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. 2021 Aug 26;9(1):51. doi: 10.1007/s40203-021-00110-0

Erythrina variegata L. bark: an untapped bioactive source harbouring therapeutic properties for the treatment of Alzheimer’s disease

Vishal S Patil 1,2,✉, Himani Meena 3, Darasaguppe R Harish 2,✉
PMCID: PMC8390608  PMID: 34532215

Abstract

A critical approach for target identification to detect the significant molecular mechanism of lead molecules via computational methods combined with in vitro procedures defines the modern strategy to combat untreatable diseases. Hence, the present investigation dealt to determine the effect of Erythrina variegata L. bark extract/fraction(s) over acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity followed by target identification and docking analysis of prime phytoconstituents. The in vitro AChE and BChE enzyme inhibitory assay were performed. Phytoconstituents from E. variegata were screened for carcinogenicity and mutagenicity and predicted for their possible targets leading to the identification of two known targets, i.e. AChE and BChE. The alkaloids with non-carcinogenic and non-mutagenic properties were studied for their main moiety responsible for the inhibitory activity. The protein models were checked in ERRAT for their quality and the homology model was created using Modeller9.10v to fill missing amino acid residues. The docking study predicted the binding affinity of bioactive molecules with identified targets using AutoDock 4.2. Molecular dynamics (MD) simulations for top hits were performed by Schrodinger Desmond 6.1v software. Chloroform fraction showed potent inhibition of AChE and BChE with IC50 value of 38.03 ± 1.987 µg/mL and 20.67 ± 2.794 µg/mL, respectively. Among all the six major bioactive compounds, Erysotine and Erythraline scored the highest binding affinity with AChE and Erysodine with BChE. MD simulation for 20 ns production run demonstrated Erysotine and Erysodine stable interaction with Arg49 of AChE and Lys427 of BChE, respectively. The current data provide enough shreds of evidence supporting the utilization of indolo [7a,1-a] isoquinoline derivatives for the identification of a new drug molecule in the management of Alzheimer’s disease.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40203-021-00110-0.

Keywords: Alzheimer’s disease, Acetylcholinesterase, Butyrylcholinesterase, Erythrina variegata, In silico docking

Introduction

Alzheimer disease (AD) is a chronic neurodegenerative disease and the most common form of dementia (progressive decline in memory) in elderly individuals (Pattanashetti et al. 2017; Palle and Neerati 2017; Biradar et al. 2020) affecting 47 million people worldwide (Ulep et al. 2018) and reflecting the dysfunction of cerebral nerve cells for storing, processing and transmitting information (Fetzer 1999). Numerous hypothesis associated with AD pathogenesis such as the cholinergic hypothesis, tau protein hypothesis, amyloid cascade hypothesis, and oxidative stress hypothesis have been proposed in the last few decades. Among these hypothesis, the cholinergic hypothesis is the most accepted theory, which suggests that decreased level of Acetylcholine (ACh) in the particular regions mainly the cerebral cortex and hippocampus of the brain, and leading to the major cause for the dysfunction of learning and memory. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) are two main cholinergic enzymes that metabolize acetylcholine and are highly expressed in AD; leading to the decreased level of acetylcholine and the appearance of the symptoms associated with AD (Zeynep et al. 2017; Khan et al. 2015; Khan et al. 2014).

Conventional AChE inhibitors such as Tacrine, Rivastigmine, Galantamine, and Donepezil are used for the treatment of mild to moderate dementia in patients suffering from AD (Mehta et al. 2012). However, these molecules provide a partial improvement of memory and cognitive function and are associated with a number of side effects like bradycardia, hypotension, bronchospasm, increased respiratory secretion, decreased intraocular pressure, nausea, vomiting (Ali et al. 2015) and are reported for decreasing effectiveness as the disease progresses (Mayo clinic 2018). Carbamate inhibitors (neostigmine, physostigmine, pyridostigmine, rivastigmine) bind to the active site of both AChE and BChE; however, the covalent bond is not stable, and the carbamate moiety is hydrolytically separated from the active site after a period of time (Zawadzka et al. 2012). Metrifonate, an organophosphorus compound irreversibly inhibits both AChE and BChE; was chosen for the treatment of AD and became an exception, however it was withdrawn due to side effects (López-Arrieta and Schneider 2006). Plant-derived natural products have received considerable attention due to the abundance of multiple phytoconstituents with diverse chemical moieties delivering minimum side effects and better efficacy, galantamine and huperzine are examples of plant alkaloids (Cronin 2001; Liu et al. 1986; Ma and Gang 2008). Hence it is rational to use natural resources and identify lead molecules from folk medicine for AD treatment.

