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Introduction

Liver disease is still a worldwide health problem. Unfortunately, conventional or synthetic drugs used in the treatment of liver diseases are inadequate and some-times can have serious side effects (Guntupalli et al., 2006). Drug-induced hepatotoxicity is a major cause of new drug withdrawal from the market. It also limits further development of promising therapeutic agents even prior to clinical trials. Over-the-counter drugs are not exempt from hepatotoxic liability; for example, acet-aminophen (paracetamol), a widely used (and misused) analgesic/antipyretic agent, when taken acutely in large

doses or chronically in greater than recommended dos-ages, can lead to liver and kidney damage (Lorelle et al., 2003). In view of severe undesirable side effects of syn-thetic agents, there is growing focus to follow systematic research methodology and to evaluate scientific basis for the traditional herbal medicines that are claimed to pos-sess hepatoprotective activity (Manokaran et al., 2008). The plant Glycosmis pentaphylla (Retz.) DC (Rutaceae), commonly known as “tooth brush plant” in English and “ashvashakota” in Sanskrit, is an aromatic shrub that grows from 0.5 to 2 m in height and distributed through-out India (Kirtikar & Basu, 1998). It is common in waste

RESEARCH ARTICLE

Hepatoprotective activity of Glycosmis pentaphylla against paracetamol-induced hepatotoxicity in Swiss albino mice

Siva Shankar Nayak1, Ranjeet Jain2, and Atish Kumar Sahoo3

1Department of Pharmaceutical Chemistry, College of Pharmaceutical Sciences, Mohuda Berhampur, Orissa, India, 2Department of Pharmaceutical Chemistry, Swami Vivekananda College of Pharmacy, Indore, Madhya Pradesh, India, and 3Department of Pharmaceutical Chemistry, M.S. Ramaiah College of Pharmacy, Bangalore, India

AbstractContext: Glycosmis pentaphylla (Retz.) DC (Rutaceae) is a shrub, traditionally used to treat anemia, rheumatism, as an anthelmintic, febrifuge and vermifuge, for jaundice, and liver complaints.

Objective: This study evaluated the hepatoprotective activity of G. pentaphylla against paracetamol-induced hepatotoxicity in Swiss albino mice.

Materials and methods: Effect of methanol extract (200 and 400 mg/kg) and petroleum ether extract (200 and 400 mg/kg) were studied on paracetamol-induced (250 mg/kg intraperitoneally) hepatic damage in mice for estimating the serum marker enzymes as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin, and total protein. Then the sections of liver were observed for histopathological changes in liver architecture including necrosis, steatosis, and fatty change of hepatic cells.

Results: Mice were protected from the hepatotoxic action of paracetamol as evidenced by significant reduction in the elevated serum level of ALT (P < 0.001), AST (P < 0.001), ALP (P < 0.001), total bilirubin (P < 0.01) and an increased level of total protein (P < 0.01) with a significant reduction in liver weight (P < 0.01) when compared with paracetamol treated group and silymarin (50 mg/kg) was used as a positive control.

Discussion and conclusion: These biochemical observations were supplemented by histopathological examination of liver sections from different experimental groups and corroborated the hepatoprotective efficacy of methanol and petroleum ether plant extract. The methanol extract (400 mg/kg) of G. pentaphylla is able to alter the toxic condition of the hepatocytes so as to protect the membrane integrity against paracetamol-induced leakage of marker enzymes.

