Antioxidant And Organprotective Activities of Amaranthus Gangeticus Leaves Against Paracetamol Induced Hepatic Damage in Albino Rats

Liver is an important organ in our body.  It is one of the vital organ of metabolism and excretion.  During its normal physiological functioning it metabolizes various endogenous and exogenously administered chemicals, so as to terminate or inactivate these agents.  Hence due to this function, it protects the whole body from the various environmental and chemical challenges.  In addition to this liver has got an inbuilt mechanism to protect itself and to regenerate on several occasions, many of these hepatotoxic challenges overpower inbuilt protective mechanism and cause hepatotoxicity resulting in the hepatic necrosis and hepatitis. Amaranthus gangeticus is a edible plant used as vegetable, it is also used by native practitioner as hepatoprotective in treating various types of jaundice.  The leaves of this plant contain polyphenolic compounds like tannins and flavonoids.  These polyphenolic compounds have antioxidant property and anti-oxidants have known to possess hepatoprotective activity.  Keeping the native knowledge and the above mentioned literature information¹, this plant was selected for present study to screen the leaves of this edible plant for the presence of phytoconstituents, antioxidant and hepatoprotective activities.  This study was carried out by using 90% ethanolic extract of Amaranthus gangeticus (AG) as hepatoprotectant and paracetamol (PCM)  as hepatotoxicant.

MATERIALS AND METHODS

Collection and identification of plant: The plant was collected from Kusnoor village (Gulbarga district), Karnataka in the month of March and was authenticated by Dr. Srinath Rao, chairman, P.G. Department of Studies and Research in Botany, Gulbarga University, Gulbarga, Karnataka.  The plant was thoroughly cleaned to remove adherent soil and other impurities, the leaves were shade dried and made into a coarse powder by rubbing in the palms.

Extraction  

90 gms of shade dried leaf powder of AG was extracted in Soxhlet’s apparatus using petroleum ether for defatting and then it was extracted with
90% ethanol (AGEE). The solvent was evaporated on a water bath at a low temperature (50°C) and finally the residue was obtained.

Materials used  

Paracetamol (Esteem Pharmaceutical Pvt. Ltd. Agra), Silymarin (SD fine chemicals, Mumbai), Ready to use diagnostic kits (Aspen Labs Pvt. Ltd., Delhi-India), 90% ethanolic extract of AG, gallic acid (Rolex Chemical Industries, Mumbai).  All chemicals and reagents used were of analytical grade.

Animals used

Wistar albino rats of either sex weighing between 150-180 gms were housed in polypropylene cages and were maintained at 27° ± 2°C with 12:12 hr, light/dark cycle.  They were fed with commercial diet (VRK Nutritional Laboratory, Sangli) and water at libitum, during the experiment.  The study was permitted by Institutional Animal Ethical Committee (Reg. No. 342).

I.         Evaluation of hepatoprotective activity in PCM-induced hepatotoxicity:

The method reported by R.R. Chattopadhyay was followed².           In the dose response experiment, albino wistar rats were randomly assigned into 5 groups of 6 animals in each.

Gp-I Animals (-ve control) were administered with 1ml/kg p.o. of saline for 7 days. Gp-II Animals (+ve control) were administered with 1ml/kg p.o. of saline for 7 days. Gp-III Animals were administered with silymarin 100mg/kg p.o., for 7 days. Gp-IV Animals were administered with AGEE 200mg/kg p.o., for 7 days.

Gp-V Animals were administered with  AGEE 400mg/kg p.o., for 7 days.On 5^th day, 30 minutes  after the administration of normal saline, 100mg/kg silymarin, 200mg/kg and 400mg/kg of AGEE to Group-II, III, IV and V respectively, Paracetamol 2gm/kg was given orally. After 48 hours of paracetamol dosing, rats were sacrificed under mild ether anaesthesia and the blood samples were collected from the animals of each group through carotoid artery puncture and centrifuged immediately to get clear serum, for evaluating the serum biochemical parameters by using Aspen diagnostic kits. Liver was dissected out, the blood was blotted off, washed with saline and stored in 10% formalin and proceeded for histopathology to evaluate the details of hepatic architecture in each group microscopically.

The results are shown in table-1.      

II.        Total Phenolic Content estimation³

1.         Extraction of phenols from Amaranthus gangeticus  leaves⁴

Method: In this process, 10 gms of shade dried, leaf powder of Amaranthus gangeticus (AG) is placed with the whole of the menstrum (250 ml) in a closed vessel for 2 days.  During this period shaking is done occasionally.  After 2 days, the liquid is strained and marc is pressed.  The expressed liquid is mixed with strained liquid.  It is then filtered to make a clear liquid.  The final volume is not adjusted.  It is evaporated on a water-bath at low temperature not exceeding (50°C) and preserved.Plant extracts were prepared using two different extracting solvents :

AG_CW  : Amaranthus gangeticus shade dried leaf powder macerated for 48 hrs with      cold water (distilled water stored at room temperature).    

AG_{HW   :}AG shade dried leaf powder macerated for 48 hrs with hot water (50°C).

Total Phenolic Content

It was estimated by using Folin-Ciocalteau reagent according to the method reported by Singleton and Rossi³ using standard curve generated with Gallic acid.

Procedure: For the preparation of Calibration curve, a series of calibrated 10 ml volumetric flasks were taken and appropriate aliquots of the working standard solution of gallic acid were pipetted out. To each flask, 5ml of Folin-Ciocalteu reagent (diluted ten fold) and 4ml of sodium carbonate solution (75g/l) was mixed with appropriate aliquots of gallic acid. The absorbance was measured after 30 min. at 765nm in Shimadzu 1700 UV-visible spectrophotometer. Same procedure was applied for various extracts of Amaranthus gangeticus (1mg/ml) was mixed with the same reagent as described in the construction of calibration curve and after 30 min. the absorbance was measured for the determination of total phenolic compound. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligram per gram of sample using a standard curve of gallic acid. Total amount of phenolic compounds in Amaranthus gangeticus were done in triplicates and concentrations of total phenolic  content in various extracts were determined and expressed as gallic acid in mg equivalents per gm of sample using the standard curve generated with gallic acid.            The results obtained were compiled and represented as shown in table-2.

Statistical analysis

The data presented in table-1 (n=6) and 2 (n=3) were expressed as mean ± standard error of mean (SEM).  Significant difference among the mean were calculated at the level of p < 0.001 and analyzed by one-way analysis of variance by Dunnet’s ‘t’ test.  A value of p <  0.05 was defined as significant.

Table-1 Effect of 90% ethanolic extract of Amaranthus Gangeticus   leaves on hepatic enzymes in PCM induced hepatotoxicity

Values are the mean ± S.E.M. of six rats/treatment.

*** P<0.001 Significance compared to PCM treatment.

Table-2 Total phenol content in amaranthus gangeticus leaf extract