Exploring the inhibitory action of betulinic acid on key digestive enzymes linked to diabetes via in vitro and computational models: approaches to anti-diabetic mechanisms

Figures & data

Figure 1. Chemical structure of betulinic acid.

Figure 2. (a) α-amylase and (b) α-glucosidase inhibitory activities of betulinic acid. Data = mean ± SD; n = 3. ^abValues with different letters above the bars for a given concentration are significantly (p < 0.05) different from each other.

Table 1. IC₅₀ values of biological activities.

Biological activity	Betulinic acid	Acarbose	Orlistat	Chymostatin
Αlpha-amylase	150.12 ± 2.46	49.53 ± 0.23	–	–
Alpha-glucosidase	19.45 ± 0.74	12.32 ± 0.67	–	–
Lipase	24.05 ± 0.68	–	12.38 ± 0.14	–
Chymotrypsin	34.98 ± 1.82	–	–	24.10 ± 0.91

Values are expressed as μM. Key: – = not applicable.

Figure 3. (a) Pancreatic lipase and (b) α-chymotrypsin inhibitory activities of betulinic acid. Data = mean ± SD; n = 3. ^abValues with different letters above the bars for a given concentration are significantly (p < 0.05) different from each other.

Figure 4. Optimized structure of betulinic acid.

Table 2. Electronic and reactivity descriptors.

Medium	EHOMO	ELUMO	ΔE	I	A	η	δ	χ	Cp	ω	µD
Gphase	−7.93	1.24	9.17	7.93	−1.24	4.58	0.21	3.34	−3.34	1.21	2.97
Water	−7.99	1.21	9.21	7.99	−1.21	4.60	0.21	3.39	−3.39	1.25	4.48

G_phase: Gas phase.

Figure 5. Frontier molecular orbitals of betulinic acid in (a) gas phase and (b) water.

Figure 6. Molecular electrostatic potential surface of betulinic acid in (a) gas phase and (b) water.

Table 3. RMSD, RMSF and SASA profile of betulinic acid bound to lipase, α-amylase, α-chymotrypsin and α-glucosidase.

Enzyme	RMSD	RMSF	SASA
Lipase	1.78	1.26	17362.44
Αlpha-amylase	1.91	0.90	4180.09
Alpha-chymotrypsin	1.41	1.07	9581.37
Alpha-glucosidase	1.28	0.87	29316.68

Values are represented as the estimated averaged and expressed as Å.

Figure 7. Structural representation of alterations occurring during the binding of betulinic acid with lipase (black), amylase (red), chymotrypsin (green) and glucosidase (purple), showing the conformational stability, C-α atoms RMSD for targeted enzymes to betulinic acid bound.

Figure 8. The time evolution RMSF of each residue of the enzymes C-α atom over 200 ns for betulinic acid bound to lipase, α-amylase, α-chymotrypsin and α-glucosidase.

Figure 9. Structural representation of alterations occurring during the binding of betulinic acid, solvent accessible surface area of the four targeted enzymes.

Table 4. MM/GBSA-based binding free energy profile of betulinic acid bound to lipase, α-amylase, α-chymotrypsin and α-glucosidase.

Enzyme	$\mathbf{\Delta }{\mathit{E}}_{\mathit{vdw}}$	$\mathbf{\Delta }{\mathit{E}}_{\mathit{ele}}$	$\mathbf{\Delta }{\mathit{G}}_{\mathit{gas}}$	$\mathbf{\Delta }{\mathit{G}}_{\mathit{gas}}$	$\mathbf{\Delta }{\mathit{G}}_{\mathit{bind}}$
Lipase	−21.67 ± 0.19	−3.75 ± 0.15	−25.43 ± 0.24	9.55 ± 0.13	−15.88 ± 0.17
Αlpha-amylase	−27.12 ± 0.11	−10.46 ± 0.24	−37.58 ± 0.23	14.25 ± 0.14	−23.33 ± 0.11
Alpha-chymotrypsin	−31.09 ± 0.07	−5.89 ± 0.14	−36.98 ± 0.15	8.59 ± 0.10	−28.40 ± 0.09
Alpha-glucosidase	−28.66 ± 0.25	−9.68 ± 0.12	−38.33 ± 0.28	14.67 ± 0.09	−23.67 ± 0.25

All energy components are expressed as kcal/mol.

$\mathrm{\Delta }{\mathrm{E}}_{\mathrm{ele}}$ = electrostatic energy; $\mathrm{\Delta }{\mathrm{E}}_{\mathrm{vdw}}$ = van der Waals energy; $\mathrm{\Delta }{\mathrm{G}}_{\mathrm{bind}}$ = total binding free energy; $\mathrm{\Delta }{\mathrm{G}}_{\mathrm{sol}}$ = solvation free energy; $\mathit{Accessisdenied}$ = gas phase free energy.

Figure 10. Per-residue decomposition plots showing individual energy contributions to the binding and stabilization of betulinic acid inhibitor at the binding sites of lipase (a), α-amylase (b), α-chymotrypsin (c) and α-glucosidase (d) enzymes. This reveals that energy contributions were highest in binding sites amino acid.

Figure 11. Dynamic cross-correlation matrix analyses for Betulinic acid where (a), (b), (c) and (d) represent inhibitor binding mode at the binding site of lipase, α-amylase, α-chymotrypsin and α-glucosidase enzymes. Numbers closer to 1 indicate high correlation, while those closer to − 1 indicate anticorrelation between pairs of residues.