Keywords: Glycosmis pentaphylla, Rutaceae, paracetamol, hepatoprotective, leaves

Address for Correspondence: Dr. Atish Kumar Sahoo, Associate Professor, Department of Pharmaceutical Chemistry, M.S. Ramaiah College of Pharmacy, Bangalore-560054, India. E-mail: atish_kumar1976@yahoo.co.in

(Received 30 January 2010; revised 01 May 2010; accepted 29 May 2010)

Pharmaceutical Biology, 2011; 49(2): 111–117© 2011 Informa Healthcare USA, Inc.ISSN 1388-0209 print/ISSN 1744-5116 onlineDOI: 10.3109/13880209.2010.501084

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places, villages, road-sides, and throughout the tropical and subtropical areas of India. The whole plant is ben-eficial in anemia, jaundice, rheumatism, and cough. The leaf juice is used as an anthelmintic, febrifuge, ver-mifuge, in liver complaints, and applied in the form of paste to eczema and other skin diseases (Chatterjee & Pakrashi, 1997). In homeopathy, leaves are used in bil-iary colic, worm-colic, diarrhea, and dysentery; while the ethanol extract (50%) of aerial parts is used as spas-molytic, diuretic, and the homeopathic mother tincture of leaves is claimed to cure throat cancer (Asolkar et al., 2000). Wood brushed with water is administered inter-nally as an antidote for snakebite (Chopra et al., 1999). The plant has been reported to contain glycosminine, glycosmicine, γ-fagarine, isoarborinol, and arborin-ine (Rastogi, 1999); the leaves contain furoquinoline, methyl acridone, arborinine, arborine, mupamine, and phlobaphene (Chatterjee & Pakrashi, 1997). The litera-ture reveals that G. pentaphylla is used orally in the treat-ment of jaundice in traditional Indian medicine and, moreover, the hepatoprotective activity of the stem and leaf water extract of G. pentaphylla against carbon tet-rachloride-induced liver toxicity was reported by Mitra and Sur (1997). To date, no systematic study has been reported in an acute model regarding the paracetamol-induced hepatotoxicity in mice. In the present study, an effort has been made to establish the scientific valid-ity to the hepatoprotective property of methanol and petroleum ether leaf extracts of G. pentaphylla against paracetamol-induced hepatotoxicity in mice.

Materials and methods

Plant materialThe leaves of Glycosmis pentaphylla (Retz.) DC (Rutaceae) were collected during the month of September 2008 from the rural belt of Mohuda in Ganjam District of Orissa, India; identified and authenticated by taxonomist S.K. Dash, Department of Bioscience, College of Pharmaceutical Sciences, Mohuda, and samples were kept in the laboratory for future reference. After authentication, the col-lected plant leaves were washed and air-dried under shade, cut into small pieces, powdered by a mechani-cal grinder and passed through a 40-mesh sieve and stored in a closed container for future use.

Preparation of G. pentaphylla leaves extractsCoarsely powdered leaves (1 kg) were extracted with 1200 mL of methanol by cold maceration (Ministry of Health and Welfare, 1985). The methanol extract was dried and concentrated by evaporating the solvent completely under vacuum using rotatory evaporator (Jain Scientific Glass Works, DTC 201, Ambala canton-ment, India) to produce a brownish semisolid mass after drying (16%, w/w) and the methanol extract (GPME) was further extracted with 600 mL of petroleum

ether (60–80°C) to produce a greenish brown semi-solid mass petroleum ether extract (GPPE) after drying (5.04%, w/w) and both the extracts were stored in a des-iccator. The chemical constituents of the extract were identified by qualitative analysis and confirmed by thin layer chromatography (i.e., hR

f values). The dried

extracts were made into emulsion by triturating the accurately weighed quantity (8 g) of the extract with 1% gum acacia and suspended in distilled water at definite concentrations separately used for the pharmacological study (Sabir & Rocha, 2008).

ChemicalsParacetamol (acetaminophen) was obtained from ALPA Laboratories, Indore, India and silymarin was obtained from Micro labs, Hosur, India as free samples. Assay kits were used for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin and total protein estimations and pur-chased from Autospan Diagnostic Reagent, Surat, India. All extractive solvents are of analytical grade reagents (AR) and purchased from S.D. Fine Chemicals, Mumbai, India.

