Khaya grandifololia exerts multitarget neuroprotective potential against neurodegenerative disorders: In vitro and in silico studies

Abstract

Background

Treatment for complex multifactorial neurological disorders may benefit more from multifunctional chemicals. Dysregulation of monoaminergic pathways and neuroinflammation are typical confluence points in a range of neuropsychiatric and neurodegenerative illnesses. Polypharmacological medications that affect neuroinflammatory and monoaminergic pathways were investigated as potential targets for these diseases. The aim of this study was to investigate the in vitro and in silico multi-target neuroprotective activity of Khaya grandifololia.

Methods

Decoction and hydro-ethanolic extracts were prepared and screened for their ability to inhibit enzymes related to inflammation (15-lipoxygenase, LOX), neurodegeneration (monoamine oxidase, MAO), and protein glycation and fibrillation using enzymological fluorimetric assays and docking simulations.

Results

All extracts were able to strongly inhibit the activity of MAO as well as the glycation and fibrillation activities. Also, these extracts moderately inhibited 15-LOX activity.

Conclusion

These results extend the knowledge on the potential use of Khaya grandifololia to combat multifactorial disorders, giving new approaches into therapeutic avenues for neurodegenerative disease such as Alzheimer’s disease.

Introduction

Neurodegenerative disease (NDs) are a group of disorders including Parkinson's disease, Prion disease, Huntington's disease, Multiple sclerosis, and Alzheimer's disease, that cause the loss of structure and/or functional integrity of neurons (Sharma et al., 2023). NDs are commonly affected elderly people causing sickness and mortality. The pathological hallmark of NDs suggests its causation is the formation extracellular senile plaques (Aβ plaques), intracellular neurofibrillary tangles (NFTs), deficiency of neurotransmitters and neuroinflammation (Sharma et al., 2023). The risk factors include mutation of genes, abnormalities of chromosomes, insulin resistance (Fernandes et al., 2022).

Protein glycation is caused by a series of reactions such as the Maillard reaction, Schiff base production, and Amadori reaction. Glycation is a non-enzymatic condensation process involving a protein, lipid, or nucleic acids in the production of advanced glycation end products (AGEs). Through 3-deoxyglucosone AGE intermediates, glucose entering the polyol pathway and directly create AGEs. However, this reaction depletes NADPH and glutathione, and the resulting oxidative stress indirectly enhances AGE formation (Salahuddin et al., 2014).

Inflammation is an essential part of the vicious cycle that underlies a number of diseases, including cancer and neurological disorders. For instance, neurodegeneration triggers neuroinflammation, which in turn leads to the synthesis of neurotoxic chemicals such as cytokines, prostaglandins and nitric oxide, causing more neurodegeneration (Ugalde-Muñiz et al., 2020). As such, the treatment efficacy of a single targeting therapy may be limited. A polypharmacological molecule that affects several pathways involved in these intricate disorders may be a more successful tactic to end the vicious cycle and produce improved treatment outcomes (Hassan et al., 2022). Lipoxygenases are non-heme, iron-containing enzyme that catalyze the regio- and stereospecific insertion of oxygen (O₂) into polyunsaturated fatty acids, such as arachidonic or linoleic acid, which contain a series of cis double bonds. These molecules, which are made of membrane lipids hydrolyzed by cytosolic phospholipase A2 are essential fatty acids for humans. These conversions produce eicosanoids, or hydroperoxy fatty acids, which are then further broken down into signaling molecules like lipoxins and leukotrienes involved in the regulation of a number of inflammatory disorders (Ghansenyuy et al., 2023). The 15-lipoxygenase (15-LOX, EC 1.13.11.12) is an enzyme that regulates the production of inflammatory signaling molecules and is involved in the biosynthesis of various inflammatory diseases, including disorders of the central nervous system (CNS) like Alzheimer's, Parkinson's, and stroke (Lončarić et al., 2021; Xu et al., 2013).

