and molecules are decreased significantly in paracetamol treated animals. The declined antioxidant enzyme activity is responsible for the increased lipid peroxidation measured as thiobarbituric acid reacting substance malondialdehyde (MDA), which causing loss of membrane fluidity, membrane integrity, and finally loss of cell functions of liver (Smith et al., 1987; Halliwell and Gutteridge, 1989). This peroxidative damage to membranes results in the leakage of enzymes, and metabolites to circulation. In the present study, it was observed that, the animals treated with paracetamol showed elevated levels of serum markers such as GPT, GOT, ALP and bilirubin. Normally, GOT and ALP are present in high concentration in liver. Due to hepatocyte necrosis or abnormal membrane permeability, these enzymes are released from the cells and their levels in the blood increases. GPT is a sensitive indicator of acute liver damage and elevation of this enzyme in non hepatic diseases is unusual. GPT is more selectively a liver paranchymal enzyme than GOT (Shah et al., 2002). The rise in the SGOT is usually accompanied by an elevation in the levels of SGPT, which play a vital role in the conversion of amino acids to keto acids (Sallie et al., 1999). Oral administration of ethanolic extract of A. paniculata or S. chirayita at the doses of 100 and 200 mg/kg significantly prevented the elevation of the serum markers of hepatotoxicity and restored the tissue antioxidant levels and decreased the lipid peroxidation levels in liver. The results would suggest that the extracts protected the membrane integrity of the liver cells against paracetamol induced leakage of marker enzymes into the circulation. The elevated levels of alkaline phosphatase reflected the pathological alteration in biliary flow (Ploa and Hewitt, 1989). The paracetamol induced elevation of this enzyme in the serum is lined up with high level of serum bilirubin. Administration of the ethanol extract of A. paniculata or S. chirayita decreased the ALP activity and serum bilirubin levels and stabilized biliary dysfunction and normal functional status of the liver (Suky et al., 2011). The increase in lipid peroxidation in liver due to paracetamol administration leading to tissue damage (Muriel et al., 1992) is due to the failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with ethanol extract of A. paniculata or S. chirayita significantly reversed these changes. Administration of ethanolic extract of A. paniculata or S. chirayita effectively prevented the decrease in SOD and GPX activities which may be due to the scavenging of radicals by the extract. Thus the present study indicates that A. paniculata or S. chirayita maintain the cellular integrity of hepatic tissues and helped its regeneration. Slightly better protective effect is noticed in case of GSH and GPx activities for the extract of Swertia while the extracts of Andrographis and Swertia showed almost similar activity in the case of SOD and MDA levels. The protective ability of these plant extracts towards paracetamol induced hepatotoxicity might be attributed to the free radical
scavenging activity of the extracts. The findings suggest the therapeutic use of these plants for liver ailments for ameliorating hepatotoxicity.
5. Conclusions The present work analyzed the hepatoprotective potential of ethanolic extracts of two plants, A. paniculata and S. chirayita. The results indicated the potential of these two plant extracts to offer protection against the acute hepatotoxicity induced by paracetamol. The S. chirayita extract has been found to provide higher hepatoprotection than A. paniculata.
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