title: In vitro opioid receptor affinity and in vivo behavioral studies of Nelumbo nucifera flower.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  The white and pink flowers of N. nucifera were extracted with 95% EtOH, followed by acid-base partitioning using CHCl3 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 100mg/kg/ip doses that revealed the effect on locomotion, catalepsy, body temperature, and nociception of acidic and basic CHCl3 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 (Ki) 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 Ki 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 Ki value of 9.2 ± 1.1 μM. The acidic and basic CHCl3 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 100mg/kg.
  
  
  CONCLUSIONS
  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 CHCl3 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 CHCl3 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.
authors.affiliation: National Center for Natural Products Research, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Mallika
authors.initials: M
authors.lastname: Kumarihamy
authors.affiliation: Department of BioMolecular Sciences, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Francisco
authors.initials: F
authors.lastname: León
authors.affiliation: Department of BioMolecular Sciences, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Pettaway
authors.affiliation: Department of BioMolecular Sciences, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Wilson
authors.affiliation: Department of BioMolecular Sciences, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Janet A
authors.initials: JA
authors.lastname: Lambert
authors.affiliation: National Center for Natural Products Research, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Mei
authors.initials: M
authors.lastname: Wang
authors.affiliation: National Center for Natural Products Research, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hill
authors.affiliation: Department of BioMolecular Sciences, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: McCurdy
authors.affiliation: National Center for Natural Products Research, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA; Department of Pharmaceutics and Drug Delivery, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Mahmoud A
authors.initials: MA
authors.lastname: ElSohly
authors.affiliation: National Center for Natural Products Research, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA; Department of BioMolecular Sciences, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Cutler
authors.affiliation: National Center for Natural Products Research, School of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, MS 38677, USA. Electronic address: milias@olemiss.edu.
authors.forename: Ilias
authors.initials: I
authors.lastname: Muhammad
mesh_terms: D000701:Analgesics, Opioid / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D004305:Dose-Response Relationship, Drug; D035264:Flowers; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D031653:Nelumbo; D010147:Pain Measurement / Q000187:drug effects / Q000379:methods; D010936:Plant Extracts / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011957:Receptors, Opioid / Q000819:agonists / Q000378:metabolism*
chemical_list: D000701:Analgesics, Opioid; D010936:Plant Extracts; D011957:Receptors, Opioid
keywords: Alkaloids; Cannabinoid (CB1, CB2); Central nervous system; Centrifugal Chromatography; Nelumbo nucifera; Opioid receptors (δ, ĸ, µ).