Preliminary phytochemical analysisThe GPME and GPPE extracts were subjected to prelim-inary phytochemical screening for detection of major chemical groups. In each case test 10% w/v solution of the extracts were used unless otherwise mentioned in individual test (Patil et al., 2001). The qualitative analy-sis was performed by using Mayer’s, Dragendorff’s, or Wagner’s reagent for tests of alkaloids and terpenoids; Liebermann-Burchard reagent for test of steroids; using 1% lead acetate solution for test of saponins; using 1 mL dilute ammonia or 1 mL dilute sodium carbonate or 1 mL dilute sodium hydroxide or using 25% basic lead acetate solution for the test of flavonoids and polyphe-nols; using Fehling’s A & B solution or Benedict’s solu-tion for test of reducing sugar; also by using 1 mL of 5% ferric chloride or 10% lead acetate solution for the test of tannins.

Experimental animalsAll animal procedures were in strict accordance with the CPCSEA Guidelines (Committee for the Purpose of Control and Supervision of Experiment on Animals). Healthy Swiss albino mice of 7–8 weeks old weighing 20–35 g of either sex were used. They were obtained from Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal (Madhya Pradesh, India). The mice were housed at a temperature of 20–25°C under a 12 h light/dark cycle with 50% of relative humidity. They were fed standard animal feed (Lipton’s, India) and water ad libitum. This study complied with current ethical regulations on animal research in Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal. Approval for experimental work was as per the ethical

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committee of Jawaharlal Nehru Cancer Hospital and Research Centre (Reference No. 670/225-IAEC/2008/Project No. 50).

Acute toxicity studyThe LD

50 values of GPME and GPPE were derived as

described by Litchfield and Wilcoxon (1949). The maxi-mum non-lethal doses of both the extracts were found to be 4000 mg/kg body weight (bw), orally. The 1% gum acacia was used as a vehicle and showed no mortality. By adopting fixed dose the guideline of CPCSEA and 1/10th of LD

50 cut off values (Patil et al., 2001; Shivakumar

et al., 2006) the determination of acute toxicity of the extracts were taken as screening dose, i.e., 200, 400 mg/kg for subsequent studies (GPME-200, 400 mg/kg and, GPPE-200, 400 mg/kg).

Paracetamol-induced hepatotoxicity in mice (acute model)Animals were randomized and divided into seven groups (1–7) with six mice in each group. Animals of group 1 (control) received only vehicle (1% gum acacia in distilled water). Animals of group 2 (paracetamol control) received acetaminophen 250 mg/kg only. Animals of group 3 fed with acetaminophen 250 mg/kg and the standard drug silymarin 50 mg/kg. Animals of groups 4 to 7 were fed with a single dose of acetamino-phen 250 mg/kg; whereas groups 4 and 5 were fed with GPME, 200 and 400 mg/kg. Similarly, animals of groups 6 and 7 were fed with GPPE, 200 and 400 mg/kg. Plant extracts were administered 3 h after the admin-istration of acetaminophen. All the treatments were done by means of a gavage or gastric tube. The treat-ments were continued for seven consecutive days and on the eighth day of the experiment, the animals were anesthetized with ether and blood samples were collected by puncturing the retro-orbital plexus, and serum was separated by centrifugation at 1500 rpm for 10 min. The serum was then analyzed for alanine amin-otransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin and total protein level using assay kits according to the supplier (Autospan Diagnostic Reagent, Surat, India) specifica-tions (Sabir & Rocha, 2008). The estimation was done by using a photoanalyser instrument (FT-2; AMS); each determination was carried out with a suitable kit (Autospan Diagnostic Reagent, Surat, India) containing the necessary reagents at a predetermined wavelength for each parameter.

Biochemical estimation of marker enzymesEffect of treatment with paracetamol on the serum ALT activitiesThe estimation was done by using a photoanalyser (FT-2; AMS); serum ALT determination was carried out with a suitable kit (Autospan Diagnostic Reagent) containing the necessary reagents at a predetermined wavelength 340 nm where the rate of oxidation of NADH is measured

kinetically by monitoring the decrease in absorbance at 340 nm (Murray, 1994).

