Anticonvulsant Potential of the Essential Oil of Croton Heliotropiifolius Kunth: In Vivo and In Silico Approach

Abstract

Epilepsy is a chronic condition that significantly impacts the quality of life of many individuals, underscoring the urgent need for the identification of safe and effective anticonvulsant agents. In this context, medicinal plants have emerged as a promising source of bioactive compounds for treating epilepsy. This study involved an in vivo and in silico investigation of the anticonvulsant activity of the essential oil from the leaves of Croton heliotropiifolius Kunth (OCH). In vivo experiments revealed that the essential oil promoted a significant increase in seizure latency and survival rate in animals treated with OCH at a dose of 200 mg/kg, indicating an anticonvulsant effect. To understand the possible receptors and sites of action of the compounds in the oil, we performed a molecular docking study with GABA_A and NMDA receptors. Additionally, we calculated the electronic properties of the phytoconstituents at the B3LYP/6-311++G-(d,p)/SMD level. The results of the molecular docking studies revealed that the sesquiterpenes α-bulnesene, δ-cadinene, and β-bourbonene, which are present in OCH, have a high affinity for the GABA_A receptor, with binding energies ranging from -10.0 to -9.1 kcal/mol. These compounds primarily interact with the receptors through hydrophobic forces, highlighting the importance of interaction with Phe77 of the γ2^-(E) subunit of GABA_A. Docking analysis of NMDA revealed a higher affinity for the sesquiterpene guaiadiene, with a binding energy of -8.0 kcal/mol. Molecular dynamics simulations indicate that the α-bulnesene-GABA_A and guaiadiene-NMDA complexes remained stable over 100 ns. DFT analysis revealed that the most promising ligands are more stable and have moderate to strong electrophilicity. This research provides valuable insights for the identification of new molecules in the development of herbal medicines for the treatment of epilepsy, suggesting that the anticonvulsant effect of OCH may be related to the modulation of the GABA_A receptor or NMDA.

1

Diazepam binding sites on the GABA_A receptor: (a) An extracellular domain binding site for diazepam at the α1+(D)/γ2-(E) interface. (b) Three additional diazepam binding sites in the transmembrane domain at the β2+(C)/α1-(D), β2+(A)/α1-(B) and β2-(A)/γ2+(E) interfaces and (c) ifenprodil site on the NMDA receptor.

2

Effect of the essential oil of Croton heliotropiifolius Kunth (OCH), (n = 6/group). Leaves, at doses of 50, 100, and 200 mg/kg (v.o) on the latency to the first seizure induced by Pilorcapine (400 mg/kg, i.p.). Data are expressed as mean ± standard deviation.

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Effect of the essential oil of Croton heliotropiifolius Kunth. Leaves (OCH), at doses of 50, 100, and 200 mg/kg (v.o) on the latency to death of the animals, after the induction of convulsions by Pilocarpine (400 mg/kg, i.p.). Data are expressed as mean ± standard deviation (n = 6/group). Statistically significant difference if p < 0.05 (ANOVA followed by Tukey’s posttest), **p < 0.01 and ***p < 0.001, compared to the negative control group.

4

3D and 2D representations of ligands with the highest affinity for the α1+(D)/γ2^– (E) site (site 1) of the GABA_A receptor. The surface of the site is colored according to hydrophobicity (−3.0 to +3.0), with more hydrophobic regions in light/brown tones and less hydrophobic regions in blue. In the 3D images, the ligands occupy a predominantly hydrophobic pocket, formed mainly by Tyr, Phe, Val, and Met residues, which are also observed in the 2D representations, where alkyl and π–alkyl interactions predominate. Chains D and E correspond to the α1 and γ2 subunits, respectively.

5

3D and 2D representations of ligands with the highest affinity for the GluN1­(C)-GluN2B­(D) site (site 5) of the NMDA receptor. The surface of the site is colored according to hydrophobicity (scale from −3.0 to +3.0), where more hydrophobic regions are represented in light and brown tones and less hydrophobic regions in blue tones. In the 3D images, the ligands are accommodated in a predominantly hydrophobic pocket, formed mainly by residues Phe91, Phe53, Try87, and Ala466. These same residues are identified in the 2D diagrams, in which alkyl and π–alkyl interactions predominate. Chains C and D correspond to the GluN1 and GluN2B subunits, respectively.

6

Frontier molecular orbitals and HOMO–LUMO energy gap of the compounds with the highest affinity for the receptors were calculated at the B3LYP/6–311++G­(d,p)/SMD level.

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Molecular electrostatic potential maps of the compounds calculated at the B3LYP/6–311++G­(d,p) level with isosurface values of 0.002 au.

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RMSD plots for (A) α-bulnesene–GABA_A complex and (B) guaiadiene–NMDA complex.

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MM-PBSA analysis for (A) α-bulnesene–GABA_A complex and (B) guaiadiene–NMDA complex.