Hepatoprotective Effect of Ethanolic and Hydro Alcoholic Leaf Extract of Madhuca indica in Carbon Tetra Chloride Intoxicated Rat

Pushpendra K. Patel¹*, Jyoti Sahu¹, Narendra K. Prajapati¹, B.K. Dubey¹, A. Alia²

¹T.I.T. College of Pharmacy, Department of Pharmacology, Anand Nagar, Bhopal. M.P.

²Rajiv Gandhi College, Bhopal

ABSTRACT:

The liver is a vital organ present in vertebrates and some other animals. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemical necessary for digestion. This organ plays a major role in metabolism and has a number of functions in the body including glycogen synthesis, hormone production, and detoxification. Excess consumption of N.S.A.I.D. and alcohol causes the liver disease. Madhuca indica that is also known as mahua in different parts of the country belonging to the family Sapotaceae is having several medicinal properties. The leaves of Mahua tree contain saponin, an alkaloid, and glucoside. Sapogenin and other basic acid are found in the seeds. Various Photochemical studies on Mahua include characterization of Sapogenin, triterpenoids, steroids, saponin, flavonoids and glycosides. Madhuca indica has several pharmacological activity, and potential to provide health to the society. It is used as Anti diabetic, antiulcer, hepato protective, anti pyretic, anti fertility, analgesic, anti oxidant, swelling, inflammation, piles, emetic, dermatological, laxative, tonic, anti burn, anti earth worm, wound healing headache and many more problems. The ethanolic extract of Madhuca indica at 200 mg/kg and 400 mg/kg produce significant hepatoprotective effect against paracetamol induced hepatotoxicity. Different parameters such as physical, Histopathological and biochemical studies like ALT, SGOT, SGPT, bilirubin test had been included in the study which provides the satisfactory data to conclude the hepatoprotective effects of Madhuca indica.

KEY WORDS- Madhuca indica, mahua, hepatoprotective, CCl₄, liver disease

INTRODUCTION:

Human body posses several important organ but liver is having more importance among them. It occupies second largest passion in the body after the skin. It has a wide range of functions, including detoxification, protein synthesis, and production of biochemical necessary for digestion. Now day’s people want fast result and thus they use more N.S.A.I.D. to get relief from the pain, which causes the liver disease. According to W.H.O. about 18,000 people die every year due to liver disease. The common disease of liver are the cirrhosis, inflammation, cholestasis, and hepatitis, portal hypertension, hepatic encephalopathy, and others ¹. Liver diseases are because of toxic chemicals, excess consumption of alcohol, infection and auto immune disorders. Prolonged drug therapy, excessive use of the some of the commonly used medicines like CCl₄, diclofenac, nimesulide etc. Drug induced liver injury is one of the most common causative factor that posses a major clinical and regulatory challenge. Lipid peroxidation and other oxidative damage are the mainly responsible for the damage of liver cell. Osmotic imbalance in the cell membrane and accumulation of fluid also leads the liver injury. Now days the liver damage is one of the main leading problems in India which may be metabolic disorder to even mortality ².

Several synthetic and semi synthetic medicines are available in the market to cure the liver disease but they are not as safe as herbal medicine. Herbal medicines are the medicines which obtained from the plant source and also mention in the oldest system of medicine like Ayurveda and Unani. The drugs that are obtained from the plant or any of the plant are safe in use without much of side effects. Medicinal plants are parts of human medicine since the dawn of civilization. The traditional medicine system of India is one of the strongest medicine systems in the world. It provides identification and utilization of different plant ³. The Indian subcontinent is enriched by verity of flora, both aromatic and medicinal plants. This is due to the wide diversity of climatic condition available in India, ranging from deserts to swap lands. Numerous types of herbs have been well recognized and catalogued by botanist from the high ranges of Himalaya⁴_.

Carbon tetrachloride is colorless liquid, non inflammable and is heavier than air. Carbon tetra chloride is once widely used as a cleansing agent in house hold and as a solvent for oils. Carbon tetrachloride is very toxic and because of its use in households and industries the human is more prone to its toxic effects. The main route of exposure of human and animal are its inhalation, ingestion and absorption. After its entry into the systemic circulation it causes several toxic effects in body parts especially in the liver ^5, ⁶

Madhuca indica which is also known by the several other names in different parts of the world is belongs to family Sapotaceae is a plant of Indian origin having so many beneficial efficacy and potency but unfortunately it has not been fully utilized. It is a large, shady, deciduas tree up to 18 m high with short bole, spreading branches and large rounded crown. Bark is grey to black with vertical cracks. Previous Phytochemical studies on Madhuca indica included the characterization of Sapogenin, triterpenoids, steroids, saponin, flavonoids and glycosides Madhuca indica is having several pharmacological activities like Antidiabetic, anti inflammatory effect, analgesic, anti pyretic, anti asthmatic, anti ulcer, anti cancer, hepatoprotective activity, and anti bacterial ⁷.