Effect of treatment with paracetamol on the serum AST activitiesThe estimation was done by using a photoanalyser (FT-2; AMS); serum AST activity was carried out by suitable kit (Autospan Diagnostic Reagent) containing the necessary reagents. The rate of oxidation of NADH is measured kinetically by monitoring the decrease in absorbance at 340 nm and is directly proportional to GOT activity in the sample (Murray, 1994).

Effect of treatment with paracetamol on the levels of ALP activitiesThe level of ALP activities were carried out using the kit Autospan Diagnostic Reagent analyzed in a photoanaly-ser (FT-2; AMS). By using a specific reagent at pH 10.3, alkaline phosphatase (ALP) catalyses the hydrolysis of colorless p-nitrophenyl phosphate (p-NPP) to yellow colored p-nitrophenol and phosphate. Change in absor-bance due to yellow color formation is measured kineti-cally at 405 nm and is proportional to ALP activity in the sample (Moss & Henderson, 1994).

Effect of treatment with paracetamol on the serum bilirubin levelsSerum bilirubin levels in paracetamol-induced liver dam-age were estimated using an analytical kit from Autospan Diagnostic Reagent. The assay principle is based on the fact that total bilirubin reacts in the presence of caffeine with diazotized sulfanilic acid to form azobilirubin. The determination of direct bilirubin at 546 nm is propor-tional to the concentration of bilirubin (Willard & Meites, 1982).

Effect of treatment with paracetamol on hepatic total protein levelsThe total protein level was estimated using an analyti-cal kit from Autospan Diagnostic Biuret Reagent ana-lyzed in a photoanalyser (FT-2; AMS). Peptide bonds of proteins react with cupric ions in alkaline solutions to form a colored chelate, the absorbance of which is measured at 578 nm. The biuret reagent contains sodium potassium tartrate to complex cupric ions and maintains their solubility at alkaline pH. Absorbance data are proportional to protein concentrations (Tietz, 1983).

Histopathological studiesOne animal from the treated groups showing maximal activity as indicated by improved biochemical parameters from each test, positive control, hepatotoxin and control groups were utilized for this purpose. The animals were sacrificed and the abdomen was cut open to remove the liver; 5 mm thick pieces of the liver were fixed in Bouin’s solution (mixture of 75 mL of saturated picric acid, 25 mL of 40% formaldehyde and 5 mL of glacial acetic acid) for

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12 h, then embedded in paraffin wax and cut into 5 μ thick sections were made using microtome and stained using hematoxylin-eosin dye and finally mounted in diphenyl xylene. Then the sections were observed for histopatho-logical changes in liver architecture and their photomi-croscopic observation including necrosis, steatosis and fatty change of hepatic cells (Gray, 1964; Shai et al., 2001; Saeed & Sabir, 2002).

Statistical analysisThe results were presented as mean ± SEM and statisti-cal significance between treated and a control group was evaluated by one-way analysis of variance (ANOVA) fol-lowed by Dunnett’s t-test.

Results

Preliminary phytochemical analysisResults of different chemical tests on the GPME showed the presence of phytoconstituents, i.e., reducing sug-ars, flavonoids, polyphenols, alkaloids, tannins, and saponins, whereas GPPE showed a positive tests for the presence of alkaloids, steroids, and terpenoids.

Effect of treatment with paracetamol on serum ALT activitiesRats treated with paracetamol in group 2 alone devel-oped significant liver cell damage as was evident from

a significant (P < 0.001) increase in serum alanine aminotransferase enzyme activities. Oral administra-tion of GPME and GPPE at two different doses, 200 and 400 mg/kg (groups 4 to 7), were seen to lower significantly (P < 0.01) the activities of marker enzymes; comparable to the group 3 of standard drug silymarin (50 mg/kg; P < 0.01) as shown in Table 1.

