In vitro contraceptive activities, molecular docking, molecular dynamics, MM-PBSA, non-covalent interaction and DFT studies of bioactive compounds from Aegle marmelos. Linn., leaves

Abstract

Introduction: Bioactive molecules from natural sources having contraceptive properties were excellent alternatives for modern hormonal contraceptives. Researchers around the world were working on identifying contraceptive leads targeting the male reproductive system rather than the usual female contraceptives. The lack of proper understanding on male contraceptive protein drug targets leads to insufficient evidence on activities of identified contraceptive compounds. The proteins specific to the male reproductive system and involved in sperm-egg fusion will be an excellent drug target to identify the male non-hormonal, reversible contraceptive leads. Inhibiting sperm hyaluronidase activity by natural non-hormonal compounds will lead to reversible and non-hormonal male contraception. The Aegle marmelos Linn. is one such important medicinal plant with valuable phytocompounds, used traditionally as a potential contraceptive measure. The in vivo experiments on leaf extracts of Aegle marmelos. Linn containing terpenes, sterols, and alkaloids shows prominent contraceptive activities. Moreover, this study explores the potential ability of the leaf extract on inhibiting the sperm hyaluronidase action with additional molecular details on the interaction between sperm hyaluronidases and three phytocompounds such as aegeline, marmin, and marminol. Material and methods: The in vitro hyaluronidase inhibition assay and Computer Assisted Sperm Analysis (CASA) were used to evaluate the male contraceptive properties of the Aegle marmelos Linn. leaf extract. To identify the interaction profile of aegeline, marmin, and marmenol on sperm cell hyaluronidases the in-silico methods such as molecular docking, Non-Covalent Interaction analysis, Molecular dynamics, and Molecular Mechanics Poisson Boltzmann Surface Area were used. Results and discussion: The results of in vitro hyaluronidase inhibition assay and Computer Assisted Sperm Analysis shows the inhibition of hyaluronidase enzymatic activity and reduced sperm activities in the presence of leaf extracts. After incubation with leaf extracts for about 30 minutes time intervals show, the motility drops from progressive to non-progressive and ended up with complete immotile in 100 μg/ml concentration of leaf extract. The results of molecular docking, Non-Covalent Interaction analysis, Molecular dynamics, and Molecular mechanics Poisson Boltzmann Surface Area show that the phytocompounds marmin, and aegeline have the potential ability to inhibit sperm hyaluronidase.

Keywords: Aegle marmelos. Linn.; computer assisted sperm analysis; hyaluronidase (HAase); natural contraceptives; non-covalent interaction plot.

FIGURE 1

From left to right—Frames recorded from the CASA analysis of AMLE-added sperm cells using MMC software for (A)100 μg/ml AMLE (B) 1,000 μg/ml AMLE (C) 10,000 μg/ml AMLE (D) 1,00,000 μg/ml AMLE. The blue circles generated by the software indicate a greater number of immotile cells. To highlight the immotile sperm cells, we manually highlighted the blue marks using red circles. The cells within the blue circle mark indicate a complete loss of sperm cell activity.

FIGURE 2

(Right) Frames recorded from the CASA analysis on sperm cells without AMLEusing MMC software. The red, yellow, and black streaks are produced by the MMC softwarewhich recognizes the motility of active and progressive motility.

FIGURE 3

(Left) Image from the vitality test for 100 μg/ml AMLE using the eosin staining method. The blue region indicates the presence of dead sperm cells after the application of 100 μg/ml AMLE. This may be due to the effect of AMLE on acrosomal and cytoskeletal proteins such as hyaluronidases.

FIGURE 4

(A,B): Image showing morphological characterization for the effect of 100 μg/ml AMLE on sperm cells showing more amorphous and bent neck cells are observed after incubation.

FIGURE 5

Morphology of healthy sperm cells without AMLE.

FIGURE 6

(A–D): (Clockwise) Molecular interaction of aegeline with HYAL-2 marmenol with HYAL-3, marmin with HYAL-4, marmin with HYALPH20 in the substrate and active site region.

FIGURE 7

(A): NCI iso surface plot for the HYAL-2-aegeline, (B): NCI iso surface plot for the HYAL3-marmenol, the presence of blue regions within the plot denotes the existence of strong interactions like hydrogen bonds. The presence of red regions in the plot shows thepresence of steric effects due to strong repulsion interactions. The green regions denoted thepresence of weak bonding such as van der Waal interactions. (C): NCI iso surface plotand RDG plot for the HYAL4-marmin, (D): NCI iso surface plot for the HYAL-PH20- marmin.

FIGURE 8

(A–D) (clockwise): Molecular dynamics results such as Ligand RMSD, protein backbone RMSD, RMSF and SAS for the protein-ligand complexes.