Monoamine oxidase (MAO, EC 1.4.3.4) is an enzyme of great interest, because it catalyzes the main inactivation pathway for the catecholamine neurotransmitters, including adrenaline, noradrenaline, dopamine, and 5-hydroxytryptamine. Changes in the central nervous system's neurotransmitter levels are caused by MAO, and the biochemical pathology of numerous neurogenic illnesses associated with unbalanced neurotransmitter levels. MAO-A and MAO-B are two isoforms of MAO that have been identified through studies on the therapeutic effect of MAO enzyme inhibitors in depression. The therapeutic activity of a drug is determined by its selectivity towards certain isoforms (Kamauchi et al., 2022, Paudel et al., 2019). Tyramine, serotonin, and norepinephrine are all metabolized by MAO-A. MAO-A plays a number of important functions, such as oxidizing monoamines, promoting oxidation and aiding in the process of apoptosis (Hagenow et al., 2020). The pathophysiology of high-impact disorders such as major depressive disorders, addiction, and violent behavior involves abnormally high or low levels of MAO-A (Berlowitz et al., 2022, Soliman et al., 2012).

A neuroprotective therapy that aims to alter the etiopathogenesis and slow down the progression is crucial for more focused treatment because there is currently no clinical medication that can stop the neurodegenerative process. In the end, a new paradigm of drug development has resulted from the extensive search for a successful treatment. The multi-target drug-ligand concept is an innovative and promising approach to drug design that goes beyond the “one-molecule, one-target” (Anastassova et al., 2022).

Khaya grandifololia (KG), a plant of the family of Meliaceae, is used in traditional medicine for its pronounced effects, including the treatment of malaria, cancer, feverish illnesses, ulcers, and convulsions (Mukaila et al., 2021). Many compounds were isolated from the plant namely, 17-epimethyl-6-hydroxyangolensate, 7-deacetoxy-7-oxogedunin, 7-deacetoxy-7R-hydroxygedunin benzene, 1,1′ -(oxydiethylidene)bis, carbamic acid, (4-methylphenyl)-, 1-phenyl, and 6-phenyl, 4-(1′-oxyethylphenyl) hexene with hepatoprotective and antiviral activities (Kouam et al., 2017, Galani et al., 2016). Previous studies indicated that KG extract restores mitochondrial function, inhibits apoptosis, synaptic toxicity, and hyperphosphorylation of tau protein (Ella et al., 2020). Also, KG improves cognition and prevents scopolamine-induced impairment of brain functions by activating the cholinergic and antioxidant systems in rats (Ella et al., 2022).

In this study, we investigated the effect of extracts of Khaya grandifololia on different targets of neurodegeneration (advanced glycation end products, lipoxygenase and monoamine oxidase inhibition; anti-fibrillation activities) and molecular docking simulations.

Material and methods

Chemicals

Aβ_1–42 was purchased from rPeptide (Georgia, USA). Bovine serum albumin (BSA), sodium azide, ammonium molybdate, dextrose, xylenol orange, iron (II) sulfate, acarbose, aminoguanidine hydrochloride, nordihydroguaiaretic acid, deprenyl hydrochloride, p-nitrophenyl-α-D-glucopyranoside (pNPG), linoleic acid, kynuramine, Thioflavin T (ThT), phenol red were bought from Sigma-Aldrich (St. Louis, MO, USA). Lipoxygenase, β-glucosidase, monoamine oxidase and all organic solvents of HPLC grade were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Methods

Plant collection

The bark of Khaya grandifololia (KG) was collected in July 2019 in the city of Foumban, West Region, Cameroon. The botanical identification of the plant was done at the Cameroon National Herbarium under the voucher specimen 23434 YA.

Preparation of hydroethanolic extract and decoction of Khaya grandifololia

200 g of powder was extracted twice with 2 L of ethanol/water 65/35 (v/v) with regular agitation for 48 h. The filtered solution was pooled and evaporated to dryness using a rotary evaporator before drying in an oven at 50°C (HP-AD070, Memmert; Germany). The yield of extraction was 26.45 %.

50 g of powder was mixed with 200 mL of water and boiled at 100ºC for 30 min. The mixture was filtered and evaporated using in an oven (HP-AD070, Memmert; Germany) at 50°C. The yield of extraction was 21.72 %.