Effect of treatment with paracetamol on the serum AST activitiesIn the paracetamol treated group 2; there was a signifi-cant (P < 0.001) increase in aspartate aminotransferase activity than in that of the normal group 1. AST activity of GPME and GPPE groups of two different doses 200 and 400 mg/kg (groups 4 to 7) significantly (P < 0.01) recov-ered the level of enzyme activities which is comparable with standard drug silymarin group-3 (P < 0.01) as shown in Table 1.

Effect of treatment with paracetamol on the levels of ALP activitiesRats treated with paracetamol (group 2) alone devel-oped significant liver cell damage as was evident from a significant (P < 0.001) increase in serum enzyme lev-els of ALP compared to that of normal group 1. GPME and GPPE groups of two different doses, 200 and 400 mg/kg significantly (P < 0.01 group 4, 5, 7; P < 0.05 group 6), recovered the ALP level as compared to the

Table 1. Effect of extracts of Glycosmis pentaphylla on paracetamol (acetaminophen)-induced hepatotoxicity in mice.

Groups Treatment

Serum biochemical parameter Liver weight (g% of

body weight)ASTIU/L ALTIU/L ALPIU/LTotal bilirubin

mg/dlTotal protein

g/dlGroup 1 (Normal)

1% Gum acacia 49.50 ± 1.36 65.33 ± 1.40 83.42 ± 4.75 2.23 ± 0.08 7.1 ± 0.12 3.97 ± 0.15

Group 2 Acetaminophen (250 mg/kg,i.p)

158.33 ± 4.67a 129.42 ± 1.76a 257.83 ± 4.27a 4.02 ± 0.20a 4.6 ± 0.18a 6.02 ± 0.23a

Group 3 Acetaminophen 250 mg/kg i.p. + silymarin (50 mg/kg)

62.67 ± 2.03b 70.25 ± 1.87b 113.17 ± 3.30b 2.72 ± 0.12b 6.9 ± 0.22b 4.18 ± 0.19b

Group 4 Acetaminophen 250 mg/kg i.p. GPME (200 mg/kg)

83.17 ± 1.64b 94.58 ± 1.24b 146.00 ± 5.51b 2.63 ± 0.12b 6.4 ± 0.24b 4.37 ± 0.34b

Group 5 Acetaminophen 250 mg/kg i.p. + GPME (400 mg/kg)

70.00 ± 2.22b 79.25 ± 1.64b 108.33 ± 4.56b 2.45 ± 0.13b 6.7 ± 0.32b 4.23 ± 0.43b

Group 6 Acetaminophen 250 mg/kg i.p. + GPPE (200 mg/kg)

139.17 ± 2.74b 107.58 ± 2.39b 170.50 ± 3.50c 3.12 ± 0.12c 5.3 ± 0.33c 5.01 ± 0.42b

Group 7 Acetaminophen 250 mg/kg i.p. + GPPE (400 mg/kg)

111.83 ± 2.09b 100.25 ± 0.95b 139.17 ± 5.17b 2.80 ± 0.09b 5.7 ± 0.46b 4.81 ± 0.36b

aP < 0.001, bP < 0.01 and cP < 0.05.Values are mean ± S.E.M. number of rats=6. Statistical significant test for comparisons was done by ANOVA, followed by Dunnett’s t-test.Group-2 compared with normal group-1 with aP < 0.001.Experimental groups from-3 to 7 compared with group-2 with bP < 0.01 and cP < 0.05.

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standard drug silymarin group 3 (50 mg/kg; P < 0.01) as shown in Table 1.

Effect of treatment with paracetamol on the serum bilirubin levelsA significant (P < 0.001) increase in serum bilirubin level was seen in paracetamol-treated animals in group 2 compared to that of the normal group. Bilirubin levels for GPME and GPPE groups of two different doses 200 and 400 mg/kg significantly (P < 0.01 group 4, 5, 7; P < 0.05 group 6) recovered against the paracetamol group 2 and both doses were comparable with the standard drug sily-marin at 50 mg/kg (P < 0.01) as shown in Table 1.

