The document discusses the in vitro antidiabetic activity of various enzyme inhibitors, specifically targeting α-amylase and α-glucosidase to reduce glucose absorption in diabetes and obesity. It outlines procedures for testing these inhibitors, including the measurement of enzyme activity, blood glucose levels, and starch content in animals. The study emphasizes the significance of inhibiting starch digestion to mitigate hyperglycemia and hyperinsulinemia in diabetic patients.

1. Rohit Patel
In vitro Antidiabetic activity

2. INVITRO TEST
 Inhibition of Polysaccharide-Degrading Enzymes
 Assay for α-Amylase
 Assay for α-Glucosidase
 Everted Sac Technique for Assaying α-Glucosidase
 Assays forGLUT2TransportActivity
 Perfusion of Jejunal Loops
 Transport Activity of Brush Border Membrane Vesicles
 Apical Expression of GLUT2
 Evaluation of Glucose Absorption InVivo

3. Inhibition of Polysaccharide-
Degrading Enzymes
 Starch as the predominant ingredient of human food is rapidly
degraded in the gastrointestinal tract by salivary and pancreatic
α-amylase to maltose which is further hydrolyzed by maltase
localized in the brush border of the small intestine to glucose.
 Glucose is immediately absorbed leading to hyperglycemia and
consequently to hyperinsulinemia. Both phenomena are
undesirable in diabetics and in obese patients.
 Inhibition of the digestion of starch leads to a decrease and a
retardation of glucose absorption. In nature, α- amylase inhibitors
are found in wheat and other grains (Shainkin and Birk, 1970).
Several inhibitors of amylase and α-glucosidase have been
developed (Bischoff 1991).
 Animal experiments with high doses of absorbable α-glucosidase
inhibitors indicate that lysosomal storage of glycogen may occur
(Lembcke et al. 1991).

4. 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.

5. PROCEDURE
 Commercially available pancreatic α-amylase is used.
 Various concentrations of the α-amylase inhibitor are
dissolved in 1ml 20mM Sörensen buffer, pH 6.9 and
10mM NaCl. 0.1ml pancreatic α-amylase solution in
0.4% BSA is added.
 After prior incubation at 25°C, the enzymatic reaction
is started by addition of 1.0ml soluble starch solution.
 The reaction is stopped after 10 min with 1.0ml of
dinitrosalicylic acid reagent.
 The mixture is heated in a boiling water bath for
10min, and after cooling measured at 546nm against
the reagent blank.

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

7. 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.
 The amount of liberated glucose is measured by the
glucose oxidase method. This study give detail about
the effect of intestinal disaccharidase inhibitors on
obesity and diabetes.

8. 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.

9. 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.

10. 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.

11. REFERENCES AND FURTHER
READING
 H. Gerhard Vogel , Drug Discovery and
Evaluation: Pharmacological Assays,P:1571-1577