HEPATOPROTECTIVE EFFECT OF NIGELLA SATIVA ON DIABETIC MICE

Sneha Navin 1 , Jaykar Jha 1 , Abhinav 1 , Arun Kumar 2 , Mohammad Ali 2 , Ashok Ghosh 2 and Ranjit Kumar 2, *. 1. Department of Biotechnology, L. N. Mithila University, Darbhanga. 2. Research Centre, Mahavir Cancer Sansthan and Research Centre, Patna. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Diabetes mellitus is a major health problem for the people of the world. Diabetes is a chronic metabolic disorder resulting from a variable interaction of hereditary and environmental factors and it is characterized by abnormal insulin secretion or insulin receptor affecting β cells of pancreas. Itis associated with a number of chronic complications including nephropathy, neuropathy, retinopathy and cardiovascular diseases. Diabetes mellitus affects a large number of people throughout the world and India also. Experts estimate that diabetic population will grow from 195 to 360 million by 2030 almost 4.5 percentage of the global population. Present study included histological and biochemical parameters of mice. Three groups of mice were prepared for comparative study on control, diabetic, and N. sativa .Diabetic models were prepared in mice by intraperitoneal administration of single dose of alloxan@120mg/kg b.w. Alcoholic extract of Nigella sativa was administered @100 mg/kg b.w/day for four and eight weeks. In diabetic group of mice glucose, creatinine, urea and SGPT were increased. Effective restoration was observed in glucose, SGPT, urea and createnine of N. sativa administered diabetic group of mice. Liver also shows effective restoration in N. sativa administered group of mice. Thus, it is concluded from study that alcoholic extract of N.sativa restores glucose level to normal. Nigella sativa acts effectively on diabetes mice on biochemical and histological parameters.

…………………………………………………………………………………………………….... Introduction:-
Incidence of diabetes is increasing worldwide at an alarming rate. Diabetes is on a rapid rise in developing nations 1 . People suffering with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 2 . The past two decades have seen an explosive increase in the number of people diagnosed with diabetes worldwide 3 . The World Health Organization has predicted that the diabetic patients will occur in the developing countries and increase 42% in the developed countries and 170% increase in the developing countries. The countries with the largest number of diabetes patients are India, China and United States 4 .
India has the largest number of people with diabetes in the world. Diabetes is a group of common metabolic disorders that share the phenotype of hyperglycemia 5 . Diabetic hyperglycaemia indicate that plasma levels of urea Materials and Methods:-Animals: -The mice (Mus musculus) were reared in animal house. The mice were selected for the study was 12 weeks old with 30±2 gm body weight (b.w). The mice were housed at controlled environmental conditions 22±2ºC, relative humidity 50±10%, and 12h dark-light cycle. All experimental were conducted as per the guidelines of CPCSEA (Committee forthe Purpose of Control and Supervision of Experiments on Animals).
Chemicals: -Alloxan, purchased by Loba chem Pvt. Ltd., Mumbai was utilized for the experimental design.
Medicinal plant used: -Alcoholic seed extract of Nigella sativa is orally administered to diabetic group of mice.Fresh seed of Nigella sativawas purchased from herbal store in Patna, India.

Study groups & sampling:-
The control group of six mice received distilled water orally. The 'treatment' groups (n=6) received alloxan 120 mg/kg b.w by intra-peritoneal method for diabetic model preparation. Nigella sativa (100 mg/kg/b.w/day) administered to diabetic mice orally through Gavage method. Mice were sacrificed after the scheduled treatment. Serum was collected for SGPT, glucose, creatinine and urea estimation. The Liver from all the mice were removed and washed three times in isotonic saline (0.85 v/w %) and fixed in neutral formalin for Light Microscope (LM) study.