Alpha-glucosidase inhibition assay

This assay was carried out according to the method described by Ademiluyi and Oboh (2013) with slight modification. 100 µL of sample extract and acarbose (positive control) at different concentrations were mixed with 100 µL of 1.0 U β-glucosidase and pre-incubated at 37°C for 15 min. After pre-incubation, 30 µL of P-nitrophenyl-α-D-glucopyranoside (pNPG) (5.0 mM in 100 mM phosphate buffer pH 6.8) was added and the mixture was further incubated at 37°C for 30 min. Absorbance was recorded at 405 using a spectrophotometer (Multiskan™ GO, ThermoFisher Scientific). The percentage of inhibition was calculated and the half-maximal inhibitory (IC50) values were obtained using GraphPad Prism 8.0.2 software.

Anti-glycation assay

This assay was performed according to the method described by Vinson and Howard (1996) with minor modifications. BSA (10 mg/mL) were prepared in phosphate buffer (0.1 M, pH 7.4) and all the drugs were dissolved in DMSO. 100 µL of extract at different concentrations was incubated with 50 µL of BSA, 50 µL of dextrose monohydrate 0.5 M and 100 µL of sodium azide (3 mM in phosphate buffer). Aminoguanidine hydrochloride was used as a positive control. A blank containing BSA solution in phosphate buffer. The mixture was incubated for 9 days at 37ºC. After incubation, fluorescence at 355, and 460 nm, excitation and emission were measured by using a plate reader (PHERAstar FSX). The percentage of inhibition was calculated and the half-maximal inhibitory (IC50) values were obtained using GraphPad Prism 8.0.2 software.

15-Lipoxygenase inhibition (15-LOX) assay

The FOX-based assay was carried out according to the method described by Waslidge and Hayes (1995), with slight modifications. 30 µL of extract at different concentrations was pre-incubated with 50 µL LOX (2500 U/mL prepared in 50 mM Tris HCl buffer, pH 7.4) at 25°C for 5 min. The control was constituted with 50 µL of 15-LOX solution and 30 µL of Tris HCl buffer. Blanks (background) contained the enzyme LOX during incubation, but the substrate (linoleic acid) was added after the FOX reagent. The positive control was Nordihydroguaiaretic acid (standard inhibitor). After preincubation, 50 µL linoleic acid (final concentration, 150 µM) in Tris HCl buffer was added. The mixture was incubated at 25°C for 20 min in the dark. After that, 100 µL of FOX reagent [sulfuric acid (30 mM), xylenol orange (100 µM), iron (II) sulfate (100 µM), methanol/ water (9:1)]. After that, the mixture was incubated for 30 min at 25°C in the dark. The absorbance was measured at 560 nm using a microplate reader (Multiskan™ GO, ThermoFisher Scientific). The percentage of inhibition was calculated and the half-maximal inhibitory (IC50) values were obtained using GraphPad Prism 8.0.2 software.

Monoamine oxidase inhibition (MAO) assay

The MAO-A inhibitory activity of crude extracts of Khaya grandiololia was tested according to the procedure described by Liu et al., 2018. 100 µL of extract at different concentrations was incubated with 50 µL of MAO-A (5 U/mL) in a 96-well plate at 37ºC for 10 min. Thereafter, 50 µL of kynuramine (50 µM) was added and the mixture was incubated at 37ºC for 30 min. The reaction was stopped by adding in each well 80 µL of NaOH (2 N). The fluorescence intensity was measured using a microplate reader (PHERAstar FSX, 485/520 λex/em). Deprenyl hydrochloride was used as a positive control. The percentage of inhibition was calculated and the half-maximal inhibitory (IC50) values were obtained using GraphPad Prism 8.0.2 software.

ThT assay of inhibition of Aβ aggregation

Aggregation of Aβ₄₂ was evaluated by the Thioflavin T (ThT) assay as previously described by Boubakri et al., 2020 with minor modifications. Aβ₄₂ (5 μM) diluted to 15 µM (monomeric peptide concentration) in 20 mM phosphate buffer pH 7.4 was incubated alone or with different concentrations of extract of KG or phenol red (standard) at 25°C for 24 h. Thereafter, 1.0 mM ThT solution adjusted to 20 µM final concentration was added and incubated again for 30 min. After incubation, the maximum intensity of fluorescence was read at 485 nm using a microplate reader (PHERAstar FSX); excitation wavelength was 440 nm. Buffer fluorescence was subtracted from samples. The percent inhibition of Aβ aggregation was calculated and the half-maximal inhibitory (IC50) values was obtained using GraphPad Prism 8.0.2 software.

