In silico docking and ADMET studies on clinical targets for type 2 diabetes correlated to in vitro inhibition of α-glucosidase and pancreatic α-amylase by curcumin, 18α-glycyrrhetinic acid, rosmarinic acid, and quercetin

Abstract

Type 2 diabetes mellitus (T2DM) is a chronic disease characterised by prolonged hyperglycaemia due to the inability of the liver, muscle, and fat cells to absorb glucose following insulin stimulation. Several therapeutic targets have been identified and this includes the inhibition of α-amylase and α-glucosidase in the small intestine, promotion of glucose uptake by responsive tissue and the inhibition of hepatic lipid accumulation. The in silico enzyme inhibitory abilities of 1070 compounds found in 30 commercially available herbs, spices, and medicinal plants were assessed using docking analysis with Maestro from Schrodinger and AutoDock vina from DIA-DB; four compounds (18α-glycyrrhetinic acid (18α-GA), curcumin, quercetin and rosmarinic acid) with docking scores more negative or similar to acarbose were selected for further analysis. In silico ADMET properties were obtained using canvas QikProp, pkCSM online tool, and the results were compared with acarbose. In vitro biochemical assays were used to confirm docking studies; these were the dinitrosalicylic acid (DNSA) and para-nitrophenyl-D-glucopyranoside (pNPG) assays for α-amylase and α-glucosidase inhibition, respectively. The IC50 of each compound was determined after 48 hrs exposure with the Sulforhodamine B (SRB) assay in the C2C12, HepG2 and Caco-2 cell lines, representing the muscle, liver, and intestinal tissue respectively. Using the 2-[N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) assay, the ability of the compounds to promote glucose uptake was determined in the C2C12 and HepG2 cell lines. The reduction of lipid accumulation associated with non-alcoholic liver disease (NAFLD), a feature of T2DM was evaluated in the HepG2 cells exposed to oleic acid (OA). Effects were compared with known drugs, acarbose for α-amylase and α-glucosidase inhibition and metformin for glucose uptake and lipid accumulation studies. Herbs and spices with high levels of these compounds were then identified. As green tea was also a rich source of compounds with antidiabetic effects, using UPLC-MS the levels of quinic acid, epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) in five green tea Camellia sinensis, tea brands were determined. The aim was to identify, related to anti-diabetic effects the best tea that inhibits α-amylase and α-glucosidase. The relationships between in silico and in vitro inhibition results correlated well; a more negative docking score in silico correlated with a lower inhibition constant (Ki) in vitro. For α-glucosidase, the Ki values of curcumin, 18α-GA, and quercetin were significantly lower (p < 0.05) than that of acarbose, while there was no significant difference (p > 0.05) between acarbose and rosmarinic acid. These compounds were then identified as potential inhibitors of α-glucosidase. For α-amylase, the Ki values of curcumin, 18α-GA, quercetin, and rosmarinic acid were significantly higher (p < 0.05) than acarbose. An IC50 could be determined for 18α-GA, quercetin and rosmarinic acid in the C2C12, 18α-GA and rosmarinic acid in the HepG2 and curcumin and rosmarinic acid in the Caco-2 cell lines. All other compounds showed no cytotoxicity. Generally, at the concentrations used to evaluate glucose uptake and lipid accumulation the compounds were not cytotoxic. The compounds and metformin did not promote glucose uptake under the experimental conditions used but did significantly reduce (p < 0.05) the accumulation of OA induced lipid droplets in HepG2 cells. Herbs and spices including green tea were identified as rich of these compounds and contained EGCG and ECG, known antidiabetic compounds. Further evaluation of five green tea brands confirmed α-amylase and α-glucosidase inhibition with Dilmah tea having the best inhibition. Other unidentified compounds in green tea may also contribute to activity. Key findings were that these compounds are present in herbs, spices, and teas, which are cost-effective, easily cultivated, and readily available plant products that can contribute to the alleviation of T2DM symptoms. Curcumin, 18α-GA, quercetin, and rosmarinic acid inhibited α-glucosidase, and reduced the accumulation of OA induced lipid droplets in HepG2 cells with the potential to alleviate the consequence of prolonged hyperglycemia such as the development of NAFLD. Curcumin is found abundantly in turmeric and EGCG in green tea; a dose of 1.3 g of turmeric or 1.2 g of green tea is equivalent to a 50 mg acarbose dosage per meal. One teabag contains 2.5 g of green tea; therefore, one cup of green tea per meal may be helpful in preventing prolonged hyperglycemia.