MATERIALS AND METHODS:

Collection and identification of plant material

Madhuca indica (mahua) were collected from the Raisen road, Bhopal, Madhya Pradesh in the month of January-February 2012 and authenticated by Dr. Jia Ul Hasan, faculty of botany, Safia College of Science, Bhopal and voucher specimen (322/bot/safia/2012) was deposited in T.I.T. College of Pharmacy Anand Nagar Bhopal. India.

Preparation of leaf extract

After the Authentification of leaf part of Madhuca indica, fresh and healthy leaves were dried under shade to prevent the loss of active constituents, and then they were crushed manually by hand to obtained the coarse powder and part other than leaves were removed. The powder was then passed to 40 mesh size sieve and extracted with-

1. Hydro alcoholic solution in ratio of 70 percent water and 30 percent ethanol.

2. Extraction using 95 % ethanol

Both extractions were performed using soxhlet extraction apparatus at 40⁰C until the completion of extraction cycle. After extraction process extract were dried initially at room temperature and then at the water bath to evaporate the solvent and to obtain the dry extract. The extract was store in well closed container and store in the vacuum desicator.

Drugs and chemicals

Standard drug- Standard hepatoprotective drug silymarin was obtained from sapience laboratory Bhopal.

Diagnostic reagents kit - diagnostic reagent kits for Alkaline Phosphate, Bilirubin, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) were purchased from market, manufactured by Span diagnostics Ltd. Surat, India

Selection of Animals

Wister rats of both sexes ranging from 100- 200 gm were obtained from the animal house of Sapience Laboratory, Bhopal (CPCSEA Approved and ISO 9001:2008 Certified) Bhopal, Madhya Pradesh, India. They were maintained under the standard laboratory conditions in standard polypropylene cages for 12 h light/dark cycle and provided food and water ad libitum. They were acclimatized to the environments before 14 days of experiments to be start. The experimental protocol was approved by institutional animal ethics committee registration no. 1413/a/11 (CPCSEA)

Acute toxicity

The acute toxicity studies were carried out in adult albino rats weighing 100-200 gm, by up and down method as per OECD 425 guidelines ⁸. Overnight fasted animals received test drug (both ethanolic and hydro alcoholic) at a dose of 100, 200, 400, 800, 1000, 2000 mg/kg body weight orally. Then the animals were observed continuously once in a half an hour for next 4 hours and then after 24 hours for general behavior, neurological and autonomic profiles and to find out mortality. The extracts were found safe up to dose of 2000 mg/kg body weight and none of the animal was died, so out of them 200 and 400 mg/kg dose were selected for the administration.

Induction of hepatotoxicity in animals

For the study of hepatoprotective effect of Madhuca indica ethanolic leaf extract, hepatic injury in all groups except in standard control, was induced by single oral administration of CCl₄mixed with olive oil as a vehicle in 1:1ratio ( 2 ml/kg body weight).

Assessment of hepatoprotective activity

In order to assess the hepatoprotective activity of leaf extract of Madhuca indica the Wistar rats of both sexes weighing 100-200 gm. were maintained in animal care facility and they were divided into 7 groups of 6 animals each and provided a dose of 200 and 400 mg/kg body weight by oral route.

Table 1:- Effect of ethanolic and hydro alcoholic leaf extract of Madhuca indica against CCl₄ induced hepatotoxicity

Group	Treatment Mg/kg	ALT/SGPT (IU/L)	AST/SGOT (IU/L)	Bilirubin
1	Vehicle Control	64.08+ 11.18	82.71+7.29	0.23+0.03
2	Toxic CCL₄	210.02+ 5.83	246.4+6.98	0.60+0.03
3	CCl₄ + Silymarin	141.9+ 9.20	119+0.43	0.29+0.03
4	CCl₄ + ET. 200	181.9+ 4.46	227+8.92	0.49+0.04
5	CCl₄ + ET. 400	158.0+ 3.96	204.1+7.12	0.40+0.10
6	CCl₄ + HA 200	187.4+ 5.54	202.9+6.52	0.45+0.05
7	CCl₄ + HA. 400	112.1+5.79	154.2+5.89	0.43+0.04

* E.T. = Ethanolic Extract. H.A. = Hydro alcoholic extract. CCl₄ = Carbon tetra chloride

Figure 1:- Histopathology of liver treated with Madhuca indica leaf extract

Preparation of groups for experiments

Group 1:- Control group- receive only normal diet and water.