In silico Monoamine oxidase and lipoxygenase inhibition (MAO) assay

Software used

The following software’s Python 2.5, and 2.7, Molecular Graphics Lab Tools (MGL), AutoDockTools-1.5.7, Discovery Studio Visualizer 2.5.5, and ChemDraw were used.

Preparation of the ligand

Two-dimensional structures (2D) of compounds of interest (17-epimethyl-6-hydroxyangolensate, 7-deacetoxy-7-oxogedunin, 7-deacetoxy-7R-hydroxygedunin benzene, 1,1′ -(oxydiethylidene)bis, carbamic acid, (4-methylphenyl)- and 1-phenyl, and 6-phenyl, 4-(1′-oxyethylphenyl) hexene) and drug reference (Nordihydroguaiaretic acid, Baicalein and Dreprenyl) compounds were drawn using ChemDraw software. Three dimensional structures (3D) were obtained using ChemDraw 3D software and energy minimization was performed using the Molecular Mechanics (MM2) force field (Fig. 1).

Fig. 1.

compounds of interest and reference drugs.

Preparation of the target enzyme

The crystal structure of 15-Lipoxygenase protein (ID: 4NRE) and Monoamine oxidase A (ID: 2Z5X) was downloaded from the Research Collaboratory for Structural Bioinformatics (RCSB) protein database. The preparation of the target proteins with the AutoDock tools involved the addition of all hydrogen atoms to the macromolecule, a necessary step for the correct calculation of partial atomic charges.

Three-dimensional affinity grids of 15-Lipoxygenase contained the amino acids of its important for catalysis [His 373, His 378, His 553 (Kobe et al., 2014)] of size 40 × 40 × 44 Å with spacing of 0.375 Å, the macromolecule with Y and Z coordinates of 3.82, −49.281 and −17.654.

Three-dimensional affinity grids of Monoamine oxidase A contained the Catalytic Triad [Tyr-69, Asn-181, Phe-208, Val-210, Gln-215, Cys-323, Ile-325, Ile-335, Leu-337, Phe-352, Tyr-407 and Tyr-444 (Son et al., 2008)] of size 56 × 62 × 48 Å with spacing of 0.375 Å, the macromolecule with X, Y and Z coordinates of 38.853, 28.461 and −13.833.

Docking simulations

The molecular docking used the Lamarckian genetic algorithm method, which performed a total of 2,500,000 energy calculations for every run and 10 total generations. The algorithm method works using traditional force fields, providing empirical free energy functions and a binding energy constant (µM) using the following equation:

∆G = ∆Gvdw + ∆Ghbond + ∆Gelec + ∆Gconform + ∆Gtor + ∆Gsol

The typical molecular mechanics terms are the van der Waals interaction (ΔGvdw + desolv), electrostatic interaction (∆Gelec), torsional energy (∆Gtor), desolvation upon binding, and the hydrophobic effect (+∆Gsol) (Morris et al., 1998). The other docking simulations were carried out with AutoDock default parameters. Discovery Studio Visualizer software was used to visualize the protein–ligand interactions in 3D and 2D.

Statistical analysis

Results were expressed as mean value ± SD (n = 3). Regression analysis was performed to calculate the dose-response relation between the extracts. Linear regression analysis was performed to find out the correlation coefficient. Statistical significance was evaluated employing t-test and P < 0.05 which were considered to be significant.

Results

Alpha glucosidase inhibition

In the present study, the extracts of KG were evaluated for their inhibitory effect on α-glucosidase enzyme by in vitro method at different concentrations. As shown in Fig. 2, the different extracts exhibited a higher inhibition potential. The different crude extracts inhibited the enzyme α-glucosidase (KGHE, IC50 28.27 µg/mL; KGD, IC50 27.29 µg/mL) but there was no statistical difference compared to acarbose (IC50 21.61 µg/mL) (Table 1).

Fig. 2.

The anti‑glucosidase inhibition exhibited by extracts of Khaya grandifololia. KGD: Khaya grandifololia decoction; KGHE: Khaya grandifololia hydroethanolic extract; acarbose (positive control).

Table 1.

Inhibitory potential of extracts Khaya grandifololia on α-glucosidase activity, protein glycation, 15-LOX activity, MAO-A activity, ThT activity.

