Blood glucose lowering of STZ induced diabetic rats

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4.8.3.2. Blood glucose lowering of STZ induced diabetic rats:

Diabetes was induced by intraperitoneal injection of STZ and fasting blood glucose was measured after 48 h to determine the diabetes status in rats. Animals having fasting blood glucose between 300 to 450 mg/dl were selected for the study. Selected animals were divided into groups having six animals in each group. Rats of experimental groups were orally administered the fine suspension of the desired test samples (made in 1.0% gum acacia) at 250 mg (in case of extract), 100 mg (in case of fractions) and 30 mg (in case of pure compounds) per kg b.w. Animals of the control group will be given an equal amount of 1.0% gum acacia. The dose of standard antidiabetic drugs in this protocol was 100 mg/kg b.w. of metformin. Blood glucose level of each animal was measured by glucostrips at 0, 30, 60, 90, 120, 180, 240, 300 min and at 1440 min post test sample/standard drug or vehicle treatment

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The percentage blood glucose lowering by test substance or standard drug was determined by plotting blood glucose versus time and calculating the area under curve (AUC) between 0-300 min and 0-1440 min and comparing the AUC of test substance treated/standard drug treated groups to that of sham treated control group.

4.8.3.3. Inhibition of postprandial rise in hyperglycemia post sucrose load in STZ-

induced diabetic rats:

Diabetes was induced in rats with intraperitoneal injection of STZ as mentioned above. After 48 hours of injection diabetes was confirmed by monitoring fasting blood glucose with the help of glucometer. Animals showing fasting blood glucose in the range of 140-270 mg/dl were selected and grouped consisting 5 or 6 animals in each group as per the availability of the animals. The animals of experimental groups were orally administered with desired test samples prepared in the form of fine suspension in 1% gum acacia at the dose of 250 mg/kg b.w. in the case of crude powder and extracts and 100 mg/kg b.w. in case of fractions. The control group were administered in equivalent manner with the vehicle i.e., 1% gum acacia. After 30 minutes of sample treatment the animals were administered with sucrose solution at the dose of 2.5 g/kg b.w. Blood glucose was continuously monitored at regular interval of 30, 60, 90, 120,180, 240, 300 and 1440 minutes post sucrose load.

The percentage inhibition of postprandial blood glucose by test substance or standard drug was determined by plotting blood glucose versus time and calculating the area under curve (AUC) between 0-300 min and 0-1440 min and comparing the AUC of test substance treated/standard drug treated groups to that of sham treated control group.

4.8.4. Multiple dose effect

4.8.4.1. Experimental design for low dosed STZ-induced and high fructose fed low dosed STZ-induced diabetic rats:

The STZ-induced diabetic animals were subjected to dosing with test samples and vechile, respectively for 30 days. The OGTT of each animal was carried out on days 7th, 14th, 21st and 28th. The animals were bled on days 10th, 20th and 30th post treatment, plasma separated and their lipid profiles i.e. total triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol were estimated. The hepatic and renal function markers were assayed on Semiautoanalyzer (Iris Health Care) using assay kits and instructions as provided by the manufacturer. Insulin estimation was done by ELISA kit supplied by Mercodia.

4.8.4.2. Experimental design for C57BL/KsJ (db/db) mice:

db/db mice showing random blood glucose levels above 250 mg/dl on three different time intervals within a gap of 7 days were selected, and grouped each consisted of 5 to 6 animals. The treatment with vehicle and test substances was continued for 15 days and during which OGTT was performed on days 10th and 14th, respectively. On the day of termination of study the animals were bled, sacrificed and their organs were quickly excised collected in liquid nitrogen and stored at -800 C till further processed. The blood collected from retro-orbital pluxes into EDTA-coated tubes was centrifuged: plasma was separated and insulin, cholesterol, triglycerides and HDL cholesterol content estimated therein.