Erythrina variegata L. also known as Erythrina indica L., belongs to the family Fabaceae and reported for the presence of Erysodine, Erysotine, Erysotrine, Erysovine, Erythraline, and Erythratidine as the major alkaloids (Kumar et al. 2010; Lim 2014; Ghosal et al. 1972; Chawla et al. 1988) with the moiety of indolo [7a,1-a] isoquinoline. The bioactive compounds with indolo [7a,1-a] isoquinoline moieties were documented and recorded for their inhibitory effect on AChE and BChE (Majinda 2018). Alkaloids derivatives (Erythrine byproducts) of Erythrine species are reported for the treatment of neurodegenerative disorders (Hussain et al. 2018).

However, there is a paucity of scientific reports regarding the in vitro and in silico docking study of indolo [7a,1-a] isoquinoline derivatives from Erythrina variegata for inhibition of AChE and BChE in the management of AD. Hence, the experimental study is based on the inhibitory effect of different bioactive fractions of Erythrina variegate on AChE and BChE and document the efficacy of indolo [7a,1-a] isoquinoline derivatives to protect against neurodegenerative disorders, proven with in silico approach.

Materials and methods

Chemicals

Acetylcholine iodide, Butyrylcholine iodide, Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE), 5,5′–dithiobis[2-nitrobenzoic acid] (DTNB] were procured from Sigma-Aldrich, USA. Donepezil was received as a gift sample from Apotex Research Pvt. Ltd, Bangalore.

Plant material and extract/fraction(s) preparation

The bark of Erythrina variegata was collected from Belagavi, Karnataka, India, in September/2018; authenticated by an experienced taxonomist from ICMR-National Institute of Traditional Medicine, Belagavi; deposited herbarium for the same accession number: RMRC-1404 for future reference. The bark was washed under running water to remove adhered dust material, shade dried, crushed into the coarse powder, and subjected for the maceration using 70 % V/V ethanol for seven days with occasional shaking. The mixture was then filtered and concentrated using a rotary evaporator under reduced pressure. The concentrated crude extracts of the bark portion were further subjected to fractionation using n-hexane (3 × 150 mL), ethyl acetate (3 × 150 mL), and chloroform (3 × 150 mL). The fractionated extract was stored in an airtight light resistance glass container for further use.

Enzyme preparation and test solutions

The enzyme AChE and BChE of 6.67 U/mL stock solution were prepared by dissolving in 20mM sodium phosphate buffer (pH 7.6), and the solutions were stored in − 80 °C for further use. The stock solutions of plant extracts and standard drug donepezil were prepared. From the stock solutions prepared 10, 20, 40, 80, 160 and 320 µg/mL of plant extracts and 1, 2, 4, 8, 16 and 32 µg/mL of donepezil.

In vitro anticholinesterase assay

An enzymatic inhibition protocol (Ellman’s method) was followed and determined the inhibitory efficacy of plant crude extract on the enzymatic activity of AChE and BChE (Ellman et al. 1961). Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates, respectively for AChE and BChE enzymes. A 1.7 mL of 50 mM Tris HCl buffer (pH 8.0) was dispensed in a test tube and added a range of plant extract concentrations with a final volume of 250 µL and donepezil drug. The above mixture was then added with 10 µL of AChE and BChE enzyme separately and 20 µL of DTNB and incubated for 15 min. After incubation, the mixture was subjected to spectrophotometric analysis at the volume of 10 µL each, AChE and BChE, and recorded the absorbance at 412 nm every 45 s for 3 min. The enzyme inhibition was calculated from the rate of change in absorbance with time. The IC50 was calculated between the inhibition percentage v/s extract concentrations.

%Inhibition=100-(Change of sample Abs/Change of blank Abs)×100

Selection of phytoconstituents and target prediction

The phytoconstituents from E. variegata were identified for indolo [7a,1-a] isoquinoline moiety (Fig. 1) and predicted for mutagenicity, carcinogenicity in mice, rat and their different strains using PreADMET (https://preadmet.bmdrc.kr/toxicity/). The compounds scoring negative results were selected to predict their possible targets using SwissTargetPrediction (http://www.swisstargetprediction.ch/). The drug-likeness character (Lipinski’s rule of five) of each drug molecule was predicted using MolSoft (http://molsoft.com) webserver.

