Abstract
Human pancreatic α-amylase (HPA) inhibitors offer an effective strategy to lower postprandial hyperglycemia via control of starch breakdown. Limonoids from Azadirachta indica known for their therapeutic potential were screened for pancreatic α-amylase inhibition, a known anti-diabetic target. Studies were carried out to reveal their mode of action so as to justify their hypoglycemic potential. Of the nine limonoids isolated/semi-synthesized from A.indica and screened for α-amylase inhibition, azadiradione and exhibited potential inhibition with an IC50 value of 74.17 and 68.38 μM, respectively against HPA under in vitro conditions. Further screening on AR42J α-amylase secretory cell line for cytotoxicity and bioactivity revealed that azadiradione and gedunin exhibited cytotoxicity with IC50 of 11.1 and 13.4μM. Maximal secreted α-amylase inhibition of 41.8% and 53.4% was seen at 3.5 and 3.3μM, respectively. Michaelis-Menten kinetics suggested a mixed mode of inhibition with maltopentaose (K i 42.2, 18.6 μM) and starch (K i ′ 75.8, 37.4 μM) as substrate with a stiochiometry of 1:1 for both azadiradione and gedunin, respectively. The molecular docking simulation indicated plausible π-alkyl and alkyl-alkyl interactions between the aromatic amino acids and inhibitors. Fluorescence and CD confirmed the involvement of tryptophan and tyrosine in ligand binding to HPA. Thermodynamic parameters suggested that binding is enthalpically and entropically driven with ΔG° of -21.25 kJ mol-1 and -21.16 kJ mol-1 for azadiradione and gedunin, respectively. Thus, the limonoids azadiradione and gedunin could bind and inactivate HPA (anti-diabetic target) and may prove to be lead drug candidates to reduce/control post-prandial hyperglycemia.
Highlights
Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in insulin secretion, insulin action, or both leading to chronic hyperglycemia
Porcine pancreatic α-amylase (PPA) an enzyme having a high degree of sequence, structural and functional similarity with Human pancreatic α-amylase (HPA) as mentioned earlier was used for preliminary screening of compounds possessing α-amylase inhibitory property [11,32,33]
Very few reports are available on the individual secondary metabolites or their mode of action for hypoglycemic property [48,49,50,51,52,53].This prompted us to ascertain their efficacy for therapeutic applications as inhibitors of HPA, a potent antidiabetic target
Summary
Diabetes mellitus (DM) is a metabolic disorder resulting from a defect in insulin secretion, insulin action, or both leading to chronic hyperglycemia. Inhibition of carbohydrate degrading enzymes significantly reduces post prandial increase in blood glucose after a meal by delaying starch hydrolysis [8] This suppression of post prandial hyperglycemia delays the progression of vascular complications associated with DM [9]. One such enzyme, human pancreatic α-amylase (HPA, α1,4-glucan-4-glucanohydrolase, E.C. 3.2.1.1) plays a pivotal role in DM. By inhibiting HPA in the small intestines, the rate of hydrolysis of starch is decreased delaying the digestion process This spreading of digestion process reduces the amount of glucose generated and released in the blood and is one of the effective strategies in lowering post prandial hyperglycemia
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