for analysis of the inhibition activity of a component in foods

α-Glucosidase Inhibitory Activity Assay Kit

α-Glucosidase decomposes disaccharides into glucose, and increases blood glucose levels. Therefore, an increase in blood glucose can be suppressed by inhibiting the activity of α-Glucosidase. α-Glucosidase inhibitors have been discovered in various plants and their use is being explored for foods that will not cause an increase in blood glucose levels. This kit can, in combination with LabAssay™ Glucose (sold separately), measure the α-Glucosidase inhibitory effect of food products and the like.

Kit Components
Principle
Procedure 1
Procedure 2
Calculation
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Related Information

Kit Components

Reagent name	Pkg. size	Number of the vial	The principal ingredient
Enzyme*	1 mL	2	α-Glucosidase
Buffer	For 50 mL	2	Maleic Acid/Disodium Maleate
Substrate 1	For 20 mL	2	D-(+)-Maltose
Substrate 2	For 20 mL	2	Sucrose
Positive control	100 mg	1	Acarbose

* Agglomerates may be found, but there is no problem with quality. Please use it according to the procedure of the instruction manual.

Other supplies required

LabAssay™ Glucose
96 well transparent microplate
Plate reader (with an absorption filter at 505 nm)
Micro-pipettes and pipette tips
Incubator (capable of keeping at 37°C)

Principle

Principle of the Assay

Procedure 1

Reagent Preparation

Preliminary Test for Determination of the Dilution Rate

	Maltose	Sucrose
Substrate solution	Maltose solution 50 μL	Sucrose solution 50 μL
Purified water	25 μL
Warm for 3 minutes at 37°C
Enzyme	Diluted enzyme solution 25 μL (Example: 5, 10, 20 fold diluent)	Diluted enzyme solution 25 μL (Example: 1 (neat), 2.5, 5 fold dilution)
Mix, and keep accurately for 30 minutes at 37°C
Purified water	400 μL
Heat immediately in boiling water for 3 minutes, then keep in ice

【Quantification of glucose】
1. Aliquot 100 mL of enzyme reaction solution and diluted glucose standard solution (Concentration: 15, 10, 5, 2.5 mg/dL) into 96 wells microtiter plate, add 150 mL of chromogen reagent (included LabAssay™ Glucose), then mix thoroughly.
2. Warm for 10 minutes at 37°C, and measure the absorbance at 505 nm.
3. Determine the dilution rate as the amount of produced glucose becomes the following concentrations.
　　For maltose: 10 mg/dL, For sucrose: 5 mg/dL

Note:
1) The package insert written in Japanese is attached. If you need the package insert in English, please contact us.
2) For the determination method of glucose, please refer the package insert of LabAssay™ Glucose.

Procedure 2

Reagent Preparation

Sample Preparation

	Maltose	Sucrose
Substrate solution	Maltose solution 50 μL	Sucrose solution 50μL
Sample or positive control solution (purified water as the negative control)	25 μL
Warm for 3 minutes at 37°C
Diluted enzyme solution	Diluted enzyme solution 25 μL (At the dilution rate determined by the preliminary test)	Diluted enzyme solution 25 μL (At the dilution rate determined by the preliminary test)
Mix, and keep accurately for 30 minutes at 37°C
Purified water	400 μL
Heat immediately in boiling water for 3 minutes, then keep in ice

Blank Preparation

	Maltose	Sucrose
Substrate solution	Maltose solution 50 μL	Sucrose solution 50μL
Sample solution or purified water	25 μL
Purified water	400 μL
Diluted enzyme solution	Diluted enzyme solution 25 μL (At the dilution rate determined by the preliminary test)	Diluted enzyme solution 25 μL (At the dilution rate determined by the preliminary test)
Heat immediately in boiling water for 3 minutes, then keep in ice

Glucose Quantification

Perform the same method using LabAssay™ Glucose as the preliminary test.

Calculation

IC₅₀ Calculation

A) Set the yield of glucose of negative control as 100% α-Glucosidase activity. Calculate the specific activity of each concentration of samples.

B) Plot the concentration of sample/positive control at X-axis of semi logarithmic paper and specific activity of α-Glucosidase activity at Y-axis. The concentration of positive control should be the final concentration in enzyme reaction solution. Calculate the concentration at the point of the specific activity is 50% (IC₅₀) from approximate curve.

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