Investigation of Antioxidant activity of Butea monosperm barks

 

Pradeep Kumar Samal*

SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh.

*Corresponding Author E-mail:- samalpharmacology@rediffmail.com

 

 

ABSTRACT:

The objective of this study was to investigate the antioxidant activity of Butea monosperma barks against Paracetamol induced  acute hepatotoxic model.  Butea monosperma barks were dried in shade, powdered with help of mechanical grinder and extracted with methanol. Preliminary phytochemical study shows the presence of Alkaloids, Carbohydrates, Tannins, Amino acids and Flavanoids. The methanolic extract of Butea monosperma  (MEBM) showed a significant dose dependent (250 mg and 500 mg/kg p.o.) antioxidant activity. The degree of antioxidant activity was measured by using serum  biochemical parameters like hepatic antioxidant enzymes like SOD, CAT, GSH and LPO. MEBM at a dose level of 250mg/kg and 500mg/kg produce significant (P<0.001) antioxidant activity by decreasing the level lipid peroxidation activity (LPO), while it significantly increased the levels of Reduced Glutathione (GSH), superoxide dismutase (SOD) and Catalase activity (CAT) in a dose dependent manner. The effect of MEBM was comparable to that of standard drug Silymarin.

 

KEYWORDS: Butea monosperma, LPO, SOD, GSH, CAT

 

 


1. INTRODUCTION:

Free radicals, from both endogenous and exogenous sources are implicated in the etiologic of several degenerative diseases such as coronary artery diseases, stroke, rheumatoid arthritis, diabetes and cancer.1 High consumption of fruits and vegetables is associated with low risk for these diseases, which is attributed to the antioxidant vitamins and other phytochemical.2-4 There is a great deal of interest in edible plants that contain antioxidants and health promoting phytochemicals, in view of their health implications.

 

Liver is often abused by environmental toxins, poor eating habits, methanol and over the counter drug use, that damage and weaken the liver leading to important public health problems like hepatitis, cirrhosis and methanolic liver diseases.5 The conventional drugs used in the treatment of liver diseases viz., corticoasteroids, antiviral and immunosuppressant agents are sometimes inadequate and may lead to serious adverse effects. In India, numerous medicinal plants and their formulations are used for liver disorders in traditional systems of medicine. It is cardinal to treat liver disorders since it directly affects the biochemistry of the cell through participating events like oxidative stress, redox change, etc.6 which induces lipid peroxidation.

 

Peroxidation of membrane phospholipids not only alters the lipid mileu and structural as well as functional integrity of cell membranes, but also affects the activities of various membrane-bound enzymes including total ATPase, Mg2+ATPase, Ca2+ATPase and Na+K+ATPase.7-8 In view of lack of synthetic agents for the treatment of hepatic disorder, there is a growing focus to evaluate traditional herbal medicines for hepatoprotective activity.9 Therefore; there is a need to develop satisfactory hepatoprotective drugs.

 

Butea monosperma (Fabaceae) better known as Palash by the locals is rich in flavanoids. Most flavanoids are

bioactive compounds due to the presence of phenolic group in their molecule. Among the various medicinal properties

attributed, the flower is of greater therapeutic value in the treatments of liver disorders and indigestion. 10, 11

 

2. MATERIALS AND METHODS:

2.1 Plant Materials: - The barks  of Butea  monosperma  (Fabaceac) were collected  in September 2007 and was taxonomically identified and authenticated. The Plant material was authenticated by Dr. H.B. Singh, Head, Raw Material Herbarium and Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi. (Ref. NISCAIR/RHMD/consult/-2010-11/1482/80 dated 11/08/10. After authentication, the plant materials were washed  thoroughly with water and dried   in shade   for about five weeks. The dried materials were then ground to coarse powder by using by a mechanical grinder. This powder material was again dried in the shade at about 20°C for one week and store in air tight containers for further studies.