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4.8.5. Oral glucose tolerance test (OGTT):

Overnight fasted rats were administered with glucose by oral route at the dose of 3.0 g/kg b.w. and blood glucose was measured at 30, 60, 90 and 120 min time intervals from the tail vein. Effect on oral glucose tolerance was obtained by calculating area under curve for the values of blood glucose between 0-120 min.

4.8.6 Biochemical analysis:

Blood from each animal was always collected at the desired time intervals in EDTA-coated tubes. The plasma was separated for the study of lipid profile, insulin, and hepatic and renal function markers. Levels of triglycerides, total cholesterol, HDL-cholesterol, hepatic transaminases (AST and ALT), kidney function markers i.e., urea, uric acid and creatinine were measured on Semi autoanalyzer using kits provided by the manufacturer. Plasma insulin level was measured using Mercodia insulin ELISA kit.

4.8.6.1. Plasma Lipid Profiling

4.8.6.1.1. Estimation of plasma Triglycerides

Test principle: Triglycerides levels in the plasma were estimated using the diagnostic kit based on the enzymatic method described by McGowan et al., (1983). Triglycerides in the samples was hydrolyzed by microbial lipoprotein lipase to glycerol and free fatty acid. Glycerol was converted by glycerol kinase into glycerol 3-phosphate (G-3-P) and ADP, which was oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate and hydrogen peroxide. In this reaction hydrogen peroxide was produced in equimolar concentration to the level of triglycerides present in the sample. H2O2 reacts with 4-aminoantipyrene and 4-chlorophenol in the presence of peroxidase to produce red quinoneimine coloured dye. The intensity of this dye was proportional to the concentration of triglycerides in the sample.

Procedure

10 µl of plasma sample mixed with 1000 µl of enzyme reagent and incubated for 5 min at 37ºC. At the same time blank and standard solution was prepared. The absorbance of sample against reagent blank was read at 540 nm. The activity was calculated by using the formula.

4.8.6.1.2. Estimation of plasma Total Cholesterol

Test principle: Total cholesterol in the plasma samples was estimated by the enzymatic method described by Allain et al., (1974). Cholesterol esters were hydrolyzed by cholesterol esterase to free cholesterol and free fatty acids. The free cholesterol produced and pre-existing ones were oxidized by cholesterol oxidase to cholesteol-3-one and hydrogen peroxide. Hydrogen peroxide formed reacted with 4-aminoantipyrine and phenol in the presence of peroxidase to produce pink/red colored quinonimine dye. The intensity of color produced was proportional to the cholesterol concentration.

Procedure

1000 µl of enzyme reagent and 10 µl of plasma was mixed well and kept at 37ºC for 5 min at room temperature and the absorbance was measured by using spectrophotometer at 505nm.

4.8.6.1.3. Estimation of plasma high density lipoprotein (HDL-C):

Test principle: HDL-C in the plasma samples was estimated by the enzymatic method described by Friedewald et al., (1972). Lipoproteins are particles comprising of lipids, phospholipids and apoproteins. There are four distinct groups of lipoproteins: Chylomicrons, Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL) and High Density Lipoproteins (HDL). HDL’s role in lipid metabolism is the uptake and transport of cholesterol from the peripheral tissues to the liver and low HDL-Cholesterol levels are associated with an increased risk of coronary artery disease.

Procedure

To 10 µl of plasma sample 1000 µl of reagent 1 was added and mixed well and incubated at 37ºC for 5 min. To this mixture 250 µl of reagent 2 was added and incubated for 5 min at 37ºC and the absorbance was read at 505 nm.

4.8.6.1.4. Estimation of plasma Low density lipoprotein (LDL-C)

Test principle: The usual lipoproteins breakdown pathway proceeds from chylomicrons to intermediate-density lipoproteins to LDL. The LDL then reacts with the cell membrane receptors that move cholesterol into the cell membrane receptors that move cholesterol into the cell for storage or conversion to other compounds. In the Dialab method, non LDL-Lipoproteins are enzymatically processed, while LDL is selectively protected (in the first incubation step with Reagent 1). In the second step LDL is released and selectively determined. The estimation of LDL-C in plasma involves the following enzyme catalyzed reactions:

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