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Ethnopharmacological relevance Nelumbo nucifera Geartn., known as sacred lotus, has been used traditionally in South East Asia as a traditional medicine for various CNS disorders including stress, fever, depression, insomnia, and cognitive conditions. Aim of the study To investigate the in vitro cannabinoid and opioid receptor binding affinities, and in vivo behavioral actions of Nelumbo flower extracts and to isolate the potential compounds to treat CNS associated disorders. Materials and methods The white and pink flowers of N. nucifera were extracted with 95% EtOH, followed by acid–base partitioning using CHCl 3 to give acidic and basic partitions. These partitions were subjected to Centrifugal Preparative TLC (CPTLC) to yield benzyltetrahydroisoquinoline (BTIQ) alkaloids and long chain fatty acids, identified by physical and spectroscopic methods. In addition, EtOH extracts and partitions were analyzed for chemical markers by UHPLC/MS and GC/MS. In vitro neuropharmacological effects were evaluated by cannabinoid (CB1 and CB2) and opioid [delta ( δ ), kappa ( ĸ ), and mu ( µ )] competitive radioligand binding and GTPγS functional assays. The in vivo behavioral effect was studied through the use of the mouse tetrad assay at 10, 30, 75 and 100 mg/kg/ip doses that revealed the effect on locomotion, catalepsy, body temperature, and nociception of acidic and basic CHCl 3 partitions, fractions, and compounds. Results Three aporphines, nuciferine ( 1 ), N- nor -nuciferine ( 2 ), asimilobine ( 3 ), and five BTIQs, armepavine ( 4 ), O-methylcoclaurine ( 5 ), N-methylcoclaurine ( 6 ), coclaurine ( 7 ), neferine ( 10 ), and a mixture of linoleic and palmitic acids (LA and PA), were identified and evaluated for cannabinoid and opioid receptor displacement activities. Compounds 5 – 7 showed binding affinities for the ĸ opioid receptor with equilibrium dissociation constant ( K i ) values of 3.5±0.3, 0.9±0.1, 2.2±0.2 μM, respectively. Compound 10 displayed affinities for δ -and µ - opioid receptors with K i values of 0.7±0.1 and 1.8±0.2 μM, respectively, and was determined to be a weak δ agonist by GTPγS functional assay. The mixture of LA and PA (1:1) showed an affinity for δ opioid receptor with a K i value of 9.2±1.1 μM. The acidic and basic CHCl 3 partitions, compounds 1 and 7, and 5 – 7 mixture were subjected to the tetrad assay, of which the acidic partition displayed decreased locomotion and increased catalepsy, antinociception, and hypothermia in animal at doses of 75–100 mg/kg/ip, and also showed clonic-tonic seizures upon touch at 100 mg/kg. Conclusion Bioassay-guided isolation revealed compounds 5 – 7 , 10 , and the mixture of LA and PA displayed various degrees of opioid receptor radioligand displacement affinities. The in vivo tetrad assay of acidic CHCl 3 partition, enriched with aporphines 1 and 2 , displayed actions on all four points of behavioral parameters. It can be concluded that the in vivo mild canabimimetic-type effect observed for the CHCl 3 partition is likely mediated through other CNS mechanisms since the extracts, partitions, and isolated compounds had no affinity for the in vitro CB1 and CB2 receptors. This work, along with traditional use and the reported bioactivities of the BTIQ alkaloids, suggested further studies on N. nucifera are needed to understand the roles that the extracts and/or individual compounds might contribute to the behavioral effects. [ABSTRACT FROM AUTHOR]