Extracts/Compounds	Half-maximal inhibitory (IC50) values (μg/mL)
	α-Glucosidase	Protein glycation	15-LOX	MAO-A	ThT
KGD	27.29 ± 0.45a	42.67 ± 0.33a	182.63 ± 4.83a	90.67 ± 2.40a	35.36 ± 0.70a
KGHE	28.27 ± 1.46a	30.31 ± 0.45a	169.77 ± 8.49a	106.90 ± 2.24a	34.37 ± 0.38a
Acarbose	21.61 ± 0.38a	-	-	-	-
Aminoguanidine	-	15.27 ± 0.37b	-	-	-
Nordihydroguaiaretic acid	-	-	48.79 ± 2.22b	-	-
Deprenyl				205.37 ± 4.82b
Phenol Red	-	-	-	-	33.56 ± 0.18a

KGD: Khaya grandifololia decoction; KGHE: Khaya grandifololia hydroethanolic extract. Results are expressed as the mean ± SD of triplicate experiments. Values with same superscripts down the column are not statistically different p < 0.05.

Advanced glycation end-products

The AGE fluorescence-based assay was used to assess the in vitro anti-glycation potential of varying concentrations of extracts. After incubation of different samples with the BSA in phosphate buffer, the percentage of inhibition was calculated. The results obtained showed that the plant extracts inhibit glycation in dose dependant manner (Fig. 3). Hydroethanolic extract of KG (KGHE) inhibited the glycation (IC₅₀ 30.31 µg/mL) more than decoction extract (KGD) (IC₅₀ 42.67 µg/mL) but lower than aminoguanidine used as the positive control (IC₅₀ 15.27 µg/mL) (Table 1).

Fig. 3.

The anti‑glycation activity exhibited by extracts of Khaya grandifololia. KGD: Khaya grandifololia decoction; KGHE: Khaya grandifololia hydroethanolic extract; AGH: Aminoguanidine hydrochloride (positive control).

15-Lipoxygenase inhibition

In this study, we used the ferrous oxidation-xylenol orange (FOX) assay method to test the 15-lipoxygenase inhibitory potential of extracts of KG. As shown in Fig. 4, all the extracts investigated had a certain level of inhibition of 15-lipoxygenase. To determine the most potent inhibitor against the 15-LOX, the IC₅₀ was obtained for each extract. The IC₅₀ of the hydroethanolic extract is 169.77 µg/mL while the decoction extract is 182.63 µg/mL and the nordihydroguaiaretic acid (reference inhibitor) is IC₅₀ 48.79 µg/mL.

Fig. 4.

The 15-LOX inhibition potential of extracts of Khaya grandifololia. KGD: Khaya grandifololia decoction; KGHE: Khaya grandifololia hydroethanolic extract; NA: Nordihydroguaiaretic acid (positive control).

Monoamine oxidase inhibition

The extracts of Khaya grandifololia were evaluated for their potential to inhibit monoamine oxidase A (MAO-A). As presented in Fig. 5, all the extracts demonstrated good inhibition against the MAO enzyme with a range of inhibition between 5 % and 80 %. To better determine which extract had stronger inhibitory activity, the IC50 was determined. The IC50 of KGD was determined to be 90.67 µg/mL while KGHE was 106.90 µg/mL, indicates a more potent and better inhibitory action of KGD than deprenyl used as a positive control (Table 1).

Fig. 5.

Monoamine oxidase inhibition of extracts of Khaya grandifololia. KGD: Khaya grandifololia decoction; KGHE: Khaya grandifololia hydroethanolic extract; DNPL: Deprenylhydrochloride (positive control).

ThT inhibition

To check the effect of extracts on Aβ₄₂ aggregation, we performed the ThT assay and the results are presented in Fig. 6. The different extracts inhibit Aβ₄₂ aggregation with percentages of inhibition ranging from 20 % to 90 % at tested concentrations. As shown in the Table 1, based on IC50 values calculated, there is no difference on the activity of different extracts and the positive control.

Fig. 6.

Inhibition of Aβ₄₂ aggregation by extracts of Khaya grandifololia. KGD: Khaya grandifololia decoction; KGHE: Khaya grandifololia hydroethanolic extract; DPNL: Deprenylhydrochloride (positive control).

