Evaluation of Hepatoprotective activity of Ethanolic Extract of Nymphaea alba Linn Flower in experimental rats

Objective: In the present study Hepatoprotective activity of Ethanolic Extract of flower of Nymphaea alba Linn were investigated. Material & Method: The hepatoprotective activity of Ethanolic Extract of flower of Nymphaea alba were evaluated by Carbon Tetra Chloride & Paracetamol induced hepatotoxicity in experimental rats. The Etnanolic Extract of Nymphaea alba (200 & 400 mg/kg) treat the hepatotoxicity and produced significant inhibition in the wet liver weight and wet liver volumes and the serum enzymes (SGOT, SGPT & ALP), bilirubin and cholesterol levels. Result: Preliminary phytochemical analysis of Ethanolic Extract of Nymphaea alba revealed that the presence of various phytoconstituents Alkaloids, Carbohydrates (Polysaccharides), Glycosides, Steroids, Flavonoids and Tannin & Phenolic compound. The ethanolic extract (200 & 400 mg/kg) showed significant reduction in SGOT, SGPT, ALP, Bilirubin & cholesterol level as compared to control group. Maximum inhibition was obtained at dose 400 mg/kg of Ethanolic Extract of Nymphaea alba in both model. Conclusion: The present study suggests that Ethanolic Extract of flower of Nymphaea alba Linn possess strong and potential Hepatoprotective activity in both model. So it has immense scope as an effective source to develop drug for the treatment of liver related diseases. These results may further suggest that ethanolic extract was found to possess hepatoprotective activity.


Introduction
Liver is the largest gland and heaviest gland of the body weighing about 1.4 kg in an average adult. The liver is the main site of metabolism for drugs and other exogenous compounds. The liver plays a central role in metabolism of large number of organic and inorganic chemicals and drugs. The main drug metabolizing system resides in the microsomal fraction of the smooth endoplasmic reticulum of the liver cells via p-450 cytochrome and cytochrome reductase enzyme system.
Nymphaea alba Linn (Nymphaeaceae) is Generally found in tanks and ponds throughout the warmer parts of India and Africa.. All parts of the plants are used in folk medicine. It grows in water from 30-150 centimeters deep and likes large ponds and lakes. The leaves may be up to thirty centimeters in diameter and they take up a spread of 150 centimeters per plant. It is an aquatic herb with perennial rhizomes or rootstocks anchored with mud. It is globally distributed in Europe, North Africa, Southwest Asia, India, China and Russia. It is rich in tannic acid, gallic acid, alkaloids, sterols, flavonoids, glycosides, hydrolyzable tannins and high-molecular-weight polyphenolic compounds. All the parts of the plant have medicinal uses in traditional system of medicine. It is used as an aphrodisiac, anodyne, antiscrophulatic, astringent, cardiotonic, demulcent, sedative and anti-inflammatory. Further, it also produces calming and sedative effects upon the nervous system, and is useful in the treatment of insomnia, anxiety and similar disorders 3-8.

Collection, identification and authentication of plant
The plant Nymphaea alba Linn (Flower) were collected from Sarasbag, Pune, Maharashtra, during the month of June-2012 The plant material was identified and authenticated by Prof. P. Jayaraman (Ph.D.), Director-Plant Anatomy Research Centre (PARC) Tambaram. The voucher specimen number is PARC/2012/1702 (a) and it was submitted to the laboratory of Department of Pharmaceutical Science, Shri Venkateshwara University Gajraula, Amroha (Uttar Pradesh) for future references.

Collection and maintenance of experimental animals
Wistar albino rats of either sex weighing between 150-250 gm of either sex were used. Institutional Animal Ethics Committee of Nagaji Institute of Pharmaceutical Science, Gwalior approved the experimental protocol; animals were maintained under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA Reg.No.-1498/PO/a/11/CPCSEA). The animals were housed in Poly propylene cages and maintained at 24˚C ± 2˚C under 12h light/ dark cycle and were feed ad libitum with standard pellet diet and had free access to water.

Acute Toxicity Studies
The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Co-operation and Development (OECD) received from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). One-tenth of the median lethal dose (LD 50 ) was taken as an effective dose. Acute toxicity study was done as per OECD, 2006 Guidelines. Acute oral toxicity tests found the LD 50 of the Plant extract to be >2,000 mg/kg. The animals were observed for signs of toxicity such as hyperactivity, grooming, convulsions, sedation, and hypothermia continuously for 2 hours, and for mortality up to 24 hours, after administration of the doses 9-10 .

Extraction Method
The flower of Nymphaea alba were shade dried and reduced to coarse powder in a mechanical grinder. The powdered material obtained was then subjected to successive extraction by Hot Percolation Method using petroleum ether, chloroform, and Ethanol solvents in a soxhelet extractor. The different extracts obtained were evaporated at 45˚C to get a semisolid mass. The extracts thus obtained were subjected to phytochemical analysis. The percentage yield of EENA was found to be 11 w/w %. 11

Phytochemical Analysis of the Extracts
Preliminary phytochemical analysis of EENA revealed that the presence of various phytoconstituents Alkaloids, Carbohydrates (Polysaccharides), Glycosides, Steroids, Flavonoids and Tannin & Phenolic compound 12-13 . 14 In order to assess Hepatoprotective action of plant extract In Albino wistar rats, the rats were divided into the following groups each containing 6 rats (n=6):

