Anticholinesterases activity of Murraya koenigii (L.) Spreng. and Murraya paniculata (L.) Jacq. essential oils with GC/MS analysis and molecular docking

Abstract GC/MS analysis of Murraya koenigii (L.) Spreng. and Murraya paniculata (L.) Jacq. leaves revealed the identification of 73 components, with an evident greater contribution of monoterpenes hydrocarbons to their total volatiles. α-Pinene (37.5%) and β-caryophyllene (27.4%) were the most abundant compounds in M. koenigii leaves and β-phellandrene (40.7%) in M. paniculata leaves, using headspace. β-Phellandrene (33.7%) was the major constituent by M. koenigii leaves where germacrene D (23.8%), and δ-elemene (22.0%) were predominant in M. paniculata leaves, using steam distillation. M. koenigii leaves oil showed quite remarkable cholinesterase inhibitory activity, where oil of M. paniculata leaves showed strong inhibitory activity against AChE (IC50=13.2 ± 0.9 µg/mL) and BChE (IC50=5.1 ± 0.3 µg/mL). Germacrene D, α-zingiberene, and δ-elemene showed higher affinity to BChE than AChE as revealed from docking scores (S = −5.65 to −6.03 Kcal/mol) for BChE and (S = −5.56 to −6.25 Kcal/mol) for AChE. Graphical Abstract


Introduction
Globally, more than fifty million people are diagnosed with dementia, with more than ten million new cases each year.two out of every three dementia patients are from countries with low or middle incomes.alzheimer's disease is the most common type of dementia, comprising about 60-70% of all cases (World Health organization 2022).the american Geriatrics Society's Beer's list considers donepezil as a high-risk medication in older adults due to increased rates of orthostatic hypotension and bradycardia among this population (Gust et al. 2020).as one of the major sources of drug discovery, natural products and isolated compounds have been widely investigated in efforts to develop more effective agents for the management of alzheimer's disease (Chen et al. 2021).
Murraya koenigii (L.) Spreng. is commonly known as curry-leaf in the family Rutaceae distributed in the tropical and sub-tropical countries.the leaves are rich sources of minerals, vitamins, carbohydrates, proteins, essential oil, terpenoids, and alkaloids (Rani et al. 2017).Murraya paniculata (L.) Jacq.(also known as Cholcas paniculata L., Chalcas exotica L., and Murraya exotica L.) is commonly known as Chinese box, kamini, orange jasmine, or mock orange as a tropical, evergreen plant with very small, white, and scented flowers.polymethoxylated flavonoids and their glycosides, coumarins, indole alkaloids, and essential oils were reported in the plant (Sonter et al. 2021).Essential oils of Rutaceae family are well-known for their potential bioactivities, e.g.anti-inflammatory, cytotoxic, antimicrobial and antioxidant (nahar et al. 2021).
in our study, we investigated the essential oils from the studied plants for their in-vitro cholinesterase inhibitory potential for the first time to support the market with new safe drugs for the treatment of alzheimer's disease.the chemical profile of essential oils of Murraya koenigii (L.) Spreng.from Egypt was studied for the first time in our study.

Dynamic headspace method
it resulted in the detection of different volatile profiles than that of the hydro-distilled oils with different metabolites and different relative percentages (mohammadhosseini et al. 2017).Higher proportions (better recovery) of α-pinene and β-caryophyllene in M. koenigii leaves in the headspace analysis than hydro-distillation (table S1) were recorded.the leaves of M. paniculata headspace volatiles showed characteristic profiles of monoterpenes comparable to the hydro-distilled profiles, especially with respect to their richness in α-thujene, α-pinene, and β-phellandrene, these metabolites not detected at all in hydro-distilled volatile oil (table S1).
the composition of the essential oils of M. koenigii leaves revealed that α-pinene, β-pinene, β-phellandrene, β-caryophyllene and α-selinene were the major constituents, where the chemical diversity of the oils from different locations were reported (Rao et al. 2011).Furthermore, the composition of the hydro-distilled essential oils of M. koenigii leaves in our study showed great similarity with that reported by Rao and coworkers.Where, the high percent of β-caryophyllene in headspace method was in agreement with Sukkaew et al. (2014) (Sukkaew et al. 2014).it is worth to highlight that it is the first time to investigate the Curry leaves from Egypt. the major chemical components of the hydro-distilled M. paniculata leaves were germacrene d, δ-elemene, and β-caryophyllene, where these components represented the typical strong odor components of M. paniculata oil. the findings of the current study in Egypt agreed with those of a previous study on the chemical composition of essential oil of M. paniculata leaves (Rodríguez et al. 2012, Zaatout et al. 2022).moreover, monoterpenes hydrocarbons are the major in hydrodistilled M. kongeii oil (78.2%), but sesquiterpene hydrocarbons are the major in hydro-distilled M. paniculata oil (77.6%). in headspace, M. kongeii sample showed that monoterpenes and sesquiterpenes hydrocarbons were the predominant (46.1, and 42.1%, respectively), and the remaining constituents were categorized as sesquiterpenes hydrocarbons (10%).Furthermore, monoterpenes hydrocarbons dominated the M. paniculata leaves (88.8%).