Molecular docking score

In silico study of interest compounds against 15-lipoxygenase and monoamine oxidase-A

The docking scores of compounds of interest on 15-lipoxygenase and monoamine oxidase-A are presented in Table 2. Compounds likely to bind to a target with the lowest binding energy (AG) are the ones with the strongest and most possible binding affinity. From this perspective, a binding energy is all the weaker than its affinity and its power. The compounds investigated presented docking scores between 1453.54 and −2.02 kcal/mol including the best binding affinity obtained with compound 1. On the other hand, with MAO-A, the investigated compounds presented docking scores between −4.47 and −8.92 Kcal/mol with the best docking score obtained with 6-phenyl, 4-(1 oxyethylphenyl) hexene.

Table 2.

Molecular docking scores of the compounds against 15-lipoxygenase and monoamine oxidase.

Compound	15-lipoxygenase (15-LOX)		Monoamine oxidase A (MOA-A)
	Binding Energy (Kcal/mol)	Inhibition Constant (kI) (µM)	Binding Energy (Kcal/mol)	Inhibition Constant (kI) (µM)
1	−2.02	33000	−7.92	1.55
2	+3.89	/	−8.37	0.735
3	+10.39	/	−8.92	0.287
4	+1391.22	/	−4.47	525.65
5	+938.04	/	−7.01	7.31
6	+1453.54	/	−6.55	15.79
7	+7.12	/	/	/
8	−1.72	5312	/	/
9	/	/	−5.91	46.22

1: Benzene, 1,1’- (oxydiethyldiene)bis; 2: Carbamic acid, (4-methyl-1-phenyl)-1-phenyl; 3: 6-phenyl, 4-(1’oxyethylphenyl) hexene; 4: 17-epi-methyl-6-hydroxyangolensate; 5: 7-deacetoxy-7-oxogedunin; 6: 7-deacetoxy-7R-hydroxygedunin; 7: Nordihydroguaiaretic acid; 8: Baicalein; 9: Dreprenyl

Profiles of amino acid residues in the 15-lipoxygenase interacting with compounds of interest

The analysis of the interaction profiles of the potential inhibitor candidates compared to the reference (compound 8) showed no positive interaction with the histidine triad involved in the mechanism of action of the enzyme (Table 3, Fig. 7 A-B).

Table 3.

Profiles of amino acid residues important for 15-lipoxygenase that interact with compounds of interest.

Compounds	His 373	His 378	His 553
1	-	-	+
8	+	+	+

1: Benzene, 1,1’- (oxydiethyldiene)bis; 8: Baicalein;

Fig. 7.

A. 3D and 2D views of the molecular interactions between the amino acid residues of 15-Lipoxygenase and Compound 1. B. 3D and 2D views of the molecular interactions between the amino acid residues of 15-Lipoxygenase and Compound 8.

Profiles of amino acid residues in the MOA-A interacting with compounds of interest

Analysis of the 2D interaction profile of the compounds investigated and the amino acid residues of the active pocket of MOA-A showed that all the compounds investigated established hydrogen and hydrophobic bonds specifically with residues Tyr-69, Tyr −407 and Tyr-444 (Table 4, Fig. 8). However, among these compounds only compound 3 has, in addition to the latter, established a pi-pi stacked type interaction with the Phe 352 residue.

Table 4.

Profiles of amino acid residues important for MOA A that interact with compounds of interest.

Compounds	Residual amino acids from the active sites (Tyr-69, Asn-181, Phe-208, Val-210, Gln-215, Cys-323, Ile-325, Ile-335, Leu-337, Phe-352, Tyr-407 and Tyr-444)
1	Tyr−69, Phe 352 and Tyr−407
2	Tyr−69, Phe 352 and Tyr−444
3	Tyr−69,Phe 352,Tyr 407 and Tyr−444
4	Tyr−69, Tyr−407 and Tyr−444
5	Tyr−69,Tyr−407 and Tyr−444
6	Tyr−69,Tyr−407 and Tyr−407
7	Tyr−69 and Tyr−407

1: Benzene, 1,1’- (oxydiethyldiene)bis; 2: Carbamic acid, (4-methyl-1-phenyl)-1-phenyl; 3: 6-phenyl, 4-(1’oxyethylphenyl) hexene; 4: 17-epi-methyl-6-hydroxyangolensate; 5: 7-deacetoxy-7-oxogedunin; 6: 7-deacetoxy-7R-hydroxygedunin; 7: Dreprenyl

Fig. 8.