Results:-
Fasting level of glucose was observed in every group of mice. Level of glucose in control group was 99.00 ± 2.30 mg/dl. In diabetic group it was 201.3 ± 12.55 mg/dl. While it was 135.0 ± 3.46 mg/dl and 106.3 ± 2.18 mg/dl in Nigella sativa4 weeks and 8weeks administered group of mice (Graph: I).
Serum Glutamate-Pyruvate Transaminase (S.G.P.T) level in control group of animal was 22.00 ± 3.21 U/ml. Diabetic group of mice S.G.P.T level was 312.3 ± 4.09 U/ml. While itwas 255.0 ± 3.78 and 192.3 ± 7.44 U/ml in Nigella sativa four weeks and eight weeks administered group of mice (Graph: II).
Level of Creatinine in control group of mice was 0.74 ± 0.06 mg%. In groupof diabeticmice creatinine level was 1.97 ± 0.04 mg%. Creatinine level was 1.71 ± 0.01 and 1.42 ± 0.02 mg% in Nigella sativa four weeks and eight weeks administered group of mice (Graph: III).
Urea value in control group of mice was 20.33 ± 2.33 mg/dl. While in diabetic group of mice was 46.73 ± 0.079 mg/dl. Urea level was 39.37 ± 0.33 and 32.40 ± 1.29 mg/dl in Nigella sativafour weeks and eight weeks administered group of mice (Graph: IV).

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In control group of mice, well-organized hepatic cells were observed. Distinct cytoplasmic and nuclear material was also observed. Central vein was well organized with its intact cell wall ( Figure: 1). Liver of diabetic mice shows vacuolization. Clustered and multilobed nuclei were observed. Degenerated cytoplasmic materials were observed. Clustered nuclei were also shows on periphery on central vein (Figure: 2). Diabetic mice followed by four weeks of Nigella sativa administration shows vacuolization in hepatic cells. Clustered nuclei were shows in hepatic cells. Many vacuolated spaces were observed with degenerated central vein (Figure:3). Diabetic mice followed by 8 weeks of Nigella sativa administration shows restoration in nuclear material were observed to least extend. Restored cytoplasm was observed in liver cells. Least vacuolization were also observed ( Figure: 4).   In diabetes, Oxidative stress was thought to be a result of free radicals generated during autoxidation of glucose 15 . In diabetes blood urea level increases & reason behind this is due to increased catabolism of both liver & plasma proteins. This elevation of plasma level of urea & creatinine is marker of renal dysfunction 16 . In our study we have observed in urea and creatinine level about threefold increases in diabetic group of mice.

Graph -I: Glucose level in serum of mice
Another study based on carbon tetrachloride induced rats has shown that CCl4 treatment increased the lipid peroxidation and liver enzymes, and decreased the antioxidant enzyme levels and furthermore, Nigella sativa treatment decreased the elevated lipid peroxidation, liver enzyme levels and increased the reduced antioxidant enzyme levels 17 . In present study S.G.P.T increases 13 folds in diabetic group of mice. Hepatic cellswere observed in degenerated condition with elongated and fragmented nuclear material. Many vacuolated spaces were also observed in cytoplasm of hepatic cells in diabetic group of mice.
NS alcoholic extract maintained the levels of AST, ALT and ALP close to normal against D -Galactosamine induced toxicity 18 . In present study Nigellasativacauses effective restoration in urea and creatinine level. S.G.P.T. level was restored effectively in N. sativa administered group of mice.
Nigella sativa causes effective restoration in malathion induced increases in AST, ALT, and lipid peroxidation. Nigella sativa also improvement role in liver function tests, lipid peroxidation, and antioxidant enzymes alteration induced by malathion 19 . Nigella sativaadministered group of mice shows effective restoration in SGPT level up to normal.NS showed hepatoprotective effects against isoniazid induced hepatotoxicity in rabbits and there are no histopathological or biological abnormalities were observed 20 . Nigella sativais efficient cytoprotectiveagainst CCl4induced hepotoxicity, possibly via inhibition of the production of oxygen free radicals that cause lipid peroxidation 21 . In present study Nigellasativa causes restoration in hepatic cells. Central veins and sinusoids were also restored effectively. Restoration is observed in both cytoplasmic and nuclear material.

Conclusions:-
It is concluded from study that N.sativa acts effectively on SGPT level. Urea, uric acid and creatinine level were almost normal after N. sativa administration. It maintains normal morphology of hepatic cells with least vacuolization. Nigella sativa is effective cytoprotective agent on hepatocyte of diabetic mice as well as maintains biochemical parameters to normal level. It is evident from study that Nigella sativa acts well against diabetes.