A cularine-type isoquinoline alkaloid from the root part of Cissampelos pareira

Abstract One cularine-type alkaloid (1) was isolated along with two known compounds (2 and 3) from the roots of Cissampelos pareira. The isolated compound’s chemical structures were elucidated by 1 D and 2 D NMR spectroscopy and mass spectrometry analysis. This is the first report on the cularine-type alkaloid from the genus Cissampelos. Compound (2) was reported for the first time from C. pareira. In silico molecular docking analysis showed various interactions of compounds with the active site residue of GABAA receptor, which could help to understand their putative mode of action in neurodevelopmental malfunctions and in anaesthesia. Graphical Abstract


Introduction
The genus Cissampelos (Menispermaceae) comprises about $37 species of climbing shrubs distributed in subtropical parts of America, Asia, and East Africa (Mukerji and Bhandari 1959;Singh et al. 2010).Some of these species are used in different traditional systems of medicines as a remedy for various diseases (Mukerji and Bhandari 1959;Khare 2007;Singh et al. 2010), while some are also used as curare for arrows poison (Quattrocchi 1912).The rhizomes of C. mucronata and C. owariensis are utilized to prepare arrow poison in Nigeria (Semwal et al. 2014).The roots and leaves of C. mucronata are used for menstrual problems, gastrointestinal disorders, treating malaria, and venereal diseases in Tanzania and Nigeria (Tor-anyiin et al. 2003;Nondo et al. 2011).C. glaberrima and C. ovalifolia are used to manage cough, convulsions, epilepsy, delirium, madness, febrifuge, as analgesic, and are also used as a tonic and narcotics (Giorgetti et al. 2011).The C. capensis roots extract is incorporated in obesity while the roots are utilized as a tincture, purgative, and emetic (Van Wyk and Gericke 2000;Van Wyk 2008;Afolayan and Mbaebie 2010).Only C. pareira is found in India and is wellknown for its wide use in the treatment of various ailments in Indian Traditional system of medicine (Mukerji and Bhandari 1959).This plant is used for various therapeutic purposes, such as for the treatment of indigestion, abdominal pain, pruritus, wound healing, inflammation, piles, malaria, skin rashes, fever, and breast milk secretion disorders (Amresh et al. 2007;Khare 2007;Sudhakaran 2012;Singh et al. 2018).A literature survey revealed that C. pareira possesses isoquinoline alkaloids belonging to different classes: bisbenzylisoquinoline, benzylisoquinolines, aporphines, protoberberines, and tropoloisoquinolines, etc. (Kumari et al. 2021).Alkaloids belonging to any of these classes possess diverse pharmacological activities.Hence, the current work was aimed to explore the chemical diversity of isoquinoline alkaloids, which resulted in the isolation of cularine-type alkaloid from the roots of C. pareira.However, there are few reports on cularine-type alkaloids from plants and on their physiological effects, which mainly include anaesthetic effects, their effects on smooth muscles, and blood pressure (Castedo and Suau 1986).In the current study, virtual docking analysis was also carried out with GABA A (gamma-aminobutyric acid) receptor as it is one of the targets of the anaesthetic drugs.