Carbon tetrachloride induced hepatotoxicity
Group-I and Group-II were served as Normal control and disease control respectively. Group-III, IV & V corresponded to reference standard (Silymarin-100 mg/kg/day), p.o. Ethanolic Extract of Nymphaea alba Linn (EENA) 200 mg/kg & 400 mg/kg respectively.
The treatment lasted for 7 days and on seventh day's night all the animals were fasted for 12 hrs. Then all the animals except those in group -I were treated with 1 ml of CCl 4 in liquid paraffin (1:1). 24 hrs after CCl 4 administration, blood sample were collected for the estimation of biochemical parameter (SGPT, SGOT, ALP, Total bilirubin and total cholesterol). 14 In order to assess Hepatoprotective action of plant extract in Albino wistar rats, the rats were divided into the following groups each containing 6 rats (n=6):
The treatment was carried out for 7 days and on seventh days night all the animal were fasted for 12 hrs. Then all the animals except those in group -I were treated with paracetamol (2 mg/kg, p.o.) in sucrose solution (40 % v/v) in three divided doses.48 hrs after paracetamol administration, blood sample were collected for the estimation of biochemical parameter (SGPT, SGOT, ALP, Total bilirubin and total cholesterol).

Assessment of hepatoprotective activity 15-16
After 24 hours of the last treatment, the rats were anaesthetized with ether 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 30° C for 45 minutes. Serum portion was Separated from each sample by centrifugation at 2500 rpm at 30°C for 10 minutes and subjected to biochemical investigation to assess liver function on the basis of determination of total bilirubin, Cholesterol, Alanine amino transferase (ALT/SGPT), Aspartate amino transferase (AST/SGOT), and alkaline phosphatase (ALP) level.

Histopathological studies 17
The animals were sacrificed and the abdomen was cut open to remove the liver. The liver was fixed in Boucin's solution (mixture of 75 ml of saturated picric acid, 25 ml of 40% formaldehyde and 5ml of glacial acetic acid) for 12h, then embeded in paraffin using conventional methods and cut into 5μm thick sections and stained using haematoxylin-eosin dye and finally mounted in di-phenyl xylene. The sections were then observed under microscope for histopathological changes in liver architecture and their photomicrographs were taken.

Statistical analysis
The results were reported as Mean ± SEM of different observations. Experimental data were analyzed using oneway analysis of variance (ANOVA) to compare the difference between the control and treated values. Different value of P was considered significantly. Graph Pad Prism Version was used for statistical calculations

Effect of Ethanolic Extract of Nymphaea alba Linn on CCl 4 induced hepatotoxicity
CCl 4 treated animal showed significant elevation of serum biochemical parameter such as SGPT, SGOT, ALP, total bilirubin and total cholesterol. The liver weight and the ratio of liver weight to body weight were increased compared with normal control group, and the pathological lesions of the liver were evident. Pre-treatment with silymarin-100 mg/kg p.o. and EENA at 200 mg/kg and 400 mg/kg p. o. for 7 days had produced significant protective effect on CCl 4 -induced hepatic damage by maintaining the morphological changes (liver weight and liver to body weight ratio) and normalizing the elevation of serum biochemical parameter and therefore inhibited the Histopathological abnormalities caused by CCl 4 . EENA showed dose dependent protection against CCl 4 induced hepatic damage. (Table 1)

Wet liver weight and Wet liver volume
CCl 4 and paracetamol treatment in rats resulted in enlargement of liver which was evident by increase in the wet liver weight and volume. The groups were treated with Silymarin and Ethanolic Extract of Nymphaea alba Linn (flower) showed significant restoration of wet liver weight and wet liver volume nearer to normal. The EENA at 200mg/kg and 400mg/kg body weight showed reduction of wet liver weight and wet liver significantly at p<0.05. (Table 3 and Table 4) )

Discussion
In the present study, CCl 4 & paracetamol was employed as toxic agents and the protective role of Nymphaea alba flower against the CCl 4 & paracetamol induced hepatotoxicity was studied. The extent of toxicity was estimated by histopathological studies and biochemical enzyme markers like SGOT, SGPT, ALP and Serum Bilirubin levels etc. The ethanolic extracts of flower at dose of 400 mg/kg demonstrated a significant reduction in the serum enzymes and bilirubin levels.
In case of toxic liver, Wet liver weight and Wet liver volumes are increased. In this case water is retained in the cytoplasm of hepatocytes leading to enlargement of liver cells, resulting in increased total liver mass and volume. It is reported that liver mass and volume are important parameters in ascertaining the hepatoprotective effect of the drugs. So in this study treatment with ethanolic extract of Nymphaea alba flower significantly reduced the wet liver weight and wet liver volumes of animals and hence it possesses statistically significant(p<0.05) hepatoprotective activity.
In the current study treatment of animals with EENA significantly (p<0.05 & p<0.01) decreased the levels of SGOT, SGPT, ALP in serum Enzyme which is an indicative of hepatoprotective activity 18-20 .

Conclusion
The results of study demonstrate that EENA Flower possesses hepatoprotective property due to the decrease in the serum levels of these enzymes and recovery of hepatocyte shapes. Further studies are required to identify, isolate, characterize and evaluate the active principal responsible for hepatoprotective activity of plant. The hepatoprotective effect of EENA was confirmed by the following measures. The isolated livers from the toxicant (CCl 4 & paracetamol) treated animals exhibited increase in wet liver weight and wet liver volume. EENA showed marked change in level of serum marker enzymes such as SGPT, SGOT, SALP, total bilirubin and total cholesterol , due to its hepatoprotective effect.