Group 2:- Toxic group CCl₄ treated rats- received 2 ml/kg carbon tetra chloride in olive oil (1:1 Ratio) orally on 8^thday.

Group 3:- Standard group- treated with silymarin 100 mg/kg once daily for 8 days and 2ml/kg carbon tetra chloride on the 8^th day.

Group 4:- Received ethanolic extract of Madhuca indica 200 mg/kg daily and 2ml/kg carbon tetra chloride on the 8^th day.

Group 5:- Received the ethanolic extract of Madhuca indica 400 mg/kg daily and 2ml/kg carbon tetra chloride on the 8^th day.

Group 6:- Received hydro alcoholic extract of Madhuca indica 200 mg/kg daily and 2ml/kg carbon tetra chloride on the 8^th day.

Group 7:- Received hydro alcoholic extract of Madhuca indica 400 mg/kg daily and 2ml/kg carbon tetra chloride on the 8^th day.

Blood sampling

After 24 hours of last treatment, the rats were anaesthetized with anesthetic chloroform and blood samples from each animal of all groups were collected by retro-orbital plexus puncture in sterilized centrifuge tubes. The blood samples were then allowed to coagulate at 30⁰C for 45 minutes. Serum portion was separated from each sample by centrifugation at 25000 rpm for 20 minutes ⁹

Biochemical analysis of serum samples

Serum samples collected from different groups were analyzed for Aspartate Transaminase (AST), Alkaline Transaminase (ALT) and Total Bilirubin using procedure and packaged kits made by Span diagnostics Ltd. Surat, India. The absorption was recorded using given nm in spectrophotometer.

Liver Histopathological assessment

10% formalin was freshly prepared and the right liver lobe of all groups were fixed in it for 48 hours and subsequently dehydrated in alcohol, cleared with xylem and embedded in paraffin wax, and then section have been prepared.

Statistical analysis

Results were expressed as means of SEM (n=6). Statistical analysis was performed with one way ANOVA followed by Turkey-Kramer multiple comparison test. P value less than 0.05 was considered to be statistically significant (P<0.05)

RESULT:

Acute Toxicity

The acute oral toxicity studies performed on the animals showed no animal died even at 2000 mg/kg and hence the ethanolic and hydro alcoholic extract of Madhuca indica leaf was treated as non toxic. Therefore as per CPCSEA guidelines 420 it was thought that 2000 mg/kg was the LD₅₀ cut off dose. And out of them 200 and 400 mg/kg body weight dose has been selected for the oral administration to the experimental rats.

Serum biochemical parameters

Different serum biochemical parameters have been performed for the evaluation of hepatoprotective activity of Madhuca indica ethanolic and hydro alcoholic leaf extract. The parameters include SGOT, SGPT, and total serum Bilirubin. The values thus obtained are compared with the control, toxic and standard group and the result was satisfactory and hence it was safe to use as a hepatoprotective. The results are presented in table 1.

Histopathology

Livers of animals treated with CCl₄on gross examination seen with scattered yellow and white areas attributed to fatty and necrotic changes. Examinations of the liver tissues of the animals treated with Madhuca indica ethanolic extract and silymarin exhibited recovery towards normal in a dose related manner. Light microscopic examination of stained slides of vehicle control animals showed normal architecture of the liver depicting hepatic cords and plates radiating out from the central vein, few portal triads with mild lympho mononuclear infiltrate were also seen in the sections. Few normal scattered Kuffer cells were also appreciated. Microscopic examination of liver sections of the animals treated with CCl₄ alone exhibited centrilobular necrosis, micro vesicular and macro vesicular fatty changes, scattered lympho mononuclear infiltrate in hepatic parenchyma. Treatment with Madhuca indica ethanolic extract exhibited dose-dependent reversal of these changes induced by CCl₄with few foci of necrosis of hepatocytes and fatty changes and signs of regeneration activity as in figure 1

Group 1 is control, show the normal liver structure, group 2 is toxic group showing the rupture in the central vein and other cell, group 3 is for silymarin and 4,5 are the ethanolic extract at different dose, and 6,7 for the hydro alcoholic extract, showing recovery from hepato toxicity.