 

2.2 Drugs and Chemicals: -

Analytical grades Chemical were used in this study. Silymarin (Micro labs, Bangalore) was purchased from local market. Chemical like  methanol (CDH, Mumbai), Anesthetic ether (CDH, Mumbai) and Paracetamol (Ranbaxy, Delhi), Thiobarbituric acid, Trichloro acetic acid, H2O2, Epinephrine, EDTA, Tric Hcl buffer, GSH, NADP and other phytochemical reagents were obtained from Institute. 

 

2.3 Animals: -

Each experiment had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. The approval of the Institutional Animal Ethical Committee (IAEC) of School of pharmaceutical sciences, S.O.A. University, Bhubaneswar was taken prior to the experiments (1171/c/08/CPCSEA). All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals.

 

2.4 Preparation of plant Extracts: -

About 300 g of the barks  powder of Butea monosperma  was extracted with 1.2 L of methanol using Soxhlet apparatus for 72 hrs at 40-50°C. The extract was concentrated to ¼ of its original volume by distillation as it was adapted to recover the solvent, which could be used again for extraction.12       

 

2.5 Acute toxicity study (AOT): -

Acute oral toxicity study was performed according to the procedure OECD guideline no. 425.13  AOT was performed on Swiss albino mice and the animal were kept fasting for overnight providing water ad libitum, after which the methanolic extract of Butea monosperma  (MEBM) was administered orally 5000 mg/kg and observed the mortality of animals.

 

2.6 Preliminary Phytochemical Analysis: -

The extracts obtained were subjected to various chemical tests to detect the chemical constituents present in extracts of Butea monosperma.14-17

 

2.7 Assessment of liver function: -

The methanolic extract of Butea monosperma was evaluated for their hepatoprotective activity by using Paracetamol induced acute hepatotoxicity model. Either sex of Wistar rats, weighing (180-220) was divided into 5 groups consisting of 6 animals in each group. Group 1 received distilled water (6 ml/kg, p.o.) for 7 days. Group 2 were treated with vehicle (0.5% of Tween 40, 1 ml/kg, p.o.) for 7 days. Group 3 received silymarin (50 mg/kg, p.o.) for 7 days. Group 4, 5 pretreated with methanolic extract of Butea monosperma  250 mg and 500 mg/kg body weight respectively for 7 days. Food was withdrawn 16 hrs before administration to enhance the acute liver toxicity.  Group 2, 3, 4 and 5 were treated with Paracetamol was administered (500 mg/kg, p.o.) was administered on 7th day after 1 hrs of extracts treatment. After the treatment animals were sacrificed by cervical decapitation under excessive anaesthesia on the 8th day. Immediately after sacrifice, the liver was dissected out, washed in the ice-cold saline, and the homogenate was prepared in 0.1M Tris–HCl buffer (pH 7.4). The homogenate was centrifuged and the supernatant liquid was used for the assay of marker enzymes, namely namely lipid peroxidation activity(LPO).18, Reduced Glutathione (GSH).19, superoxide dismutase (SOD).20 and Catalase activity (CAT).21

 

2.8 Statistical analysis: -

The experimental results were expressed as the Mean ± SEM for six animals in each group. The biochemical parameters were analysed statistically using one-way ANOVA followed by Tukey Kramer’s post hoc test. P value of < 0.05 was considered as statistically significant.

 

3. RESULTS:

Preliminary phytochemical studies with extract revealed the phytoconstituents like cardiac glycoside, carbohydrates, phytosterols, saponins, phenolics and tannins. Different doses of methanolic extract of Butea monosperma barks  (MEBM) was screened in albino mice for their acute oral toxicity. No mortality was recorded till 2000 mg/kg body weight. Hence the extract was found to be safe up to the dose levels of 2000 mg/kg. So 1/10th and 1/20th of these dose i.e. 250 and 500 mg/kg body weight of MEBM for oral dose was select as therapeutic dose for pharmacological activity screening.

 

Fig: 1  LPO  effect of the Butea monosperma barks extracts in Paracetamol induced hepatotoxic rats.

