Nisha Joseph
Post Graduate and Research Department of Botany, Catholicate College Pathanamthitta, Kerala, India.
S. Deepthi
Post Graduate and Research Department of Botany, Catholicate College Pathanamthitta, Kerala, India.
Gokul G. Nair
Post Graduate and Research Department of Botany, Catholicate College Pathanamthitta, Kerala, India.
Merin Grace Jiji
Post Graduate and Research Department of Botany, Catholicate College Pathanamthitta, Kerala, India.
In this post, we present a brief overview of our recently published book chapter titled “Evaluation of α-Amylase Inhibitory Activity of Aqueous Extracts of Selected Plants”
Aerial shoots of Aerva lanata, fruits of Emblica officinalis and Momordica charantia, pseudostem of Musa paradisiaca, and leaves of Psidium guajava were collected from Pathanamthitta, Kerala. The plants were identified based on Bentham and Hooker’s system of classification.
Preparation of Plant Extracts
The plant parts were shade-dried and powdered finely. 10 g of the dried powder was boiled with 100 mL of distilled water for 30 minutes. The mixture was filtered using filter paper, and the filtrate was collected and dried in a hot air oven. The dried extract was scraped and stored in an Eppendorf tube. To prepare the aqueous extract, 0.1 g of the dry extract was dissolved in 10 mL of distilled water, resulting in a 1% (w/v) solution.
Estimation of Total Phenols
Total phenolic content was determined using the Folin–Ciocalteu method. In brief, 0.1 mL of the extract was mixed with 0.5 mL of Folin–Ciocalteu reagent and 5.0 mL of sodium carbonate solution. The reaction mixture was incubated at room temperature for 30 minutes, and absorbance was measured at 640 nm using a spectrophotometer. Gallic acid served as the standard for calibration.
Estimation of Tannins
Total tannin content was estimated following the described method. One milligram of each extract was dissolved in 10 mL of methanol–water (7:3 v/v). To this, 0.5 mL of Folin’s phenol reagent (diluted 1:2) and 5 mL of 3.5% sodium carbonate solution were added. After 5 minutes, the colour intensity was measured at 640 nm.
Assay of α-Amylase Inhibition
In vitro α-amylase inhibition was assessed using the standard method. A reaction mixture containing 100 μL of test extract, 200 μL of α-amylase enzyme, and 100 μL of 2 mM phosphate buffer (pH 6.9) was incubated for 20 minutes. Subsequently, 100 μL of 1% starch solution was added. For control samples, the enzyme was replaced with buffer. After 5 minutes of incubation, 500 μL of dinitrosalicylic acid reagent was added to both test and control mixtures, followed by heating in a boiling water bath for 5 minutes. Absorbance was recorded at 540 nm. Appropriate reagent blanks and inhibitor controls were included in all assays. Metformin [3-(diamino methylidene)-1,1-dimethylguanidin], a known antidiabetic agent which reduces glucose absorption, lowers liver glucose production and improves insulin sensitivity, is used as the positive control in this study. The percentage inhibition of α-amylase activity was calculated using the formula:
% α amylase inhibition = (Abs100% control − Abs Sample) / (Abs100% Control) × 100
Statistical Analysis
Each treatment was replicated three times, and data were expressed as mean ± standard error (SE). Statistical analysis was performed using SPSS software (free trial version), including one-way analysis of variance (ANOVA) followed by Duncan’s New Multiple Range Test (DNMRT) for mean comparison.