In Vitro and in-silico Anti-diabetic Evaluation of the Combination ofAnnona squamosa Linn., Leaf Extract and Oleanolic Acid

Background: Diabetes mellitus (DM) is a metabolic disorder caused by insufficient insulin production from pancreatic β-cells or insulin resistance; its prevalence rapidly increases worldwide. Increasing reports indicate that most plant bioactive agents exhibited alternative and safe effects in managing DM. Objective: The study aims to evaluate the in vitro antioxidant and anti-diabetic efficacy of the combination of Annona squamosa Linn. (AS) leaf extract and Oleanolic acid (OA) using in vitro and in-silico approaches. Methods: The leaf of AS was extracted by soxhlet extraction using n-hexane and methanol. The methanol extract of AS (MEAS) was subjected to GC-MS analysis. Quantification of total phenolic and flavonoid content and OA were carried out by HPLC and HPTLC analysis, respectively. In vitro antioxidant (DPPH, NO, and H2O2) and anti-diabetic (α-amylase and α-glucosidase) potentials of MEAS, OA, and a combination of MEAS and OA (MEAS + OA) were studied at different concentrations using ascorbic acid and acarbose as standard, respectively. An in-silico study determined their binding interactions with α-amylase (PDB ID-1B2Y) and α-glucosidase (PDB ID-3W37). Conclusion: We found that the combination of MEAS + OA exhibited the highest in vitro antioxidant and anti-diabetic activities compared to MEAS and OA. It concluded that OA has a significant role in potentiating the anti-diabetic effect of A. squamosa. Results: GC-MS analysis of MEAS revealed three major bioactives like bicyclo[7.2.0]undec-4- ene, 4,11,11-trimethyl-8-methylene-,[1R-(1R*,4Z,9S*)]-, germacrene D and undecane. The highest amount of phenolic (tannic acid and gallic acid) (150 μg/ml) and flavonoid (rutin and quercetin) (40 μg/ml) compounds were found in MEAS. OA was quantified as 356.74 ng/ml in MEAS by HPTLC. The significant inhibitory effects of MEAS, OA, and (MEAS + OA) on free radicals and α-amylase and α-glucosidase were observed concentration-dependent. However, MEAS + OA exhibited a greater percentage of inhibition than MEAS and OA alone. The in-silico analysis revealed highest docking-score of OA (-9.8 & -8.8), Germacrene D (-7.5 & -6.5) and Bicyclo[ 7.2.0]undec-4-ene, 4,11,11-trimethyl-8-methylene-,[1R-(1R*,4Z,9S*)]-, (-7.0 & -6.4) against IB2Y and 3W37 proteins, respectively.