 

Fig: 2. GSH effect of the Butea monosperma barks extracts in Paracetamol induced hepatotoxic rats.

 

Fig: 3  sod  effect of the Butea monosperma barks extracts in Paracetamol induced hepatotoxic rats.

 

Fig: 4  cat  effect of the Butea monosperma barks extracts in Paracetamol induced hepatotoxic rats.

 

The effects of CETI and METI on rat liver lipid peroxidation(LPO), glutathione(GSH) and enzyme antioxidants namely SOD and CAT levels are shown in Fig. 1 to 4. The levels of LPO were considerably increased in rats treated with paracetamol as compared to the normal rats. Treatment with MEBM (250 mg and 500 mg/kg/day) resulted in a significant decrease (P<0.05) in levels of LPO and brought them near to normal level. A significant decrease in the activities of glutathione and enzyme antioxidants (SOD and CAT) were noted after single administration of  paracetamol. Upon administration of  MEBM  the activities of glutathione and enzyme antioxidants were significantly (P<0.05) reversed to near normal. The effects of MEBM were comparable with that of standard reference drug silymarine.

 

4. DISCUSSION:

Paracetamol (acetaminophen) is a commonly and widely used analgesic and antipyretic agent. Hepatotoxic doses of acetaminophen deplete the normal levels of hepatic glutathione, when NAPQI covalently binds to cysteine groups on proteins to form 3-(cystein-S-yl) acetaminophen adducts22. The glutathione protects hepatocytes by combining with the reactive metabolite of paracetamol thus preventing their covalent binding to liver proteins 23. Lipid peroxidation has been postulated to the destructive process of liver injury due to acetaminophen administration. In the present study the elevations in the levels of end products of lipid peroxidation in the liver of rat treated with paracetamol were observed. The increase in malondialdehyde (MDA) levels in liver suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals. Treatment with MEBM significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection by MEBM is due to its antioxidant effect. The non-enzyme antioxidant, glutathione is one of  the most abundant tripeptides present in the liver. Its functions are mainly concerned with the removal of free radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals, and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase and GST24. In our present study the decreased level of GSH has been associated with an enhanced lipid peroxidation in paracetamol-treated rats. Administration of  MEBM significantly increased the level of glutathione in a dose-dependent manner. The enzyme antioxidant defense system is the nature protector against lipid peroxidation. SOD, CAT and GPx enzymes are important scavengers of superoxide ion and hydrogen peroxide. These enzymes prevent generation of hydroxyl radical and protect the cellular constituents from oxidative damage25. In the present study, it was observed that the MEBM significantly increased the hepatic SOD activity in paracetamol induced liver damage in rats. This show MEBM can reduce reactive free radicals that might lessen oxidative damage to the tissues and improve the activities of the hepatic antioxidant enzyme. Catalase (CAT) is an enzymatic antioxidant widely distributed in all animal tissues and the highest activity is found in the red cells and in the liver. CAT decomposes hydrogen peroxide and protects the tissue from highly reactive hydroxyl radicals26. Therefore the reduction in the activity of these enzymes may result in a number of deleterious effects due to theaccumulation of superoxide radicals and hydrogen peroxide. Administration of MEBM increased the activities of CAT in paracetamol-induced liver damage in rats to prevent the accumulation of excessive free radicals and protected the liver from paracetamol intoxication.

 

The qualitative analysis of Butea monosperma  barks  extract indicated the presence of flavonoids, polyphenols, are natural antioxidants but have also been reported to significantly increase SOD, Glutathion and catalase and decrease MDA level.

 

5. ACKNOWLEDGEMENTS:

The authors wish to thank Prof. J.S. Dangi, Head of the Institute for facilities and Mr. Karteek Patra for technical assistance.

 

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Received on 11.04.2013          Modified on 15.05.2013

Accepted on 20.05.2013         © RJPT All right reserved

Research J. Pharm. and Tech 6(6): June 2013; Page 610-613