Effect of treatment with paracetamol on hepatic total protein levels and liver weightRats treated with paracetamol group 2 alone developed significant liver cell damage as was evident from a signifi-cant (P < 0.001) decrease in the total protein levels when compared to normal group 1. But the administration of GPME and GPPE groups of two different doses 200 and 400 mg/kg have showed the significant (P < 0.01 groups 4, 5, 7; P < 0.05 group 6) recovery of total protein level as compared to the standard drug silymarin (50 mg/kg; P < 0.01) as shown in Table 1, whereas there was a signifi-cant reduction (P < 0.01) of liver weight of groups 4 to 7, when the GPME and GPPE groups of two different doses 200 and 400 mg/kg was given along with paracetamol.

Histopathological studiesHistopathological examination of liver sections of control group 1 and standard group 3 showed normal cellular architecture with distinct hepatic cells, sinu-soidal spaces and central vein (Figures 1A and 1C). Disarrangement of normal hepatic cells with centri-lobular necrosis, vacuolization of cytoplasm and fatty degeneration were observed in the paracetamol treated mice of group 2 in Figure 1B. The liver sections of the mice treated with GPME (200 and 400 mg/kg) of groups 4 and 5 showed signs of protection as was evident by the absence of necrosis and vacuoles (Figures 1D and 1E) while the liver sections of the mice treated with GPPE (200 and 400 mg/kg) of groups 6 and 7 showed reduction in fatty degeneration and absence of necrosis (Figures 1F and 1G).

Discussion

In the acute toxicity studies, oral administration of both GPME and GPPE of G. pentaphylla did not produce any mortality in mice up to a dose level of 4000 mg/kgbw. This may be due to the broad non-toxic range of the plant, where the plant extract showed a high LD

50 and relative

safety. Paracetamol (acetaminophen)-induced hepatic injuries are commonly used models for hepatoprotec-tive drug screening (Plaa & Hewitt, 1982). It is a well-known antipyretic and analgesic agent, which produces hepatic necrosis at higher doses because of the reaction

metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which causes oxidative stress and glutathione depletion (Boyd & Bereczky, 1966). Introduction of cytochrome or depletion of hepatic glutathione is a prerequisite for paracetamol-induced hepatotoxicity (Moron et al., 1979; Gupta et al., 2006). Assessment of liver function can be made by estimating the activities of serum ALT, AST, ALP, bilirubin and total protein which are enzymes originally present in higher concentration in cytoplasm (Manokaran et al., 2008). When there is hepatopathy, these enzymes leak into the blood stream which serves as an indicator for the liver damage (Nkosi et al., 2005).

The abnormally high level of serum ALT, AST, ALP, total bilirubin and decrease in total protein content observed in group 2 in our study are the consequence of paracetamol-induced liver dysfunction and denotes the damage to the hepatic cells (Table 1). Treatment with GPME and GPPE at dose levels of 200 and 400 mg/kg reduced the enhanced level of serum ALT, AST, ALP, total bilirubin and increased the total protein level which seemed to offer protection and maintain the functional integrity of hepatic cells. The elevated levels of serum marker enzyme activities such as ALT, AST enzyme levels are a direct measure of hepatic injury and they show the status of liver. The elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integ-rity of cell membrane in liver. Thus the lowering of the enzyme content in serum is a definite indication of the hepatoprotective action of a drug.