Fig. 1.

Fig. 1

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2D chemical structures of a Erysodine, b Erysotine, c Erysotrine, d Erysovine, e Erythraline, f Erythratidine and g Donepezil retrieved from the chemical database, PubChem

Preparation of ligand

All the 2D structures of the selected six compounds were retrieved from the PubChem database, imported into MarvinSketch, converted into three-dimensional structures, and minimized under the MMFF94 force field. The conformation having the lowest energy was chosen for the docking study; the number of torsions was set to each ligand was kept for default.

Preparation of macromolecule

Human Acetylcholinesterase (PDB ID: 4PQE) and Butyrylcholinesterase (PDB ID: 4XII) crystallographic structure were retrieved from RCSB Protein Data Bank (https://www.rcsb.org/). Missing amino acid residues were added via the homology modeling using Modeller9.10. Water molecules and other heteroatoms were removed using Discovery Studio 2017 to avoid docking interference. The protein was added with a hydrogen atom on a polar bond and 6.5 and 9.78 kcal/mol Kollman charges were added as a default to AChE and BChE respectively.

Homology modeling of acetylcholinesterase

The homology modeling was performed for AChE having PDB id: 4PQE due to its missing amino acid residues within the crystal structure. The query sequence of Acetylcholinesterase (Accession Number: AAA68151.1) was retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/). The Acetylcholinesterase (PDB ID: 4PQE) having 100 % sequence identity and 88 % query cover was chosen as the template for model building using Modeller9.10. Initially, 5 different models were generated, as a result, the model having the least Discrete optimized protein energy (DOPE) score was chosen for further study. The quality of the model target protein was checked using online servers Procheck, Errat, and Saves (https://saves.mbi.ucla.edu/). The overall quality of each protein molecule was determined by Errat and the orientation of each amino acid within the protein is determined via the Ramachandran plot.

Ligand–protein docking

MGLTools 1.5.7, an open-source software suite containing AutoDock 4.2 tool was utilized to perform docking of phytocompounds with AChE and BChE. Ten different structural confirmations of ligand molecules were obtained during docking simulation. The docking poses having the lowest binding energy was completed with the target protein to visualize the ligand–protein interaction in Discovery studio 2017.

Molecular dynamics (MD) simulation studies

To check the interaction stability of top hits complexes i.e. Erysotine with AChE and Erysodine with BChE, a 20 ns MD simulation was carried out using Schrodinger Desmond version 6.1 (Bowers et al. 2006; Patil et al. 2021). A pre-set Simple Point Charge (SPC) water model was utilized as the solvent in a cubic box with 10 Å × 10 Å × 10 Å dimensions as the periodic boundary. The Erysotine–AChE system was neutralized by adding 5 Cl counterions and the Erysodine–BChE system with 14 Cl counterions. The SHAKE algorithm was also utilized to constrain the geometry of water molecules, as well as the lengths and angles of heavy atom bonds. To calculate the long-range interactions between the molecules, the Particle Mesh Ewald method was used. The cut-off for Lennard-Jones interactions was set at 10. The system was minimized for a 100.0 ps production run. Finally, the NPT ensemble was used to maintain the pressure of 1.01325 bar and temperature of 300 K using the Thermostat “Nose–Hoover chain” method with 1.0 ps relaxation time and the Barostat “Martyana–Tobias–Klein” method (with 2.0 ps relaxation time). The short-range Coulombic cut-off radius was set at 9.0 Å. The whole run’s trajectory sampling was captured at a 10.0 ps interval up to 20 ns. The root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (rGyr) parameters were considered to examine the stability and residue-wise interaction fluctuations.

Results

In vitro anticholinesterase activity

The AChE and BChE enzyme inhibitory activity of E. variegata extract fractionation and clinically proven drug donepezil were shown in Table 1. Among the different extracts, chloroform fraction showed potent inhibition of AChE and BChE with IC50 Value 38.03 ± 1.987 µg/mL and 20.67 ± 2.794 µg/mL respectively; n-hexane fraction showed potent inhibition of BChE with IC50 Value of 42.96 ± 5.562 µg/mL and lowest AChE and BChE inhibitory activity is exhibited by crude extract and ethyl acetate compared to standard drug donepezil.

