In Vitro and in Silico Analysis of α -Amylase Inhibitory Activity of Ethanolic Extract of Adhatoda vasica Leaves

Abstract

Objective: Diabetic individuals have a higher probability of suffering from illness and death due to small blood vessel-related problems such as retinopathy, neuropathy, nephropathy, and stroke than other complications. There are many synthetic anti-diabetic agents available, but these can be expensive and have undesirable pathological effects. The enzyme α-amylase (hydrolase), catalyzes the hydrolysis of starch to maltose and glucose via the cleavage of α-1,4-glucosidic linkages. Diabetes mellitus patients may benefit from a therapeutic strategy that involves slowing the hydrolysis of starch by inhibiting the activity of α-amylase. Thus, looking for cost-effective, natural, and safe antidiabetic agents is essential. This study aims to screen phytoconstituents and evaluate the in-vitro and in-silico α-amylase inhibitory activity of the ethanolic extract of Adhatoda vasica leaves.

Methods: The extraction of Adhatoda vasica leaves was performed using ethanol via the Soxhlet extraction process. Different concentrations (100 μg/mL to 1000 μg/mL) of ethanolic extract, Acarbose, and Sitagliptin, were prepared and evaluated for α-amylase inhibitory activity using the spectrophotometric method. Molecular docking (AutodockVina 1.2.0) and toxicity profiling (SToPToX web server) studies were performed.

Results: The ethanolic extract of Adhatoda vasica leaves showed the highest percentage inhibition against α-amylase (56.763 ± 0.0035) at a concentration of 1000 μg/mL. The in-silico study supported this inhibitory activity. Vasicoline (C5) and Quercetin (C9), the active constitute of Adhatoda vasica, showed the best binding energies of -8.3 and -8.0 Kcal/mol, respectively against α-amylase enzyme (PDBID: 4W93). A toxicity study revealed the safety profile of the plant extract.

Conclusion: It was concluded that Adhatoda vasica leaves possess some bioactive compounds that are responsible for controlling blood glucose levels, and their identification, purification, and isolation may lead to the development of new therapeutic agents with fewer side effects than the available drugs.

Keywords: adhatoda vasica; anti-diabetic activity; molecular docking; α-amylase.

Figure 1.

Chemical constitutes (C1-C7 alkaloid and C8-C12 flavonoids) of plant Adhatoda vasica and 3D structure of co-crystal native ligand (Acarbose C13) and reference compound Sitagliptin (C14).

Figure 2.

Average percentage inhibition of α-amylase activity at different concentrations of the plant extract and standard drugs used and error bar diagram of % inhibition ±SD. This diagram represents the error bar diagram of the average percentage inhibition at a concentration of 100 to 1000 μg/mL.

Figure 3.

The 3D structure of superimposed ligands (C1–C12) with co-crystal native ligand (C13) used to calculate the RMSD value.

Figure 4.

The 2D molecular interaction of Vasicoline (C5), Quercetin (C9), the co-crystal native ligand (Acarbose, C13), and the reference drugs Sitagliptin (C14), and Vasicin (Q1, 3D) against α-amylase.

Figure 5.

‘A’ and ‘C’ applicability domain (AD) for acute inhalation and acute oral toxicity of compound C5, respectively. ‘B’ and ‘C’ predicted fragment contribution for acute inhalation and acute oral toxicity of compound C5, respectively. The green color in the structure represents the functional group contributing to non-toxic properties and the brown color represents the functional group contributing to an enhancement in the toxic character.