2.6. In Vitro Antidiabetic Activity (α-Amylase Inhibition Assay)

α-Amylase Inhibition Assay (Starch-Iodine Method)

Mohammed Abu Tayab¹_’ *

¹ Department of Pharmacy, International Islamic University Chittagong, Chattogram 4318, Bangladesh

Corresponding author: abutayab.me@gmail.com

Abstract

α-amylase inhibition assay is an in vitro test for determining the potential antidiabetic activity of plant extracts and other substances, using acarbose as a standard inhibitor. The assay, known as the starch-iodine method, measures the remaining starch after incubation with α-amylase, where iodine forms a colored complex with starch, and absorbance is measured at 620 nm. Percentage inhibition of α-amylase activity is calculated relative to an enzyme control without inhibitor. Dose-response data can be constructed to estimate the IC₅₀ by regression analysis.

1. Background

In type 2 diabetes mellitus (DM2), persistent hyperglycemia is indicated by two metrics: fasting and postprandial blood glucose concentrations  (Dong et al., 2012). Elevated postprandial blood glucose levels are thought to have a significant role in the development of DM2 (Dong et al., 2012; Kim et al., 2014). Controlling postprandial hyperglycemia has been considered as an essential therapeutic technique in the treatment of DM2. The strategy involves blocking carbohydrate-hydrolyzing enzymes like α-amylase and α-glucosidase to delay glucose absorption through the intestinal epithelium (Ademiluyi and Oboh, 2013; Bhandari et al., 2008; Subramanian et al., 2008; Tundis et al., 2010). α-amylase is a 57.6 kDa calcium-dependent metalloenzyme composed of 512 amino acids arranged in a singular oligosaccharide chain (de Sales et al., 2012; Whitcomb and Lowe, 2007). Upon the binding of starch to its substrate-binding site, certain catalytic residues from its Domain A engage with starch, commencing hydrolysis into shorter oligosaccharides by severing α-1,4 glycosidic bonds (de Sales et al., 2012; Etxeberria et al., 2012). The last phase of digestion encompassed α-glucosidases that operated on oligosaccharides, breaking them down into glucose prior to absorption into the circulation (Etxeberria et al., 2012; P et al., 2011). The breakdown of this dietary carbohydrate occurs rapidly and leads to increased postprandial hyperglycemia (P et al., 2011). Acarbose, voglibose, and miglitol are often available DM2 medications of synthetic origin that manage postprandial hyperglycemia by inhibiting these enzymes (Ademiluyi and Oboh, 2013).

2. Materials and reagents

1. α-Amylase enzyme 

2. Sodium phosphate buffer (0.02 M, pH 6.9) containing 0.006 M NaCl 

3. Acarbose (standard inhibitor)

4. Soluble starch

5. Hydrochloric acid (HCl)

6. Iodine (I₂)

7. Potassium iodide (KI)

8. Distilled water

9. Plant extract

10. Test tubes and micropipettes 

3. Equipment and software

• UV-visible spectrophotometer

• Water bath or incubator set to 37°C

• GraphPad Prism version 8.0.2 for Windows, or equivalent data-analysis software 

4. Reagent preparation

1. Sodium phosphate buffer (0.02 M, pH 6.9, containing 0.006 M NaCl): Dissolve 286 mg of Na₂HPO₄·7H₂O, 129 mg of NaH₂PO₄·H₂O, and 35 mg of NaCl in distilled water and make the final volume up to 100 mL. Confirm that the buffer pH is 6.9.

2. Soluble starch solution (1% w/v): Mix 1 g of soluble starch with distilled water and make the final volume up to 100 mL.

3. Hydrochloric acid (1 M): Add 8.33 mL of concentrated HCl (12 M) to distilled water, then make the final volume up to 100 mL. Always add acid to water.

4. Iodine reagent (0.005 M I₂ and 0.005 M KI): Dissolve 127 mg of I₂ and 83 mg of KI in hot distilled water and make the final volume up to 100 mL.

5. α-Amylase solution (0.04 mg/mL): Dissolve 4 mg of α-amylase in 100 mL of the sodium phosphate buffer described above.

6. Test solutions: Prepare acarbose and plant-extract solutions at concentrations ranging from 125 to 1,000 µg/mL.

5. Procedure

1. Label tubes for the test concentrations, enzyme control, and blank.

2. For each test concentration, mix 500 µL of acarbose or plant-extract solution with 500 µL of α-amylase solution (0.04 mg/mL).

3. Incubate at 37°C for 10 minutes.

4. Add 500 µL of 1% soluble starch solution to each tube.

5. Re-incubate at 37°C for 15 minutes.

6. Stop the enzymatic reaction by adding 20 µL of 1 M HCl.

7. Cool the iodine reagent, add 100 µL of iodine reagent, and mix.

8. Measure absorbance at 620 nm using a UV-visible spectrophotometer.

6. Controls

1. Enzyme control: Replace the test solution with 500 µL of sodium phosphate buffer; this represents 100% enzyme activity.

2. Blank: Contains buffer solution only. The blank is subtracted from absorbance values.

7. Calculation

Percentage α-amylase inhibition was calculated using the formula:

% α-amylase inhibition = (Ac − As)/Ac × 100

where Ac and As denote the absorbance of the control reaction and the absorbance of the test sample, respectively.

8. Data analysis

Perform each in vitro assay in triplicate (n = 3). Construct dose-response curves by plotting percentage α-amylase inhibition against concentration (µg/mL). Determine IC₅₀ values by nonlinear regression analysis using GraphPad Prism version 8.0.2 for Windows or equivalent software.

Acknowledgments

Not applicable

Competing interests

The author declares no conflicts of interest.

References

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Bhandari, M.R., Jong-Anurakkun, N., Hong, G., Kawabata, J., 2008. α-Glucosidase and α-amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergenia ciliata, Haw.). Food Chemistry 106(1), 247-252.

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