Table 1.

In vitro acetylcholinesterase and butyrylcholinesterase inhibitory activity of different fractions of E. variegata

Extract/fractions AChE IC50 value (µg/mL) BChE IC50 value (µg/mL)
Crude extract 193.7 ± 5.568 246.9 ± 6.943
n-Hexane 77.96 ± 5.466 42.96 ± 5.562
Ethyl acetate 118.7 ± 4.667 77.87 ± 7.979
Chloroform 38.03 ± 1.987 20.67 ± 2.794
Donepezil* 2.51 ± 0.079 21.40 ± 0.582
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*Standard molecule for the treatment of AD

Selection of compound and protein

Among total bioactive phytoconstituents, 6 constituents namely Erysodine, Erysotine, Erysotrine, Erysovine, Erythraline, and Erythratidine have identified as the derivatives of indolo [7a,1-a] isoquinoline and predicted for their non-carcinogenic and non-mutagenic characteristics, which were subjected for target protein prediction to identify AChE and BChE as the common targets. A qualitative parameter “Lipinski’s rule of five” was used to understand the drug-likeness character of each drug molecule. Among all the six compounds, Erysotine scored the highest drug-likeness value (Table 2).

Table 2.

Druglikeness and similarity of indolo[7a,1-a]isoquinoline derivatives

Compounds Molecular formula Molecular weight (g/mol) HBD HBA Log P Drug likeness Score Overlay similarity
≤ 500 g/mol ≤ 5 ≤ 10 ≤ 5
Erysodine C18H21NO3 299.37 1 4 1.40 − 0.14 –
Erysotine C18H23NO4 317.38 2 5 0.78 0.21 0.955
Erysotrine C19H23NO3 313.39 0 4 1.75 − 0.26 0.845
Erysovine C18H21NO3 299.37 1 4 1.40 − 0.14 0.944
Erythraline C18H19NO3 297.35 0 4 1.84 − 0.75 0.849
Erythratidine C19H25NO4 331.41 1 5 1.13 0.04 0.937
Donepezil* C24H29NO3 379.5 0 4 4.73 1.76 –
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*Standard molecule for the treatment of AD. Overlay similarity of compounds was analyzed w.r.t Erysodine

Homology modeling of AChE and quality of protein molecules

The DOPE score of AChE modeled protein was found to be − 70180.6. Similarly, the overall quality factor studied under Errat for AChE was found to be 77.93 % and for BChE 93.793 % (Fig. 2).

Fig. 2.

Fig. 2

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(1) a Quality of homology modeled AChE (PDB ID: 4PQE), b Ramachandran plot and (2) a Quality of the protein molecule BChE (PDB ID: 4XII) b Ramachandran plot

Docking studies of compounds with AChE and BChE

Among all the six bioactive phytoconstituents, Erysotine scored the highest binding affinity of − 4.87 kcal/mol (inhibitory constant 270 µM) with AChE. Similarly, Erysodine scored highest binding affinity i.e. − 6.66 kcal/mol (Inhibitory constant 13.18 µM) with BChE. Donepezil, a standard molecule scored binding affinity, i.e. − 5.02 kcal/mol (Inhibitory constant 210.41 µM) and − 4.43 kcal/mol (Inhibitory constant 569.63 µM) with AChE with BChE, respectively. The binding energy, inhibition constant, and hydrogen bond interaction of individual compounds with each target are summarized in Table 3 and the interaction between each ligand and target is shown in Supplementary Figs. 1 and 2 for AChE and BChE, respectively. Figure 3 displays the intermolecular interactions of Erysotine and Erythraline with AChE and Erysodine with BChE.

Table 3.

