ISOLATION AND CHARACTERISATION OF ACETYLCHOLINESTERASE AND PROLYL ENDOPEPTIDASE INHIBITORS FROM SELECTED MEDICINAL PLANTS

Abstract

Acetylcholinesterase (AChE) and Prolyl endopeptidases (PEP) are involved in the catalytic hydrolysis of acetylcholine and cleavage of neuropeptides influencing the activity of the brain, leading to some symptoms of Alzheimer’s disease (AD).Inhibition of AChE and PEP is considered a promising strategy for AD management.There are no effective drugs for the management of AD which necessitated the search for new potent neurotherapeutic agents.This study, was therefore, aimed at isolating and identifying inhibitors of AChE and PEP from selected medicinal plants identified from Nigerian ethnomedicine. Ten plants selected were macerated in methanol and their extracts evaluated for AChE inhibitory activity using Ellman colorimetric in vitro assay. The most active plant extracts: Phyllanthus muellerianus (PM) leaves (FHI 111339), Tinospora cordifolia(TC) stem (FHI 112287) and Cola hispida(CH) seed (FHI 111321) were successively partitioned into n-hexane, dichloromethane, ethyl acetate and aqueous fractions. The fractionswere assessed for AChE inhibitory activity using eserine as standard. In vitro analyses were used to evaluate the antioxidant potential of the plant extracts. Active fractions were subjected to chromatographic separations to isolate and purify bioactive compounds. Structures of isolated compounds were determined by spectroscopic analyses. Compounds were screened for AChE and PEP inhibition using in vitro colorimetric assays and metal chelating potential, respectively. Bacitracin was used as standard for PEP. Molecular docking was done using software (MOE 2015.010) to anticipate the binding affinity between drug candidates with protein targetsusing PDB ID: 3IVM (Prolyl endopeptidase) and 10CE (Acetylcholinesterase). Standard curves were generated and calculation of IC50 values was done using Microsoft Excel. Data were analysed using One way ANOVA followed by Dunnet’s Multiple Comparison testat α0.05

Methanol extracts of PM (IC50 of 1.29±0.70 mg/mL) and CH (IC50 of0.87±0.40 mg/mL) gave promisingAChE inhibitory activity at 5 mg/mL. Ethyl acetate fraction of PM (0.74±0.12 mg/mL) and CH(0.66±0.24 mg/mL) gave the highestAChE inhibitory activity compared to eserine. The dichloromethane fraction of TC exhibited good metal chelating activity (IC50 of 0.20±0.08 mg/mL). Beta-sitosterol, daucosterol, 1-octacosanol, rel-(2s,3s,4r,16e)-2-[(2'r)-2'-hydroxynonadecanoylamino]-heneicosadec-16-ene-1,3,4-triol, four clerodane diterpenoids and five alkaloids were isolated from TC.Two steroids, 5-hydroxymethylfurfural, and 2-hydroxyquinoline-4-carboxylic acidwere isolated from CH. Stigmasterol and β-sitosterol glucoside were isolated from PM. Oxoglaucine (Oxoaporphinoid alkaloid) from TC exhibited the highest AChE inhibitory activity (IC50 of 0.80±0.09 mg/mL) compared to eserine (IC50=0.53±0.34 mg/mL) at 1 mg/mL, while stigmasterol from PM demonstrated the highest PEP inhibitory activity (IC50= 0.77±2.9 mM) compared to bacitracin (IC50= 0.13±1.5 Mm) at 1 mM.Oxoglaucine also gave the highest metal chelating potential (IC50= 0.22±0.00 mg/mL) compared to EDTA (IC50 of 0.05±0.11 mg/mL). Molecular docking revealed hydrophobic, hydrogen bonding and π -stacking interactions among tested compounds and the active site of AChE andPEPresidue, which indicates their ability to mitigate the symptoms associated with AD.

Oxoglaucine and stigmasterol isolated from Tinospora cordifoliaand Phyllanthus muellerianus, respectivelyshowed high acetylcholinesterase and prolyl endopeptidase inhibitory activities. The two biomolecules could serve as potential leads for novel drug development for the management of Alzheimer’s disease.