Cholinesterase inhibitory activity
there has been much research on the psychological impacts of aromatic herbs, essential oils, and hydroalcoholic extracts.natural products have promising anti-alzheimer activity, among these are the plants dominated monoterpenes (Wojtunik-Kulesza et al. 2021).aChE/BChE inhibitors are most desirable because they improve cognition with minimal side effects.oil of M. paniculata leaves was the most potent selective BChE inhibitor with an iC 50 of 5.1 ± 0.3 µg/mL and BChE selectivity index (Si, iC 50 of aChE/iC 50 of BChE) of 2.6 (table S2).

Molecular docking study
to rationalize the biological evaluation results, molecular docking studies were carried out using molecular operating Environment (moE, 2019.0102)software.docking of the major constituents of both oils; germacrene d, α-pinene, α-zingiberene, β-caryophyllene, β-phellandrene, and δ-elemene (their percentage approximately equal or more than 10%), within the active site of aChE and BChE was carried out to explore their possible binding modes.the co-crystal structures of recombinant human aChE (rhaChE) in complex with donepezil (pdB:4EYZ) (Cheung et al. 2012) and human BChE (hBChE) in complex with tacrine (pdB:4dBS) (nachon et al. 2013) were downloaded from the protein data Bank.the aChE and BChE active sites consist mainly of three subsites: a peripheral anionic site (paS), a mid-aromatic gorge and a catalytic active site (CaS).the docking scores and binding interactions of germacrene d, α-zingiberene, and δ-elemene, which exhibited good affinity and interactions in comparison to the corresponding ligand, were presented in tables (S3-S5).
investigation of the docking results revealed that donepezil interacts with the active site of aChE through π-π interaction and π-H interaction with the amino acids trp86, trp286, and tyr341.moreover, it forms H-bond with phe295 at paS and H 2 o-mediated H-bond with asp74 and tyr337 (Figure S1). the docked compounds interacted with aChE active site with reasonable binding affinity (S = −5.56 to −6.25 Kcal/mol), in addition to their ability to form π-H interaction with the amino acids trp86, trp286, tyr337, phe338 and tyr341.moreover, all of them were incorporated into H-bonding with at least one amino acid of the active site (Figure S2, table S4).one the other hand, tacrine binds to the active site of BChE with binding score of −6.72 Kcal/mol through π-π interaction with trp82 amino acid. in addition, it forms H-bond with His438 and H 2 o-mediated H-bond with asp70, Ser79, and thr120 (Figure S3). the docked compounds showed almost the same binding score range as that for aChE (S = −5.65 to −6.03 Kcal/mol), but they showed higher affinity to BChE than aChE in terms of docking scores and that confirms the biological evaluation results.also, all compounds can form π-H interaction with different amino acids such as trp82, tyr332, phe392, trp430, and His438.moreover, they were able to form at least 3 H-bonds with the active site amino acids and this may explain the enhanced affinity compared to aChE (Figure S4).

Experimental
See supplementary file.

Conclusion
our research provides promising evidence for the efficacy of Murraya species aromatherapy in the treatment of dementia as aChE and BChE inhibitors.Furthermore, using multiple methods (headspace and steam distillation) revealed a significant chemical diversity.inhibitory studies using isolated bioactive components from those potent essential oils could provide critical information for acceptable pharmacological outcomes.the molecular docking study highlighted the potent activity of germacrene d, α-zingiberene, and δ-elemene with high binding scores in comparison to the native ligands' donepezil and tacrine.a more basic study is required to realize the pharmacological mechanisms underlying the studied essential oils.to facilitate clinical evaluations and recommendations, we propose additional investigations, including in vivo and clinical trials with determination of the therapeutic index and quantal dose correlations.

Disclosure statement
no potential conflict of interest was reported by the authors.

Funding
the author(s) reported there is no funding associated with the work featured in this article.