Binding energy, Inhibition constant, and Hydrogen bond interaction of the compounds with AChE and BChE

Compound name Acetylcholinesterase (PDB ID: 4PQE) Butyrylcholinesterase (PDB ID: 4XII)
Binding energy (kcal/mol) IC50 (µM) Hydrogen bond interaction Non HBI (no. of interactions) Binding energy (kcal/mol) IC50 (µM) Hydrogen bond interaction Non HBI (no. of interactions)
Erysodine − 4.49 511.73 Glu35 Ile51, Leu39 (2) − 6.66 13.18 Lys427 Met81, His126 (2), Leu448, Val127, Lys427, His77 (3), Pro429
Erysotine − 4.87 270.35 Glu35, Arg52 Leu39 (3) − 5.94 44.59 His77 Glu80, Leu125 (2), Val127, Met81, His126, His77 (2)
Erysotrine − 3.76 1750.00 Arg49 Asp92, Thr94, Ile51 (2) − 5.92 45.85 Val127 Glu80 (2), Leu125, Met81 (2)
Erysovine − 4.26 750.96 Glu35 Arg52, Arg34 − 5.9 47.60 Lys427 His77 (2), Lys427, Leu448, His126 (2), Met81, Val127
Erythraline − 4.86 271.64 Arg47 Trp87, Gly45, Ser88, Arg47 (2) − 5.62 76.06 Hıs77, Val127 Leu125 (2), Met81, Asn83, Glu80 (2)
Erythratidine − 3.93 1320.00 Arg47 Asp36, Ala37 (2), Leu48, Val41, Leu40 − 6.16 30.37 – His77, Leu125, Met81 (2), Glu80
Donepezil* − 5.02 210.41 – Val91, Ser88 − 4.43 569.63 Asp87 Met16, Tyr61, Leu29, Leu18, Ala27, Thr86, Asp63
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HBI hydrogen bond interaction, IC50 50 % inhibitory constant (Along with binding energy, the IC50 value for each compound is predicted from AutoDock 4.2 software)

*Standard molecule for the treatment of AD

Fig. 3.

Fig. 3

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Intermolecular interactions of (1) Erysotine, (2) Erythraline with acetylcholinesterase (PDB ID: 4PQE) and (3) Erysodine with butyrylcholinesterase (PDB ID: 4XII). a 2D representation and b 3D representation

Molecular dynamics simulation studies

Erysotine and AChE complex

Erysotine and AChE complex MD system consisted of 55,159 atoms with 15,608 water molecules. The ligand RMSD in complex with protein value was observed within the range of 1.808 Å to 11.569 Å from 0 to 20 ns. The average RMSD of ligand w.r.t. protein was 7.24 Å and ligand w.r.t. ligand was 0.43 Å. The ligand RMSD from 2.5 to 8 ns and 16 to 20 ns was found to be stable and a fluctuation was observed from 8 to 16 ns. The C-α and backbone RMSD fluctuation was to be within 1.123–2.866 Å and the average RMSD was found to be 2.25 Å (Fig. 4_1A). The average RMSF value of atoms w.r.t. protein was 3.45 Å and ligand was 0.19 Å. The average RMSF difference of both protein-ligand atoms was 3.26 Å (Fig. 4_1B).

Fig. 4.

Fig. 4

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(1) Erysotine with AChE and (2) Erysodine with BChE stability at 20 ns MD production run. A RMSD and B RMSF, respectively

Further, Erysotine–AChE complex intermolecular interaction analysis concluded that Arg49 to form a stable hydrophobic bond for around 36 %, hydrogen bond for 3 %, and water bridge for 4 % of the interaction fraction. Asp36 formed a water bridge for around 26 % of the interaction fraction. Arg52 formed hydrogen bond and water-bridge for around 6 % of the interaction fraction. The compactness of the Erysotine–AChE complex over 20 ns was analyzed. The rGyr deviation was found within 3.15–3.25 Å, which indicates the higher compactness of Erysotine with AChE. Figure 5_1 illustrates the residue-wise ligand contacts, interaction fractions, and rGyr of Erysotine in complex with AChE.

Fig. 5.

Fig. 5

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(1) Erysotine with AChE and (2) Erysodine with BChE interactions at 20 ns MD production run. A, B, D Ligand–Protein contacts, C rGyr

Erysodine and BChE complex

Erysodine and BChE complex MD system consisted of 62,358 atoms with 18,007 water molecules. The ligand RMSD in complex with protein value was observed within the range of 0.656–8.75 Å from 0 to 20 ns. The average RMSD of ligand w.r.t. protein was 4.38 Å and ligand w.r.t. ligand was 0.59 Å. The ligand RMSD from 0 to 3.5 ns at the range of 1–2 Å was stable and suddenly increased to 5 Å at 3.5 ns. Again the ligand RMSD was stable from 3.6 ns to 10 ns within the RMSD range of 3–5 Å. Further, the ligand RMSD was found stable from 10 to 20 ns at the range of 4–8.75 Å. The C-α and backbone RMSD fluctuation was to be within 0.816–1.795Å and the average RMSD was found to be 1.53 Å (Fig. 4_2A). The average RMSF value of atoms w.r.t. protein was 2.18 Å and ligand was 0.19 Å. The average RMSF difference of both protein-ligand atoms was 1.98 Å (Fig. 4_2B).

