Invitro antidiabetic activity like\ Assay for α-Amylase Assay for α-Glucosidase Evaluation of Glucose Absorption In Vivo

1. Rohit Patel
Invitro antidiabetic activity

2. INTRODUCTION
 Antidiabetic 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.

3. Assay for α-Amylase
PURPOSE AND RATIONALE
 α-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.

4. PROCEDURE
 The 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.

5. Assay for α-Glucosidase
PURPOSE AND RATIONALE
 Inhibition of glucosidase can be measured in vitro
using glucosidase from porcine small intestinal
mucosa.

6. 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.

7. Evaluation of Glucose Absorption
In Vivo
PURPOSE AND RATIONALE
 The 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.

8. 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.

9. EVALUATION
 The 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.

10. CONCLUSION
 Thus 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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