Molecular docking
The binding affinity for reference ligands propofol, amobarbital, and benzodiazepine are À5.79,À5.52, and À4.81, respectively.The comparison of docking scores of the standard ligands and the isolated compounds revealed that compounds 2 and 3 have higher binding affinity with dock scores of À7.07 (Glide binding energy: À44.26 kcal/ mol; Supporting information Figure S12, Table S2) and À6.81 (Glide binding energy: À27.33 kcal/mol; Supporting information Figure S13, Table S2), respectively, as compared to standard ligands.The docking score of compound 1 was found to be À5.57(Glide binding energy: À33.34 kcal/mol; Supporting information Figure S11, Table S2), which was higher than amobarbital (dock score À5.52) and benzodiazepine (dock score À4.81) but lesser than the propofol (docking score À5.79).This docking analysis revealed that compound 2 showed H-bond interactions with Gln:229, Asn:265, and pipi stacking interaction with Phe:289 (Supporting information Figure S12); compound 3 showed three H-bond interactions with Ile:228 and Asn:265 (supporting information Figure S13, Table S2).Interactions observed for compound 1 (Supporting information Figure S11) include only one pi-cation interaction between the nitrogen of alkaloid and phenyl ring of Phe:289.From docking analysis it could be concluded that the hydrogen bonding and pi-pi stacking interactions act synergistically in compound 2 for effective binding with the receptor.The possible outcome from this study is that further functionalization and derivatization of compound 1 may improve its binding affinity with GABA A receptor.However, the score for all isolated compounds is either better or comparable to the standard molecules indicating their potential as anaesthetic agents.

General
Optical rotations of compounds were measured on Anton Parr MCP 100 Modular Circular Polarimeter.The melting points of all isolated compounds were measured on a Brønsted Electrothermal 9100.Mass spectra were recorded on Water Q-ToF-Micro Micromass and high-resolution 6560 Ion Mobility Q-TOF LC/MS (Agilent, Santa Clara, USA).NMR spectra were obtained on a Bruker-Avance 600 MHz instrument. 1 H and 13 C NMR of all pure compounds can be found in the supporting information.IR was analysed by Shimadzu IR Prestige-21 with ZnSe single reflection ATR accessory.UV-Vis spectra were recorded on Shimadzu UV-Vis spectrometer-2600.

Collection and authentication of plant material
The roots of C. pareira were collected in January 2019 from Palampur (32.12 N 76.53 E), Himachal Pradesh, India.Taxonomy experts did the authentication of plant material in CSIR-IHBT, Palampur.The sample was deposited to the herbarium, CSIR-IHBT, Palampur, HP-176061, India, with specimen voucher number PLP 16688.

Molecular docking
Molecular docking could give a basic idea of the potency of compounds for particular diseases by providing insights into the binding patterns of compounds with targeted proteins.Based on literature reports (Castedo and Suau 1986), the anaesthetic effect of the isolated cularine isoquinoline alkaloid (1) along with other isolated compounds (2 and 3) was studied using in silico tools.The in silico docking was performed with GABA A receptor (PDB ID: 6X3X) to find the binding sites of the compound to the receptor.Amobarbital, benzodiazepine, and propofol were taken as reference ligands.Some anaesthetics act by enhancing the GABA transmission via the GABA A receptor, which is a ligand-ion gated channel and a popular candidate for the anaesthetic action (Pleuvry 2008).The 2 D structures of compounds were made with ChemDraw 20.0 and were converted and minimized into 3 D by LigPrep Schr€ odinger 2022 (Schr€ odinger release 2022-1).The 3 D template of the human GABA A receptor alpha1-beta2-gamma2 subtype (PBD ID: 6X3X) crystallized with GABA and diazepam was retrieved from RCSB-Protein Data Bank (Kim et al. 2020).The selected protein template was minimized and optimized by the protein preparation wizard in Schr€ odinger.Then receptor grid was generated at the diazepam binding site of GABA A receptor.After that, the minimized structures of isolated compounds and reference compounds were subjected to molecular docking analysis using Glide.The extra-precision (XP) with flexible model was applied to predict the binding affinity of compounds with selected protein.

Conclusion
The phytochemical investigation of chloroform fraction of C. pareira roots led to the isolation of cularine alkaloid (1) while p-hydroxyphenethyl trans-ferulate (2) and quercitol (3) were isolated from n-butanol fraction.This is the first report on the occurrence of cularine-type alkaloid in the genus Cissampelos whereas compound 2 was isolated for the first time from C. pareira.Docking results might help further to analyze these compounds in vitro or in vivo and could also help in understanding their cellular and systemic mode of action.

Figure 1 .
Figure 1.Chemical structures of compounds isolated from C. pareira.