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Naringenin is a flavonone that is considered to have a bioactive effect on human health as antioxidant, radical scavenger, antiinflammatory, carbohydrate metabolism promoter, immunity system modulater. Â
However, because of their reactivity, free radicals can cause side reactions resulting in cell damage, which may contribute to the development of cardiovascular disease and cancer. Lipids, protein and nucleic acids are weak to free radicals.
Naringenin (the chemical name, 4,5,7- trihydroxyflavanone, yellow crystalline powder) and STZ (the chemical name, 2-Deoxy-2-[[(methylnitrosoamino)-carbonyl] amino]-D-glucopyranose) was purchased from Sigma Chemical Company (St Louis, MO, USA). All the other chemicals used were of analytical grade and were purchased from commercial sources.
Male albino wistar rats, weighing about 150-180g obtained from Central Animal House, Aptus Biosciences Pvt Ltd, India, were used for the present investigations. The animals were maintained on standard rat feed supplied by Provimi, India. The experiments were conducted according to the Ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC).
Determination of LD50 Value of Naringenin
Acute Oral Toxicity Study
The procedure was followed by using OECD guidelines 423 (Acute toxic class method). Twelve animals (Wistar Albino rats, 150-180 gm) were selected for studies.The acute toxic class method is a step wise procedure with 3 animals of single sex per step. Depending on the mortality and / or moribund status of the animals, on average 2-4 steps may be necessary to allow judgement on the acute toxicity of the test animals while allowing for acceptable data based scientific conclusion.
The method uses defined doses ( 2.5, 5, 10, 100 mg / kg body weight ) and the results allow a substance to be ranked and classified according to the Globally Harmonized System (GHS) for the classification of chemical which cause acute toxicity.
Body weight of animals before and after administration, onset of toxicity and signs of toxicity like changes in skin and fur, eyes, and mucous membrane and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behavior pattern, sighs of tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma was also to be noted, if any, was observed.
No toxicity or death was observed for these given dose levels, in selected and treated animals. So the LD50 of the aqueous solution of Naringenin as per OECD guidelines-423 is greater than 2000mg/kg (LD50 >2000mg/kg).
Hence the biological dose was fixed at 2.5, 5mg/kg body weight for the Naringenin.
The animals were fasted overnight and DM was induced by a single intraperitoneal (i.p) injection of freshly prepared STZ (55mg/kg body weight of rats) in 0.1 M citrate buffer (pH 4.5). The animals were allowed to drink 5% glucose solution overnight to overcome the drug induced hypoglycaemia (Prince PSM et.al, 1998). Control rats were injected with citrate buffer alone. The animals were considered as diabetic, if their blood glucose values were above 250mg/dL on the third day after STZ injection. The treatment was started on the fourth day after STZ injection. The treatment was continued for 7days.
The rats were divided into five groups comprising six animals in each group as follows:
Control rats given only buffer
STZ- Diabetic rats
Diabetic rats treated with Protamine zinc insulin 6 units/kg/ day
Diabetic rats treated with NAR.2.5 mg/kg/ day
Diabetic rats treated with NAR.5.0 mg/kg/ day
ORAL GLUCOSE TOLERANCE TEST (OGTT):
The overnight fasted (18 hr) rats were taken and divided into five groups and each group consists of six animals. They were provided with drinking water only. Normal saline solution was administerd to group I animals.Group II STZ Diabetic animals were received Glucose load. Group III animals were received Protamine zinc insulin 6units/kg b.w) as a standard. Naringenin solution 2.5mg/kg and 5.0 mg/kg b.w) was administered, by oral route, to group IV and IV. Glucose (2g/kg) load was fed 30 minutes after the administration of Naringenin solution. Blood was withdrawn from tail vein under mild ether anaesthesia at initial and 30, 60, 90 minutes after glucose (glucose load, 2g/kg) administration (V. Babu et al, 2003) and glucose levels were estimated using glucose strips and a glucometer. Blood glucose levels were noted and reported.
At the end of the experiment, blood was collected into heparinised tubes, and the plasma and serum were separated by centrifugation at 3000 rpm for 10 min. The clear supernatant was used for the analaysis of Various biochemical parameters. The liver and kidney were quickly removed, washed in ice-cold, isotonic saline and blotted individually on ash-free filter paper, and the organ weights were measured for preparing 10% homogenate. The tissues were then homogenized in 0.1 M Tris-HCl buffer, pH 7.4. The 10% homogenate was used for the estimation of proteins, enzymes and other parameters. Blood glucose, Urea, Uric acid and Creatinine were estimated using a commercial diagnostic kit (Ranbaxy Laboratories, New Delhi, India).
EVALUATION OF PARAMETERS
Blood Plasma parameters:
Blood glucose Urea, Uric acid and Creatinine are evaluated by using Standard diagnostic kits Purchased from Ranbaxy Laboratories, New delhi.
Blood glucose level estimation
Glucose level in plasma was estimated by glucose oxidase/peroxidase method using a commercial kit from Ranbaxy, India followed by Trinder, P. (1969) Annals.Clin.Bio chem. 6, 24.
Reagents used for estimation of blood glucose level,
Glucose standard (100 mg %)
10 ïl of plasma was added to 1.0 ml of working enzyme reagent, mixed well and incubated at 37ï‚°C for 15 min. The colour developed was read at 505 nm against blank containing distilled water instead of the sample. A standard was also processed similarly.
The level of glucose is expressed as mg/dl.
Determination of Creatinine:
The method most widely used today are based on Jaffe reaction. This reaction occurs between Creatinine and the picrate ion formed in Alkaline medium (Sodium picrate).
Creatinine + Picric acid (yellow colour)
NaOH (alkaline medium)
Creatinine picrate (Orange colour)
Absorbance was measured at 520 nm
Creatinine stock standard:
150 mg creatinine in 100 ml of water (1.5 mg/ml)
Creatinine working standard for blood (3 mg/dl):
Dilute 10 ml of stock & increase the volume upto 500 ml with water.
Add & Mix well the 0.5 ml of Serum, water 1.5 ml & picric acid 6ml.
Add 0.4 ml of 2.5 M NaOH.
Allow to stand for 20 min. Measure the absorbance in auto analyzer.
Alkaline phosphatase was estimated in plasma by commercial kit (Vital Diagnostics Pvt Ltd) followed by following method.
substrate reagent: alkaline phosphate reagent ( tablet form)
buffer - DEA buffer
One tablet of substrate reagent was dissolved in 1.1 ml of buffer reagent.
20 Âµl of plasma was added to 1 ml of working reagent mixed well. The absorbance was measured at 0, 30, 60 and 90 seconds in a auto analyzer and the mean absorbance was taken (Î”A)
The ALP was calculated by the following formula
ALP in U/L= Î”A / min X 2713
Where, 2713 = standard factor
The activity of the enzyme is expressed as U / litre of serum.
After blood sampling for the biochemical analysis, the animals were sacrificed, quickly dissected out. The splenic part of the pancreas was fixed in Bouin's fluid by total immersion for 24 hours after it was followed by Paraffin wax embedding method of Drury and Wallington (1980). Sections of 5Î¼m in thickness were produced from the tissue blocks and stained with haematoxylin and Eosin x 2200. for light microscopic examination of the pancreatic islets.
All the grouped data was statistically evaluated via the Statistical package for Social Sciences version 14.0 (SPSS Inc, Chicago, IL, USA). Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant differences test. P-values of less than 0.05 were considered to indicate statistical signicance. All the results were expressed as mean Â± SD for six animals in each group.