GPME and GPPE at a dose of 200 mg/kg (P< 0.01) and 400 mg/kg decreased significantly (P < 0.01) the activity of both AST and ALT as compared to the standard drug sily-marin. Serum ALP and bilirubin levels are also related to the status and function of hepatic cells. Increase in serum ALP level is due to increased synthesis, in the presence of increasing biliary pressure (Moss & Butterworth, 1974). In the present study, GPME and GPPE at a dose of 200 mg/kg (P < 0.01 & P < 0.05), and GPME and GPPE at a dose 400 mg/kg, were found to reduce significantly (P < 0.01) both serum ALP and bilirubin levels in the treated groups which are comparable to the standard drug silymarin (Table 1). A reduction in total serum protein (Table 1) observed in the paracetamol-treated rats may be associ-ated with a decrease in the number of hepatocytes, which in turn may result in decreased hepatic capacity to syn-thesize protein and consequently increase liver weight (Table 1). When the GPME and GPPE groups of two different doses 200 and 400 mg/kg was given along with paracetamol, a significant increase (P < 0.05 & P < 0.01) in total serum protein was observed indicating the hepato-protection activity of the extract and also accounting for the decrease in the liver weight most probably through the hepatic cell regeneration.

Hepatoprotective activity of G. pentaphylla was shown by its ability to inhibit paracetamol-induced liver dam-age in mice. The fall in serum marker level by GPME (400 mg/kg) was significantly (P < 0.01) able to alter the toxic condition of the hepatocytes as compared to GPPE

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at a dose of 200 and 400 mg/kg, so as to protect the mem-brane integrity against acetaminophen-induced leakage of marker enzymes. This means there was a potential functional change in the hepatocytes of the hepatic cells. Hence, it has been concluded that the methanol extract of G. pentaphylla (GPME) at dose levels of 400 mg/kg revealed its potential significance as a hepatoprotec-tion against acetaminophen-induced toxicity which was further supported by the presence of phytoconstituents such as flavonoids, polyphenols, tannins, and saponins. All the results can be comparable with the standard drug silymarin. A comparative histopathological study of liver from different experimental groups corroborated the hepatoprotective efficacy of methanol extract which was more effective than the petroleum ether extract of Glycosmis pentaphylla. Further detailed studies may, however, confirm the utility profile of this drug.

Acknowledgements

The authors thankfully acknowledge Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal, India.

Declaration of interest

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

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T. S. of liver of normal negativecontrol group-1

T. S. of liver of positive control group-2 T. S. of liver of standard group-3

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Figure 1. Histopathological study of rats liver treated with the methanol and petroleum ether extracts of Glycosmis pentaphylla. Photomicrographs of mice liver obtained from different treatment groups. T.S Figure (A) revealed normochromatic hepatocytes (H), occasionally apoptosis, cart wheel nucleus (CWN) and lipid vacuoles (CV). Figure (B) T.S. revealed hyper chromatic highly damaged hepatocytes (H), large numbers lipid vacuoles (LV), frequent apoptosis (Apop), necrosis (CWN). Figure (C) Standard group T.S. revealed normochromatic hepatocytes (H), occasionally apoptosis (Apop), cart wheel nucleus (CWN) and lipid vacuoles (LV). Figure (D) & (E) T.S. revealed normochromatic hepatocytes (H) with good mitotic index, occasionally lipid vacuoles (LV) without apoptosis, necrosis and cart wheel nucleus (S). Figure (F) & (G) T.S. of liver revealed hyper chromatic hepatocytes (H) with average mitotic index, lipid vacuoles (LV) and cart wheel nucleus (CWN), few apoptotic hepatocyts (AH). H and E staining (X400). Group 1(Control), Group 2 (paracetamol 250 mg/kg), Group 3 (paracetamol 250 mg/kg + silymarin 50 mg/kg), Group 4 (paracetamol 250 mg/kg + GPME-200 mg/kg), Group 5 (paracetamol 250 mg/kg + GPME-400 mg/kg), Group 6 (paracetamol 250 mg/kg + GPPE- 200 mg/kg) and Group 7 (paracetamol 250 mg/kg + GPPE-400 mg/kg), H & E staining (400X).

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