Rohit Patel

Invitro antidiabetic activity

INTRODUCTIONAntidiabetic effects can be studied invivo using animal

models or invitro using a variety test systems. Invitro tests can play a very important role in the evaluation of antidiabetic activity of drugs as initial screening tools where the screening of large number of potential therapeutic candidates may be necessary. They might provide useful information on the mechanism of action of therapeutic agent.

Biological material used in these includes perfused whole organs, isolated tissues, cell culture systems, or tissue slice preparations.

While in vivo biological systems using live animals (whole organisms) are necessary to study how such mechanisms behave under clinical or pathophysiological conditions, data from experiments carried out in in vitro systems can establish mechanisms and define toxicities.

Assay for α-AmylasePURPOSE AND RATIONALEα-Amylase activity can be measured by

determination of the reducing groups arising from hydrolysis of soluble starch by isolated pancreatic α-amylase according to the protocol of Rick and Stegbauer (1970). The reduction of 3,5-dinitrosalicylic acid to nitroaminosalicylic acid produces a color shift which is followed photometrically by changes in the absorbance at 546 nm. Inhibition of starch hydrolysis by an α- amylase inhibitor results in a diminished absorbance at 546 nm in comparison with the controls.

PROCEDUREThe effect of sample on α-amylase activity can

be studied using an enzyme-starch system.Sample is mixed by stirring with 25 mL of 4%

potato starch in a beaker; 100 mg of α-amylase is added to the starch solution, stirred vigorously, and incubated at 37°C for 60 minutes.

After the incubation period 0.1 M NaOH is added, to terminate enzyme activity. The mixture is centrifuged (3000 x; 15 minutes) and

the glucose content in the supernatant is measured at 546nm against the reagent blank.

Assay for α-GlucosidasePURPOSE AND RATIONALEInhibition of glucosidase can be measured

in vitro using glucosidase from porcine small intestinal mucosa.

PROCEDURE Glucosidase is prepared from rat or porcine small intestinal

mucosa or porcine pancreas. The inhibitory activity is determined by incubating a

solution (20 μl) of an enzyme preparation with 80mM sodium phosphate buffer, pH 7.0 (500 μl) containing 37mM sucrose or maltose, or 3.7mM isomaltose and a solution (20 ml) containing various concentrations of the inhibitor at 37°C for 20 min.

The reaction mixture is heated for 2min in a boiling water bath to stop the reaction.

After the addition of 1.0 ml of 0.1 M disodium hydrogenphosphate solution, the absorption of liberated p-nitrophenol .

The amount of liberated glucose is measured by uv-vis read absorbance at 400nm.

This study give detail about the effect of intestinal disaccharidase inhibitors on obesity and diabetes.

Evaluation of Glucose Absorption In VivoPURPOSE AND RATIONALEThe inhibition of glucose absorption can be

determined by measuring blood glucose after administration of starch or disaccharides with and without the inhibitor. In addition, non-absorbed starch or disaccharides can be determined in the intestine.

PROCEDURE Male Wistar rats are kept on a standard diet with free

access to tap water at constant temperature (24±1°C).

Sixteen hours prior to the experiment food but not water is withheld.

Groups of rats receive by stomach tube 2.5 g/kg raw starch in a water suspension without or with various doses of the α-amylase inhibitor.

After 10, 20, 30, 60, 120 and 240 min, blood is withdrawn for determination of blood glucose and non-esterified fatty acids. The animals are sacrificed after these intervals and the residual starch in the stomach and the intestine determined. Definitely more starch is found in the intestine after simultaneous application of the α- amylase inhibitor. Similar experiments are performed in dogs for determination of serum insulin. The increase of blood glucose and serum insulin as well as the decrease of NEFA are inhibited.

EVALUATIONThe values of starch content in stomach

and intestine, as well as the blood glucose, serum insulin and NEFA-values are compared between control and treated animals.

CONCLUSIONThus this review provides the information of

various invitro studies used in antidiabetic assessment, which can establish mechanisms for the antidiabetic activity of the drug.

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