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. 2022 Aug 10:9:866719.
doi: 10.3389/fmolb.2022.866719. eCollection 2022.

Inhibition mechanism of alpha-amylase, a diabetes target, by a steroidal pregnane and pregnane glycosides derived from Gongronema latifolium Benth

Oludare M Ogunyemi  1   2 , Gideon A Gyebi  3   4 , Afolabi Saheed  5 , Jesse Paul  1 , Victoria Nwaneri-Chidozie  1 , Olufunke Olorundare  5 , Joseph Adebayo  6 , Mamoru Koketsu  7 , Nada Aljarba  8 , Saad Alkahtani  9 , Gaber El-Saber Batiha  10 , Charles O Olaiya  2
Affiliations

Inhibition mechanism of alpha-amylase, a diabetes target, by a steroidal pregnane and pregnane glycosides derived from Gongronema latifolium Benth

Oludare M Ogunyemi et al. Front Mol Biosci. .

Abstract

Alpha-amylase is widely exploited as a drug target for preventing postprandial hyperglycemia in diabetes and other metabolic diseases. Inhibition of this enzyme by plant-derived pregnanes is not fully understood. Herein, we used in vitro, in silico, and in vivo studies to provide further insights into the alpha-amylase inhibitory potential of selected pregnane-rich chromatographic fractions and four steroidal pregnane phytochemicals (SPPs), viz: marsectohexol (P1), 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→14)-β-D-oleandropyranosyl]-11,12-di-O-tigloyl-17β-marsdenin (P2), 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl]-17β-marsdenin (P3), and 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-canaropyranosyl]-17β-marsdenin (P4) derived from Gongronema latifolium Benth. The results revealed that the SPPs source pregnane-rich chromatographic fractions and the SPPs (P1-P4) exhibited inhibitory potential against porcine pancreatic alpha-amylase in vitro. Compounds P1 and P2 with IC50 values 10.01 and 12.10 µM, respectively, showed greater inhibitory potential than the reference acarbose (IC50 = 13.47 µM). Molecular docking analysis suggests that the SPPs had a strong binding affinity to porcine pancreatic alpha-amylase (PPA), human pancreatic alpha-amylase (HPA), and human salivary alpha-amylase (HSA), interacting with the key active site residues through an array of hydrophobic interactions and hydrogen bonds. The strong interactions of the SPPs with Glu233 and Asp300 residues may disrupt their roles in the acid-base catalytic mechanism and proper orientation of the polymeric substrates, respectively. The interactions with human pancreatic amylase were maintained in a dynamic environment as indicated by the root mean square deviation, radius of gyration, surface accessible surface area, and number of hydrogen bonds computed from the trajectories obtained from a 100-ns molecular dynamics simulation. Key loop regions of HPA that contribute to substrate binding exhibited flexibility and interaction potential toward the compounds as indicated by the root mean square fluctuation. Furthermore, P1 significantly reduced blood glucose levels and area under the curve in albino rats which were orally challenged with starch. Therefore, Gongronema latifolium and its constituent SPPs may be exploited as inhibitors of pancreatic alpha-amylase as an oral policy for impeding postprandial blood glucose rise.

Keywords: G. latifolium; alpha-amylase; diabetes; molecular docking; molecular dynamics simulations; phytochemicals; pregnanes.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The 2D structures of marsectohexol (P1) and the pregnane glycosides, viz: iloneoside (P2), 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranosyl]-17β-marsdenin (P3), and 3-O-[6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-canaropyranosyl]-17β-marsdenin (P4) derived from Gongronema latifolium Benth.
FIGURE 2
FIGURE 2
Inhibitory activity of bioactive fractions of Gongronema latifolium Benth. against alpha-amylase. Percentage inhibitory activity of the selected pregnane-rich chromatographic sub-fractions on porcine alpha-amylase (A). Half-maximal inhibitory concentrations (IC50) of the pregnane-rich fractions (B).
FIGURE 3
FIGURE 3
Inhibitory activity of steroidal pregnane compounds (P1–P4) and acarbose against alpha-amylase. Percentage inhibitory activity (A) and half-maximal inhibitory concentrations (IC50) (B).
FIGURE 4
FIGURE 4
Redocked native compound: (A) Superimposition of selected docked conformer of co-crystallized myricetin on the retracted co-crystallized structure. (B) interacted amino acid residues with myricetin.
FIGURE 5
FIGURE 5
The 3D depiction of the interactions of acarbose and the isolated steroidal pregnanes with amino acid residues in the active site of porcine pancreatic alpha-amylase (PPA). Ligands are depicted in stick representations in colors red: acarbose (reference inhibitor); blue: P1; green: P2; pink: P3; purple: P4. Interaction types (dotted lines) and their bond lengths are depicted in green (H-bonds); light purple: hydrophobic interactions (pi-alkyl, alkyl, and pi-stacking); purple: pi-pi T-shaped interactions; yellow: pi-sulfur interactions and pi-stacking interactions. Amino acid residues are in a three-letter representation.
FIGURE 6
FIGURE 6
The 3D depiction of the interactions of acarbose and the isolated steroidal pregnanes with amino acid residues in the active site of human pancreatic alpha-amylase (HPA). Ligands are depicted in stick representations in colors red: acarbose (reference inhibitor); blue: P1; green: P2; pink: P3; purple: P4. Interaction types (dotted lines) and their bond lengths are depicted in green (H-bonds); light purple: hydrophobic interactions (pi-alkyl, alkyl, and pi-stacking); purple: pi-pi T-shaped interactions; yellow: pi-sulfur interactions and pi-stacking interactions. Amino acid residues are in a three-letter representation.
FIGURE 7
FIGURE 7
The 3D depiction of the interactions of acarbose and the isolated steroidal pregnanes with amino acid residues in the active site of human salivary alpha-amylase (HSA). Ligands are depicted in stick representations in colors red: acarbose (reference inhibitor); blue: P1; green: P2; pink: P3; purple: P4. Interaction types (dotted lines) and their bond lengths are depicted in green (H-bonds); light purple: hydrophobic interactions (pi-Alkyl, alkyl, and pi-stacking); purple: pi-pi T-shaped interactions; yellow: pi-sulfur interactions and pi-stacking interactions. Amino acid residues are in a three-letter representation.
FIGURE 8
FIGURE 8
Structural stability of apo human pancreatic amylase and in complex with the isolated steroidal pregnanes. (A) Backbone root mean square deviation (RMSD) plots. (B) Radius of gyration (RoG) plots. (C) Surface accessible surface area (SASA) plots. (D) Change in the number of hydrogen bonds.
FIGURE 9
FIGURE 9
Per residue root mean square fluctuations (RMSF) plots of molecular dynamics (MD) simulation of human pancreatic amylase (HPA) and in complex with the isolated steroidal pregnanes.
FIGURE 10
FIGURE 10
Effects of P1 (marsectohexol) from Gongronema latifolium and acarbose on the blood glucose level after the administration of 3 mg/kg starch to albino rats. Values are the mean ± SEM (n = 4), at p < 0.05.
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