Unlocking the therapeutic potential of Saussurea costus: purification and functional characterization of α-amylase inhibitors

Abstract

Introduction: Regulating the catalytic activity of alpha-Amylase enzymes can decrease glucose production during the postprandial phase, potentially offering therapeutic benefits for diabetes. This research aimed to assess the inhibition of α-amylase using crude extracts from Saussurea costus, a medicinal plant traditionally used for treating diabetes and its associated complications.

Methods: Two novel potent proteinaceous amylase inhibitors: ScAI-R and ScAI-L were purified and characterized from Saussurea costus roots and leaves.

Results: The pure inhibitors exhibited an apparent molecular weight of about 16 kDa and a high N-terminal sequence identity (81%) with the monomeric α-amylase inhibitors from Kengyili amelanthera and Triticum dicoccoides. In addition to their significant stability at extreme pH values (2.0-12.0) and temperatures (100°C), the structural integrity of both inhibitors was remarkably enhanced in the presence of divalent cations such as Mg²⁺, Ca²⁺, and Hg²⁺ at 5 mM. Interestingly, the half-maximal inhibitory concentrations of ScAI-R (IC₅₀ = 23 μg/mL) or ScAI-L (IC₅₀ = 28 μg/mL) against human salivary amylase against were comparable to that of the standard drug acarbose (IC₅₀ = 23 μg/mL). Both purified inhibitors acted as non-competitive inhibitors with K_i values of 0.38 and 0.32 µM, respectively, and displayed the highest affinities towards human salivary and pancreatic α-amylases (up to 90% inhibitory activity) and, to a lesser extent, porcine pancreatic α-amylase (∼70% inhibitory activity). Furthermore, these inhibitors exhibited efficient antimicrobial activities against Gram (-) and Gram (+) bacteria, as well as fungal strains. Cytotoxicity towards the human cancer colorectal cells LoVo and HCT-116 with an IC₅₀ of up to 50 μg/mL was also observed.

Discussion: Thus, Saussurea costus α-amylase inhibitors could be potential candidates for hyperglycemic control in diabetic and colorectal cancer patients.

Keywords: Saussurea costus; antibiotic agent; cytotoxicity; diabetes mellitus; enzyme inhibition; stability; α-amylase.

FIGURE 1

Chromatography profiles of ScAI-L (A) and ScAI-R (B) on Sepahdex-G50. (C) SDS-PAGE profile of purified ScAI-L and ScAI-R. Lane 1: molecular mass markers, Lane 2: ScAI-L; Lane 3: ScAI-R.

FIGURE 2

Multiple sequence alignment of the NH₂-terminal sequences of ScAI-R and ScAI-L with the cereal-type amylase inhibitor family using the Clustal Omega tools (A) and phylogenetic tree (B). Amino acids are color-coded to represent basic (magenta), acidic (blue), small or hydrophobic (red), and hydroxyl or amine (green). (*) Identical residues; (:) highly conserved residues; (.) significant conservation.

FIGURE 3

The effects of temperature on the activity (A) and stability (B) of ScAI-R and ScAI-L inhibitors. The results are expressed as the mean ± SD of three independent experiments.

FIGURE 4

The effects of β-mercaptoethanol on ScAI-R and ScAI-L amylase inhibitor activity. The results expressed are as the mean ± SD of three independent experiments.

FIGURE 5

The effects of pH on the activity (A) and stability (B) of ScAI-R and ScAI-L. The results are expressed as the mean ± SD of three independent experiments.

FIGURE 6

Half-maximal inhibitory concentration (IC₅₀) determination for human salivary amylase inhibition by ScAI-R and ScAI-L. The results are expressed as the mean ± SD of three independent experiments.

FIGURE 7

ScAI-R and ScAI-L activity against mammalian, bacterial, and fungal α-amylases. The results are expressed as the mean ± SD of three independent experiments.