Further, Erysodine–BChE complex intermolecular interaction analysis concluded that Lys427 to form a stable hydrogen bond for around 87 %, hydrophobic bond for 50 %, and water bridge for 5 % of the interaction fraction. His77 showed hydrophobic bond and water-bridge for around 41 and 8 % of the interaction fraction, respectively. The compactness of the Erysodine–BChE complex over 20 ns was analyzed. The rGyr deviation was found within 3.15–3.25 Å, which indicates the higher compactness of Erysodine with BChE. Figure 5_2 illustrates the residue-wise ligand contacts, interaction fractions, and rGyr of Erysotine in complex with BChE.

Discussion

Central cholinergic neuronal systems are the backbone for learning and memory (Zeynep et al. 2017) which impairment leads to the decline in memory storage or deficits in the retention of newly acquired information which is well demonstrated in Alzheimer’s disease (progressive cognitive impairment) (Zeynep et al. 2017; Khan et al. 2014; Whitehouse et al. 1981). The current pharmacotherapy of Alzheimer’s disease includes the drastic utilization of synthetic Acetylcholinesterase and Butyrylcholinesterase inhibitors, i.e. Donepezil, Rivastigmine, Memantine, Tacrine which are evident for causing a verity of adverse effect (Ali et al. 2015) and also lose the effect as the disease progress due to deteriorating brain cells and low level of acetylcholine (Mayo clinic 2018).

Herbal plants are using in day-to-day life for the treatment of many diseases and disorders. In the previous studies, many researchers suggested that plant alkaloids have a potential role in the treatment of Alzheimer’s disease (Hussain et al. 2018). Previous studies also suggested that the plant-derived alkaloids reduce the development of neurodegenerative diseases through their various mechanisms which include inhibition of Acetylcholinesterase, as NMDA antagonist, by increasing the level of GABA, by inhibiting Butyrylcholinesterase and many others (Dey and Mukherjee 2018). Alkaloids also exert neuroprotective activity in many diseases like AD, Parkinson’s disease, anxiety, depression (Hussain et al. 2018; Ng et al. 2015; Dey and Mukherjee 2018). Also, various investigations demonstrated that alkaloids as the potent inhibitor of AChE and BChE (Konrath et al. 2013). In this study, performed AChE and BChE enzyme inhibitory activity of different fractions of E. variegata crude bark extract. The chloroform fraction displayed the potent inhibitory activity of AChE and BChE and is evidenced by previous research data (Moraga-Nicolás et al. 2018).

In the present study, we identified six derivatives of indolo [7a,1-a] isoquinoline from the E. variegata classified under the category of alkaloids and predicted to be inhibitors of AChE and BChE. These derivatives are filtered moieties under the criteria of carcinogenicity and toxicity, identified from PreADMET and predicted from SwissTargetPrediction to inhibit AChE and BChE based on the blend of 2D and 3D likeness via the measurement with known ligands (Gfeller et al. 2014).

The docking study was carried for indolo [7a,1-a] isoquinoline derivatives using AutoDock 4.2 as explained by Patil et al. (2019) to assess the binding affinity and their conformational position with each target molecules. The docking study results revealed that Erysotine and Erythraline have the highest binding affinity with AChE (IC50 0.2703 mM and 0.2716 mM, respectively) and Erysodine with BChE (IC50 0.01318 mM) (Table 3). A previous study by Nassief et al. (2020) isolated, characterized, and demonstrated Erythrina alkaloid as a potent inhibitor of ACHE, in which an IC50 of Erythraline against ACHE was found to be 0.40 mM. Hence, the present study predicted IC50 of Erythraline, i.e. 0.2716 mM against ACHE corroborate reports reported by Nassief et al. In this study, a 20ns MD production runs demonstrated the intermolecular interaction stability of Erysotine with AChE and Erysodine with BChE. The previous literature reflects the inhibition of AChE and BChE are the targets in the management of AD (Ali et al. 2015); demonstrated by the various researchers via the utilization of alkaloids (Hussain et al. 2018; Nassief et al. 2020). In the current study, we demonstrated the alkaloids, derivatives of indolo[7a,1-a]isoquinoline as an inhibitor of AChE and BChE via the processer imitations.

