Cheminformatics bioprospection and experimental validation of corn silk for interventive type 2 diabetes therapeutics

Abstract

Diabetes mellitus (DM) is one of the oldest known human diseases, with type 2 diabetes mellitus (T2DM) being the most prevalent form. Type 2 diabetes mellitus (T2DM) is characterized by elevated blood glucose levels due to defective insulin production and/or resistance to insulin. If left untreated, it can lead to severe complications affecting various body systems. While synthetic medications are commonly used to treat T2DM, their associated drawbacks, such as high cost, inaccessibility and side effects, mitigate their application in managing T2DM. Consequently, there has been a growing interest in natural products with antidiabetic potential. Natural products, including medicinal plants and plantderived products, have been used for centuries, and their active compounds continue to be explored for therapeutic applications. For example, corn silk (CS), a waste material of corn cultivation, possesses several therapeutic properties, including antidiabetic potential. Although, studies reporting the promising hypoglycaemic potentials of CS exist, its exact mechanism of action remains incompletely elucidated, a research gap that was fulfilled in this study through metabolomics, cheminformatics bioprospection and in vitro experimental validation. To identify the constituents in CS, ultra-performance liquid chromatography-mass spectrometry analysis and principal component analysis was performed on its three extracts (aqueous, hydro-ethanolic and ethanolic) at two developmental growth stages (premature and mature). A library consisting of 128 metabolites was generated from all the samples of CS with qualitative and quantitative variations observed between the two growth stages of CS and the type of solvent used for extraction. Specifically, the mature CS had a higher abundance of most metabolites, with the hydro-ethanolic extract of CS being the most metabolites-rich compared to the aqueous and ethanolic extracts of CS. These metabolites were thereafter subjected to bioprospection against the therapeutic targets, such as enzymes and genes implicated in the pathogenesis of T2DM using computational techniques. The modulatory role of CS metabolites on six enzymes implicated in the pathogenesis of T2DM and its secondary complication, particularly alpha-amylase (AA), alpha-glucosidase (AG), aldose reductase (AR), dipeptidyl peptidase-4 (DPP-4), protein tyrosine phosphatase 1B (PTP1B) and sorbitol dehydrogenase (SDH), was analysed using molecular docking complemented with molecular dynamics (MD) simulation. Molecular docking analysis identified aesculin (-8.1 kcal/mol), austricin (-7.8 kcal/mol), (6E)-1-(4-hydroxyphenyl)-7- phenylhepta-4,6-dien-3-one (-9.9 kcal/mol), (-)-11-hydroxy-9,10-dihydrojasmonic acid 11-beta-D-glucoside (-8.6 kcal/mol), phaseic acid (-6.0 kcal/mol), and erythronolide B (- 9.2 kcal/mol) as compounds with the most negative scores against AA, AG, AR, DPP-4, PTP1B and SDH, respectively. However, a further insight into the binding free energy (ΔGbind) calculations of the putative leads against each enzyme over a 150-ns simulation period revealed that R-7-butyl-6,8-dihydroxy-3-[(3e)-pent-3-en-1-yl]-3,4- dihydroisochromen-1-one (BHP), 1-O-vanilloyl-beta-D-glucose (VBJ), (-)-11-hydroxy9,10-dihydrojasmonic acid 11-beta-D-glucoside (HDJ), p-coumaroyl malic acid (CMA), 2- hydroxydecanedioic acid (HDA), and (-)-11-hydroxy-9,10-dihydrojasmonic acid 11-beta-D-glucoside (HDJ) hold remarkable therapeutic promise as modulators of AA, AG, AR, DPP-4, PTP1B, and SDH, respectively. The post-MD dynamic simulation analysis and interaction plots in each case revealed the formation of thermodynamically stable complexes suggestive of the putative leads as potential modulators of the respective investigated enzymes and their possible applications in the management of T2DM and its secondary complications. Density functional theory (DFT) analysis was used to determine the molecular characteristics of the top ranked CS metabolites identified to modulate the investigated enzyme targets. Although the lower energy gaps, higher softness and lower chemical hardness of the metabolites did not correlate with their high negative binding free energy (potentially due to the observed relative residue fluctuations and increased surface area of the targets upon ligand binding), their electrophilicity indices which were above 1.5 electron volt (eV) alluded to their strong electrophilic potential. This highlights their ability to interact with amino acids with nucleophilic side chains of the target enzymes that is indicative of enhanced specificity and binding to the enzymes. Subsequently, a network pharmacology study was conducted to elucidate the relationship between CS constituents and signaling pathways implicated in T2DM. The analysis identified the cAMP pathway as the central signaling pathway, with adenosine receptor A1 (ADORA1), hydroxycarboxylic acid receptor 2 (HCAR2) and gamma-aminobutyric acid type B subunit 1 (GABBR1) as key therapeutic targets. Gallicynoic acid (-48.74 kcal/mol), dodecanedioc acid (-34.53 kcal/mol), and tetradecanedioc acid (-36.80 kcal/mol) interacted effectively with ADORA1, HCAR2, and GABBR1, respectively, relative to the reference standards (metformin and resveratrol) and formed thermodynamically stable complexes, as indicated by post-MD analysis results. These findings suggest the compounds as potential drug candidates for T2DM through modulation of cAMP pathway genes. The cAMP pathway is implicated in the pathogenesis of T2DM through various levels including glucagon and epinephrine-stimulated cAMP production, increased glucose release from the liver, modulation of insulin signaling, insulin resistance and the regulation of gut hormone secretion, including glucagon-like peptide-1. To complement and validate the results obtained through network pharmacology as a further way of elucidating the mechanism of antidiabetic action of CS, experimental validation employing the use of HepG2 cells was performed. The effect of different CS formulations on HepG2 cells was firstly assessed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) viability and glucose consumption assays, followed by real-time polymerase chain reaction (RT-qPCR) to understand the effect of CS on the expression of ADORA1 and GABBR1, the top two target genes modulated by the CS metabolites as identified by the network pharmacology study. For the MTT assay, CS extracts at concentrations 75 – 100 µg/mL promoted viability of HepG2 cells, with the ethanolic extract of the mature CS being the most viable relative to the controls (insulinand metformin-treated) and the untreated cells. Generally, higher HepG2 cell viability and glucose uptake were observed following treatment with mature CS extracts compared to premature CS. Specifically, the most significant and enhanced glucose uptake level was observed with both normal and insulin-resistant HepG2 cells following treatment with the aqueous extracts of mature CS extract compared to the controls. Furthermore, compared to the untreated cells, as well as insulin- and metformin-administered cells, treatment with CS extracts remarkably inhibited the expression of ADORA1 and GABBR1 in insulin-resistant HepG2 cells with the most prominent effect observed with the aqueous extract of premature CS. These observation with the CS aqueous extracts may be attributed to their relatively higher abundance of the profiled metabolites such as gallicynoic acid B and tetradecanedioc acid, which were more than 40% each by composition in both the mature and premature extracts. These findings regarding the high concentrations of gallicynoic acid B and tetradecanedioc acid in CS aqueous extracts are not only significant in modulating the expression of ADORA1 and GABBR1, resulting in increased glucose uptake in the treated cells but consistent with the results of MD simulation that profiled the two compounds as putative leads against the two most significant therapeutic targets in the cAMP signalling pathway associated with T2DM. Overall, the findings from this study have contributed to the elucidation of the mechanisms of antidiabetic action of CS metabolites which would be vital in the development of CS as a therapeutic agent for the management of T2DM and its associated secondary complications.