Hepatoprotective Activity of Flowers of Madhuca longifolia (Koen.) Macbr. Against Paracetamol-Induced Hepatotoxicity
M. Umadevi*, C. Maheswari , R. Jothi, Sai Kishore Paleti, Y. Srinivasa Reddy R. Venkata Narayanan
R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore-641402, Tamilnadu, India.
*Corresponding Author E-mail: umadevi.m@rediffmail.com
ABSTRACT:
The present study was conducted to evaluate the hepatoprotective activity of methanolic extract of flowers of Madhuca longifolia using paracetamol-induced liver damage in Wistar albino rats. Two doses of methanolic extract of Madhuca longifolia (100 and 200 mg/kg) were administered orally to the animals with hepatotoxicity induced by paracetamol (2 gm/kg). The methanolic extract showed significant (p<0.005) protective effect by lowering serum levels of various biochemical parameters such as serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphatase (ALKP) and total bilirubin, and by increasing serum levels of total protein and albumin in the selected model. The biochemical observations were supplemented by histopathological examination of liver sections. The present study demonstrated the hepatoprotective activity of methanolic extract of M. longifolia validating the traditional use of flowers.
KEYWORDS: Madhuca longifolia, Paracetamol, hepatoprotective
INTRODUCTION:
Madhuca longifolia (Koen.) Macbr. (Sapotaceae) is a medium sized deciduous tree occurs throughout India. The plant is commonly known as Mahua (Hindi), Indian butter tree (English) and Madhuka (Sanskrit). According to Ayurveda the flowers are sweet, cooling, oleaginous and aphrodisiac. It is good in diseases of heart, cures burning sensation, biliousness and ear complaints1. The bark is reported to possess antioxidant, hepatoprotective2 and antihyperglycemic3 activities. The flowers are reported to have analgesic effect4. The leaves possess nephro and hepatoprotective effect, as well as antioxidant activity5. The Ayurvedic Pharmacopoeia of India recommends the flower without stalk or calyx in asthma and pthisis6. However, there has been no report on the hepatoprotective activity of the flowers of this plant. Therefore, a study was conducted to investigate the hepatoprotective effect of the methanolic extract of M. longifolia against paracetamol-induced liver damage in albino rats.
MATERIALS AND METHODS:
1. Plant:
The flowers of Madhuca longifolia (Koen.) Macbr. was collected in and around Coimbatore and authenticated by a Botanist, National Institute of Herbal Science, Chennai, India.
A voucher specimen (PARC/2009/306) was deposited in the herbarium of the Department of Pharmacognosy, R.V.S College of Pharmaceutical Sciences, Coimbatore.
2. Preparation of extract:
The flowers were shade dried, milled and the coarse powder was extracted by using methanol as a solvent in the soxhlet extractor. The solvent was removed under reduced pressure to get semisolid mass. Preliminary phytochemical screening of the extract was performed using standard methods to know the phytoconstituents in the extract7. It was found that the extract contains flavonoids, saponins and phenolic compounds.
3. Animals:
Wistar albino rats weighing 150-200 g of either sex, maintained under standard husbandry conditions were used for all studies. They were fed standard rodent pellet diet and free access of water. The experiments were performed after the experimental protocol approved by the Institutional Animals Ethics Committee (IAEC1012/C/06/CPCSEA-corres-2008-09).
4. Acute Toxicity Study:
Acute toxicity study was performed for M. longifolia extract according to the acute toxic classic method as per OECD guidelines8. Female albino rats were used for acute toxicity study. The animals were kept fasting for overnight providing only water, after which the extracts were administered orally at the dose of 300 mg/kg and observed for 14 days. If mortality was observed in two out of three animals, then the dose administered was assigned as toxic dose. If the mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher dose i.e. 2000 mg/kg.
5. Paracetamol-induced hepatotoxicity:
The rats were divided into 4 groups of six each, control, paracetamol and test groups. Group I served as a normal control received a single dose of 40% sucrose solution (1 ml/rat) on day 3. Group II received a single dose of paracetamol suspension (2 g/kg) on day 3. Groups III, IV and V were given daily doses of silymarin (25 mg/kg), M. longifolia extract 100 mg/kg and 200 mg/kg, respectively for 4 days, and a single dose of paracetamol suspension (2 g/kg) on day 3. All the drugs were administered orally.
6. Assessment of liver function:
After 48 hours of administration of paracetamol, blood was collected from all groups and allowed to clot at room temperature and serum was separated by centrifuging at 2500 rpm for 10 min. The serum was used for estimation of biochemical parameters such as serum glutamic oxaloacetic transaminase (SGOT)9, serum glutamic pyruvic transaminase (SGPT)9, serum alkaline phosphatase (ALKP)10, total bilirubin11, total protein12 and albumin13 to determine the functional state of liver.
7. Histopathological study14:
The rats were sacrificed under light anaesthesia, livers excised, rinsed clean in saline and preserved in 10% formalin for histopathological study using 5 mm thick sections stained with haematoxylin-eosin dye. The sections were observed under microscope for histopathological changes in liver architecture and the photomicrographs were taken.
8. Statistical analyses:
All values are expressed as mean ± SEM. Statistical analyses were performed by Student’s t test. Values of p lower than 0.0001 were considered significant.