The previous literature points the Donepezil as the potential inhibitor of AChE over BChE (Yoon et al. 2013; Shrivastava et al. 2017; Jin et al. 2014) which is also demonstrated in the current CPU mock-ups and was found to be comparable with test compounds. In our in vitro ACHE and BCHE inhibitory study, the IC50 of Donepezil was found to be 2.51 ± 0.079 and 21.40 ± 0.582 µg/ml against ACHE and BCHE by in vitro. The previous study by Reza et al. (2018) reported the IC50 of Donepezil 17.01 ± 0.33 µg/ml. Hence, compared to Reza et al. reports, 6.77 folds decrease in the concentration of Donepezil was observed in this study. Ogura H et al. (2000) performed comparative ACHE and BCHE inhibitory activity of Donepezil and other ChE inhibitors by in vitro and reported Donepezil IC50 of 6.7 nM (2.52 ng/ml); which is 1000 folds lower than current findings. Islam et al. (2019), Moniruzzaman et al. (2015), and Abdul Manap et al. (2019) reported Donepezil IC50 of 6.31 ± 0.09 µg/ml, 31.83 ± 0.49 µg/ml, and 54.79 ± 0.01 µg/ml against ACHE by in vitro, respectively. Further, in vitro study performed by Kalidindi and Krishnamurthy (2020) reported Donepezil IC50 of 0.0366 ± 0.0094 µg/ml. Similarly, Islam et al. (2019) and Moniruzzaman et al. (2015) reported an IC50 of 11.93 ± 0.13 and 16.54 ± 0.21 µg/ml against BCHE, respectively. On looking into the overall results, variation among the IC50 values could be due to a change in the purity of Donepezil, instrumental, environmental, and experimental factors such as enzyme unit, temperature, pH, etc. The current study reported that Erysotine, Erythraline, and a standard molecule Donepezil have a higher binding affinity with AChE whereas Erysodine scored the highest binding affinity with BChE amongst the predicted phytoconstituents. In the current study, Erysotine and Erythraline were identified as the potential inhibitors of AChE and Erysodine as an inhibitor of BChE which were the derivatives of indolo [7a,1-a] isoquinoline and alkaloids from the folk medicines. The study describes the concept of identification and utilization of a new drug molecule from the traditional medicinal system with lesser side effects (predicted to be non-mutagen and non-carcinogen) and higher efficacy. This would be helpful in the improvement of the current strategy in the pharmacotherapy of AD. Although the current strategy of study is only based on the CPU limitations, it provides the important concept for the utilization of indolo [7a,1-a] isoquinoline derivatives as a dual inhibition of AChE and BChE in the management of AD; which is to be further proved via wet-lab protocols.

Conclusion

In present research data, we reported indolo [7a,1-a] isoquinoline derivatives as potential candidates for the inhibitory effect on AChE and BChE. An attenuation of AChE and BChE enzymatic activity via natural compounds can maximize the successive AD management without increasing the risk factors correlated with adverse effects. However, the present study is preliminary and further in vivo studies are needed to be carryout using well-designed wet-lab protocols.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1983 KB) (1.9MB, docx)

Acknowledgements

The authors are thankful to the Apotex Research Pvt. Ltd, Bangalore for providing donepezil as a gift sample and thankful to KLE College of Pharmacy, KAHER, Belagavi, India, and ICMR-NITM, Belagavi, India for providing necessary facilities.

Author contributions

VSP contributed to the concept, design, resources, materials, data collection and/or processing, analysis and/or interpretation, literature search, and manuscript writing. HM contributed to the concept, design, in silico docking studies, and critical review. DRH guidance to VSP on MD simulation, provided laboratory facilities, and critically revised the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declarations

Conflict of interest

The authors declared no conflict of interest.

Footnotes

Publisher’s Note

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Contributor Information

Vishal S. Patil, Email: vishalpatil6377@gmail.com

Darasaguppe R. Harish, Email: harish.dr@icmr.gov.in

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