DISCUSSION:

There are several models to evaluate the hepatoprotective activity in experimental animals these are paracetamol, carbon tetra chloride, ethanol, anti TB drugs and other NSAID drugs. CCL₄ is one of widely used and effective model for evaluation. Its hepatotoxic action is being with the changes in endoplasmic reticulum which results in loss of metabolic enzyme located in the intracellular structure. The hepatoprotective potentials of the plant depends on its chemical constituents presents in it, and upon its ability in reducing the harmful effects caused by the hepatotoxins.

The hepatoprotective mechanism of the extract of the Madhuca indica is due to more than one reason. The most important reason behind its liver protective activity is antioxidant property, as antioxidant, scavenging and regulators of intracellular content of the glutathione. Flavonoid is one of the main constituents present in the Madhuca indica leaf extract and it has been well known that the flavonoid is having potent effects as the hepatoprotective agent.^10,11 Oxidative stress usually develops when the pro-oxidative action of an inducer exceed the anti oxidant capacity of the defence system. The another possible mechanism of action of the extract of Madhuca indica may be as a cell membrane stabiliser and permeability regulator that prevent hepatotoxic agent to cross across the cell membrane, another mode of action may be as a promoters of the rRNA synthesis, they help in the synthesis in the protein in the liver cell and they usually increase the rate of new cell generation and as to repair the old and damage cell inside the hepatic cell.

CONCLUSION:

The ethanolic and hydro alcoholic extract of Madhuca indica at the increasing dose showed a significance reduction in the liver toxicity, which was induced by the CCl₄.The hydro alcoholic extract comparatively reduced the hepatotoxicity by ethanolic extract, and more potent as hepato protective. The result was satisfactory when compare with then silymarin the standard drug. The serum biochemical parameter and the histopathology of liver slide also stand with the result as the extract is useful in the treatment in the hepatotoxicity and other associated liver problems.

ACKNOWLEDGEMENTS:

Author thanks to Sapience Laboratory, Bhopal for valuable in vivo studies.

REFERENCES:

1. Anu Chaudhary, Anil Bhandari, A. Pandurangan, Hepatoprotective activity of methanolic extract of Madhuca indica on carbon tetrachloride- induced hepatotoxicity in rats. Pharmacologyonline,1;2011:873-880.

2. Anil Kumar. R Satis, T Rama, D Babul, J Samhitha, Hepatoprotective effect of Flemingia strobilifera R. Br. On paracetamol induced hepatotoxicity in rats .International Journal of Pharmatech Research,2(3);2010:1924-1931.

3. Amit Kumar, Ajay Kumar, Sanjay Kumar, Pankaj Kumar Chauman, Sandip Patil, Pooja Sharma, In vitro Evaluation of Phytochemical, antioxidant and anti microbial activity of Madhuca indica leaf extract. Asian journal of biochemical and Pharmaceutical Research, 4 (1); 2011: 175-185.

4. Agrawal SS, Paridhavi M, Herbal Drug Technology, Universities Press (India) Hyderabad, 2007

5. B.Hemabarathy, Siti Balkis Budin, Victor Feizal, hepatotoxicity in rats treated with crude extract of Alpinia galangal. Journal of Biological Science, 9(1);2009: 57-62

6. OE Etim, EJ Akpan, IF Usoh, Hepatotoxicity of carbon tetrachloride: Protective effects of gongronema latifolium, Pak. J. Pharm. Sci.21 (3); 2008:268-274.

7. Pushpendra K. Patel, Narendra K Prajapati, B.K Dubey, Madhuca indica: A review on its medicinal property. International Journal of Pharmaceutical Science and Research, 3(5); 2012: 942-950.

8. OECD guidelines for the testing of chemicals, acute oral toxicity test- up and down procedure (UDP) 425 adopted 3 October 2008.

9. Abhishek K Sah, Amol Gohate, Sujit K Rambhade, Hepatoprotective activity of Phylanthus niruli herbs and Solanum nigram stem bark against paracetamol induced hepatotoxicity. American Journal of Pharmatech Research, 2(2);2012: 535-544.

10. Arroyo Vicente, Colmenero, Ascites and hepatorenal syndrome in cirrhosis, Journal of Hepatology, 38; 2003:69-89.

11. Sadhana Sathayea, Yogita Bagula, Sanjay Guptab, Harpreet Kaura, Roopali Redkar, Experimental and Toxicological Pathology, 63, 2011, 587– 591

Received on 13.08.2012

Modified on 20.08.2012

Accepted on 02.09.2012

Research J. Pharmacology and Pharmacodynamics. 4(5): September –October, 2012, 311-314