3D and 2D views of molecular interactions of Lead compounds and reference on MOA-A.

Discussion

Neurodegenerative diseases (NDs) are more common in the elderly due to an increased prevalence with aging. Consequently, one of the main problems facing modern pharmacology is the development of disease- or gene-modifying medications to slow the progression of NDs (Paudel et al., 2019). The development of effective treatments has moved frustratingly slowly, even in the face of extensive primary research on the causes and pathogenic characteristics of NDs. The FDA-approved enzyme inhibition approach to treatment for symptomatic relapse, potential side effects and drug-drug interactions, high costs, and a low success rate in clinical trials are likely the main obstacles to the development of neuronal drugs (Paudel et al., 2019). Medicinal plants have been screened for therapeutic compounds possessing anti-amyloidogenic properties, inhibitors of acetylcholine esterase activity, Aβ peptide fibrillation inhibitors, anti-inflammatory properties, secretase inhibitors, anti-aging and antiamnesic potential (Ella et al., 2022). In this study, we have evaluated the role of Khaya grandifololia, in neurodegenerative diseases by screening the plant extract against AGEs, LOX, MAO and ThT inhibition activities.

The glycation process produces a lot of free radicals, carbonyl species, and reactive dicarbonyl species throughout its early and propagation phases. These substances have an impact on normal physiological function (Yeh et al., 2017). Amnioguanindine was the first anti-AGE drug; but due to its toxic effect, there is an increase interest on plant-based remedies with fewer side effects. Anti-glycation has gained attention recently as a potential method to delay the onset of disease and human aging (Thrikawala et al., 2018). It is evident that the alteration of AGEs causes aberrant protein deposition and accumulation. This, in turn, maintains the inflammatory response and local oxidative stress, ultimately leading to the pathological and clinical features of neurodegenerative disorders. The decrease of fluorescent intensity in the presence of extracts of KG demonstrated an inhibitory potential of the plant toward AGE formation. According to recent research, AGEs may induce neurotoxicity through oxidative stress by controlling amyloid buildup and Aβ aggregation (Ko et al., 2015). AGEs accumulate throughout life, particularly in tissues with extended lifespans. Excessive buildup is linked to diabetes, chronic renal disease, and age-related illnesses including Alzheimer's disease (AD). It is also seen in circumstances of hyperglycemia and oxidative and inflammatory stress. In the brain, AGEs colocalize with proteins linked to AD, including tau, neurofibrillary tangles, and amyloid-β (Chen et al., 2021).

Alpha-glucosidase is one of the key enzyme involved in the control and reduction of hyperglycemia. This enzyme catalyses the final step in the digestive process of carbohydrates in mammals. Hence, α-glucosidase inhibitors can retard the liberation of D-glucose of oligosaccharides and disaccharides from dietary complex carbohydrates and delay glucose absorption, resulting in reduced postprandial plasma glucose levels and suppressed postprandial hyperglycemia (Gao et al., 2008). The extracts of KG display a high potential inhibition and can be used for the control of postprandial hyperglycemia due to the presence of biactive ingredients known to inhibit digestive enzymes (Rahman et al., 2020).

In the human body, lipoxygenases (LOXs) play a critical role in inflammatory reactions by producing prostaglandin and leucotrienes. Too much reactive oxygen species (ROS) can trigger inflammation, which in turn triggers cytokine release and LOX activation. Inflammation is associated with some diseases, including cancer, stroke and cardiovascular/neurodegenerative disorders. Inhibition of LOX is considered to be a key factor in disease prevention by affecting the inflammation process (Lončarić et al., 2021). Our results demonstrated that the plant extracts of KG inhibited the activity of 15-LOX due to the presence of bioactive metabolites. Studies have shown that extracts and some isolated bioactive compounds from medicinal plants reduced inflammation by inhibiting lipoxygenase activity (Muñoz-Ramírez et al., 2020).