RESULTS:
1. Acute toxicity studies:
M. longifolia methanolic extract did not cause any mortality up to 2000 mg/kg and was considered as safe.
2. Paracetamol-induced acute hepatotoxicity:
Paracetamol intoxication in normal rats elevated the levels of SGOT, SGPT, ALKP and total bilirubin (Table 1). Whereas decrease in the levels of total protein and albumin were observed significantly indicating acute hepatocellular damage and biliary obstruction.
The rats that received the M. longifolia extract showed a significant decrease in all the elevated SGOT, SGPT, ALKP and total bilirubin levels and significant increase in reduced total protein and albumin levels. These results were comparable with the standard drug silymarin. The effect of M. longifolia extract at the dose of 200 mg/kg was found to be higher than that at the dose of 100 mg/kg. Therefore, there has been a dose related response in the liver function.
3. Histopathological studies:
Histopathological examination of liver sections of control group showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and central view (Figure 1). Disarrangement of normal hepatic cells with necrosis and vacuolization were observed in paracetamol intoxicated liver (Figure 2). The liver sections of the rat treated with silymarin and M. longifolia extract (100 and 200 mg/kg) along with paracetamol showed less vacuole formation and absence of necrosis and overall less visible changes observed (Figure 3 and 4).
Figure 1: Normal rat liver section
Figure 2: Liver section of rat intoxicated with Paracetamol
|
Group |
SGOT (IU/L) |
SGPT (IU/L) |
ALKP (IU/L) |
Total bilirubin (mg/dl) |
Total protein (g/dl) |
Albumin (g/dl) |
|
Control |
99.88 ± 2.92 |
75.37 ± 2.83 |
256.05 ± 16.25 |
1.32 ± 0.05 |
8.20 ± 0.27 |
3.98. ± 0.07 |
|
Paracetamol |
243.62 ± 10.44** |
209.47 ± 7.31** |
428.25 ± 5.79** |
3.1 ± 0.15** |
2.25 ± 0.14** |
1.86 ± 0.05** |
|
Silymarin |
105.80± 0.58* |
77.73± 0.29* |
259.59± 0.99* |
1.55± 0.02* |
7.73± 0.13* |
3.91± 0.05* |
|
M. longifolia extract (100 mg/kg) |
172.20 ± 3.16* |
131.57± 3.28* |
354.18± 4.47* |
2.42 ± 0.04* |
4.2 ± 0.14* |
2.97± 0.05* |
|
M. longifolia extract (200 mg/kg) |
110.10 ± 2.96 * |
79.36 ± 2.81 * |
264.45± 15.76* |
1.72± 0.09* |
7.18 ± 0.20* |
3.38± 0.05* |
Each value is mean ± SEM (n=6), * Significant as compared to paracetamol treated group (p<0.0001), ** Significant as compared to control group (p<0.0001)
Figure 3: Liver section of rat treated with M. longifolia extract 100 mg/kg and intoxicated with Paracetamol
Figure 4: Liver section of rat treated with M. longifolia extract 200 mg/kg and intoxicated with Paracetamol
DISCUSSION:
Acute toxicity study shows the safety of methanolic extract of M. longifolia. Paracetamol is an antipyretic and analgesic drug which produces hepatic necrosis in high doses. Toxic doses of paracetamol cause a serious hepatotoxicity. Normally paracetamol is metabolized by glucuronide and sulphate conjugation (major pathway). However, N-acetyl-p-benzoquinoneimine is a highly toxic minor metabolite formed by cytochrome P-450 enzymatic system (minor pathway). The little amount of the metabolite so formed from the therapeutic doses of paracetamol gets detoxified by conjugation with glutathione. With toxic doses of paracetamol large amounts of the toxic metabolite are formed which cannot be handled and detoxified through glutathione conjugation. Instead this metabolite forms covalent bond to hepatic and renal cellular proteins causing necrosis and renal cell death15.
Reduction in SGOT and SGPT levels towards the normal value is an indication of regeneration process in rats treated with M. longifolia extract. Reduction of ALKP level with concurrent depletion of raised bilirubin level suggests the stability of the biliary function during injury with paracetamol. The protein and albumin levels were also raised suggesting the stabilization of endoplasmic reticulum leading to protein synthesis. The histological examination of the liver sections reveals that the normal liver architecture was disturbed by hepatotoxin intoxication. In the liver section of the rats treated with M. longifolia extract and intoxicated with paracetamol the normal cellular architecture was retained, thereby confirming the protective effect of the extract. Flavonoids, saponins and phenolic compounds present in the methanolic extract may be responsible for the hepatoprotective activity. In literature many authors reported the hepatoprotective activity of phytoconstituents like flavonoids16, saponins17 and phenolic compounds18.
CONCLUSION:
The protective action of M. longifolia against paracetamol-induced hepatotoxicity validates the traditional uses of the herb for liver disorders. Therefore, an indepth study on various fractions of the methanolic extract effective as hepatoprotective entities from the plant is warranted to determine the most potent hepatoprotective fraction from M. longifolia.
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Received on 06.09.2010 Modified on 14.09.2010
Accepted on 22.09.2010 © RJPT All right reserved
Research J. Pharm. and Tech. 4(2): February 2011; Page 259-262