N-Methylneolitsine as a new and potent acetylcholinesterase inhibitor of Cissampelos pareira Linn. aerial parts: bioassay-guided isolation and quantitative densitometric analysis

Abstract The rising geriatric population is expected to increase the demand for drugs treating neurodegenerative diseases. The present work is aimed to discover acetylcholinesterase (AChE) inhibitors from Cissampelos pareira Linn. aerial parts (Family: Menispermaceae). Bioassay-guided isolation, AChE inhibition study and estimation of the therapeutic marker in different parts of raw herbs were conducted. The structure of the compound (1) was elucidated as N-methylneolitsine by using NMR (1D and 2D) and ESI-MS/MS spectral data, which is a new natural analogue of neolitsine. It showed good AChE inhibition with an IC50 value of 12.32 µg/mL. It was densitometrically estimated to be 0.074 − 0.33% in aerial parts of C. pareira, collected from various locations. The alkaloid reported here could be potentially useful for the treatment of various neurodegenerative diseases and the aerial part of C. pareira could be used as a promising ingredient for various preparations treating neurodegenerative diseases. Graphical Abstract


Introduction
alzheimer's disease is one of the major neurodegenerative diseases, characterized by progressive deterioration of memory, learning and other cognitive functions.one of the major causes of the disease is the extracellular accumulation of stable complexes of β-amyloid peptide and acetylcholinesterase (aChE) in the brain (Konrath et al. 2013;Hussain et al. 2018).
Cissampelos pareira Linn.(Family: Menispermaceae), commonly referred to as ambastha or Laghu patha, is naturally distributed throughout tropical and subtropical parts of india.the plant has long been used in various traditional therapies like ulcers, rheumatism, fever, asthma, cholera, diarrhoea, rabies, malaria, treatment of poisonous bites, worm infestation and neuroprotective activities (mukerji and Bhandari 1959; Bafna and mishra 2010; Kumari et al. 2021).Hydro-alcoholic extract of C. pareira significantly improved the memory and learning of mice and considerably reversed the scopolamine-induced amnesia at 400 mg/kg.the extract also decreased the whole brain acetylcholinesterase activity when compared to piracetam (200 mg/kg) (Kulkarni et al. 2011).a combined extract of C. pareira and Anethum graveolens was reported to use as a food supplement for protection against mild cognitive impairment in the early phase of alzheimer's disease (thukham-mee and Wattanathorn 2012).a literature survey revealed that alkaloids and flavonoids are the major constituents of C. pareira.among them, laudanosine, corytuberine, bulbocarpine, nor-n-magnoflorine, amentoflavone and cissampeloflavone have been isolated from the stem and leaves parts of C. pareira (Kumari et al. 2021).
in the present study, we reported the assay-guided isolation and characterization of the new aChE inhibitor.the compound (Figure 1) was estimated in the aerial and root parts of C. pareira collected from different locations by High-performance thin Layer chromatography (HptLC).the analytical approach by HptLC was used to determine the active components in the plant species for their chemotaxonomic importance (toniolo et al. 2014).

Results and discussion
the methanol extract of C. pareira aerial part showed aChE inhibition of 56.23% at 100 µg/mL (iC 50 89.89µg/mL) by colourimetric method (Ellman et al. 1961).it was partitioned between n-butanol and water.the freeze-dried water fraction showed the highest inhibition at 100 µg/mL with an iC 50 value of 72.95 µg/mL (Figure S2). it showed one major alkaloid spot on tLC and was purified by column chromatography and crystallization.
the molecular formula of the alkaloid, [C 20 H 20 no 4 ] + was deduced from 13 C nmR and the molecular ion [m] + appeared at m/z 338.1395 (calculated m/z 338.1392) in its HR-ESimS spectra, indicating 11.5° of unsaturation.the uV spectrum showed absorbance maxima at 232, 285 and 314 nm related to an aporphine moiety (Guo et al. 2011).the 1 H and 13 C nmR spectra revealed that the compound has three uncoupled aromatic ring protons and two distinct methyl groups attached to a nitrogen atom (table S1). the deshielded methyl groups indicate the presence of a positive charge on the nitrogen atom (Bala et al. 2019) S1). the presence of Sp 3 methine carbon at δ 67.50 is characteristic of a boldine subclass of aporphine alkaloid (Guo et al. 2011).
to confirm the neolitsine moiety, the HSQC spectrum showed the entire expected proton-carbon one-bond connectivity (table S1). the C-6 methine proton at δ 4.77 (d) showed vicinal coupling in the CoSY spectrum with the methylene group at δ 2.91 (m) and 3.41 (m), as well as an HmBC correlation with the sp 3 carbon at δ 27.95 (Figure S3).all the methylene protons showed geminal coupling in the CoSY spectrum.the key HmBC correlations of the two-methyl protons at δ 2.98 (s) and δ 3.35 (s) with the methylene group at δ 60.52 and methine group at δ 67.50 confirmed the presence of a quaternary nitrogen atom in the cyclic ring system.the C-11 proton at δ 7.53 showed HmBC correlation with the quaternary Sp 2 carbons at δ 146.8, δ 126.37 and δ 115.85.Similarly, the protons of C-3 and C-8 carbons showed two and three-bond connectivity in HmBC spectra (Figure S3).
to ascertain the compound (1) to be n-methylneolitsine, it was subjected to ESi-mS n analysis.the molecular ion peak at m/z 338 and consistent mass fragments were observed in positive mode.the prominent peak at m/z 293 might arise with the loss of (CH 3 ) 2 nH moiety.the peak at m/z 263 was observed with the loss of the formyl group from the m/z 293 fragment (Figure S4).aChE inhibitory activity of the n-methylneolitsine showed higher inhibition of 74.83% at 20 µg/mL with iC 50 of 12.32 µg/mL as compared to the initial methanol extract and fractions (table S2 and Figure S2).
an HptLC method was developed with the solvent system n-butanol: acetic acid: water (18: 06: 06, v/v/v) on tLC silica plates to estimate n-methylneolitsine in the aerial and root parts of C. pareira from various locations in india.the compound (1) showed a well-separated spot on the tLC plate with an R f value of 0.39 (Figure S5) in the aerial part.the developed method was validated for linearity, Lod, LoQ, specificity, accuracy and recovery studies (Haldar et al. 2018;mohapatra et al. 2020).the aerial parts of C.pareira L. were found to contain about 0.33 − 0.07% (w/w, on a dried basis) of n-methylneolitsine.the compound was present in a maximum amount (0.33%, w/w) of the sample collected from uttarakhand, india.

Experimental
please see the supplementary material.

Conclusion
n-methylneolitsine has a potent aChE inhibitory activity and is being reported for the first time from a natural source.this finding adds to the list of compounds, such as eserine, tacrine, donepezil, rivastigmine and galanthamine that are already known for their aChE inhibitory activity (Benzi and moretti 1997;Sung et al. 2002).the aerial parts of C. pareira Linn.could be used as a promising ingredient for various herbal preparations treating various neurodegenerative diseases.Seconeolitsine and methyl seconeolitsine were reported to be semi-synthesized from boldine and used as therapeutic agents against microbial infections resistant to other antibiotics (tirado-Vélez et al. 2021).Hence, n-methylneolitsine might have potential antimicrobial properties and it would be studied for anti-bacterial and anti-viral activities in future.