Monoamine oxidases (MAO) are neurotransmitter-catabolizing enzymes, which play a role in the pathophysiology of neurological diseases. So, MAO-Inhibitors are established in the pharmacotherapy to manage neurological diseases (Hagenow et al., 2020). To date, the main idea is that pharmacological blockade of the breakdown of serotonin, dopamine, and other MAO substrates underlies the antidepressant properties of MAO inhibitors by modulating some of the neurochemical deficits (Berlowitz et al., 2022). It was noticed that the extract KGD offered more inhibition potential. Furthermore, previous studies report that several compounds from herbal medicines possess significant MAO inhibitory effects (Ayeni et al., 2023).

The aggregation of Aβ monomers to form oligomers and fibrils (aggregates) induces neurotoxicity. So, the use of compounds or extracts inhibiting Aβ aggregation is considered a good approach to the therapy and prevention of neurodegenerative disease (Dhouafli et al., 2018). In the present study, we found that the ethanolic extract of Khaya grandifololia dose-dependently reduces ThT-positivity of amyloid aggregates grown from the Aβ peptide, indicating that the extract inhibits amyloid fibril formation and that this effect could be associated with its richness in phenolic compounds. Our results are of interest, showing that this plant is a rich source of biomolecules able to interfere with Aβ aggregation. Recently, the study proves that medicinal plants containing phenolics are abrogated the aggregation of amyloid beta protein and reduced its toxic effect (Boubakri et al., 2020).

AD is a multifactorial progressive and chronic disorder. The paradigm "one drug—one target" is insufficient to treat a large number of multifactorial diseases caused by multifunctional pathophysiological processes. So, the development of multitargeting compounds with additive or synergistic effects is extremely relevant (Proschak et al., 2019). Khaya grandifololia contain many biological compounds such as phenolic compounds, flavonoids, saponins, alkaloids, triterpenes, glycosides, and tannins (Ella et al., 2022, Kouam et al., 2022). These bioactive constituents are multitarget-directed ligands able to counteract oxidation stress (glycation), neurotranmitters degradation (MAO activity) and inflammation (LOX activity) (Fernandes et al., 2022).

Steady advances in computational approaches such as pharmacodynamic prediction represent effective tools for explaining theurapeutic potential of plant extracts, and screening and selecting new candidate molecules for medication (Akamba et al., 2023, Piebeng et al., 2024). In this light, in silico stimulation of Khaya grandifololia identified isolated compounds has been carried out. the results showed that, benzene, 1,1′ -(oxydiethylidene)bis has a better docking score than reference compounds and interacts with His 553 important for catalytic activity (Kobe et al., 2014). this result may explain the potential of Khaya grandifololia extracts to inhibit 15-lipoxygenase. However, compounds 2–7 recorded an energy (+3.89 to +1453.54 kcal/mol) which suggests non-realizable interactions. Molecular stimulation of Khaya grandifololia compounds on MOA-A showed that these had scores ranging from −4.47 and −8.92. Only compound 17-epimethyl-6-hydroxyangolensate recorded a score below the reference (Dreprenyl). All Khaya grandifololia ligands target more residues of the MAO-A active site than the reference compound, which targets only two (Tyr-69 and Tyr-407). A striking fact is that compound 6-phenyl, 4-(1′-oxyethylphenyl) hexene targets Tyr-69, Phe 352, Tyr 407 and Tyr-444 all from the active site of MOA-A. These results may explain the in vitro inhibitory potential of Khaya grandifololia extracts on MOA-A activity.

Conclusion

This study demonstrated the importance of extracts of Khaya grandifololia as multi-target agents, showing the benefits plants against hallmarks of neurodegenerative disease, including 15-LOX, MAO, as well as glycation and β-aggregation inhibition. The molecular docking interactions revealed that compounds isolated from this plant might play significant roles in neuroactive ligand–receptor interactions in the major neurodegenerative pathway. However, in vivo studies are needed to confirm these results.

CRediT authorship contribution statement

Frederic Nico Njayou: Supervision, Writing – review & editing, Conceptualization. Bruno Dupon Akamba Ambamba: Writing – review & editing, Methodology, Data curation, Visualization, Formal analysis. Fils Armand Ella: Writing – original draft, Formal analysis, Conceptualization, Writing – review & editing, Methodology, Data curation. Paul Fewou Moundipa: Writing – review & editing, Conceptualization, Supervision.

Funding

This study did not receive any funding.

Ethical statement

Not applicable.

Conflicts of Interest

The authors of this study declare no conflict of interest.