title: A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study.
abstract: BACKGROUND
  The leaf hydroalcoholic extract of Cissampelos sympodialis Eichl. (Menispermaceae) has shown promising activity in different animal models of asthma. Several alkaloids have been identified in the extract, including warifteine and methylwarifteine (bisbenzylisoquinoline), as well as milonine (morphinandienone).
  
  
  OBJECTIVE
  To develop and validate an analytical method for the simultaneous quantitation of the bioactive markers of C. sympodialis hydroalcoholic leaf extract and to apply the method to a seasonal (phenological) study of the concentration of the alkaloid markers.
  
  
  METHODS
  The method used reversed phase high performance liquid chromatography with UV detection and calibration by standard addition. Separation was achieved using a C₁₈-column (250 × 4.6 mm, 5 µm) and a mobile phase consisting of a mixture of 0.05% aqueous (Et)₃NH₂ (A):MeOH(B) in gradient mode at a flow rate of 1.0 mL/min.
  
  
  RESULTS
  The method proved to be linear in the concentration range tested (2-100 µg/mL, r² > 0,99), precise (RSD ≤ 15%), accurate (85-115%), selective and robust. Detection limits for warifteine, methyl-warifteine and milonine were 0.39, 1.10 and 1.77 µg/mL respectively. The highest concentration of total alkaloids (determined as the sum of the three alkaloids) in the hydroalcoholic extract of the leaves was 2.9 ± 0.2 mg/g extract (n = 3), prior to fruit development. Both warifteine and methylwarifteine were detected in the total alkaloid fraction of the ripened fruits.
  
  
  CONCLUSIONS
  The results demonstrated that significant variations in the concentration of the biomarkers occurred throughout the vegetative cycle. The lowest concentration of the alkaloids in the leaves coincided with their appearance in the ripened fruits.
authors.affiliation: Centro de Biotecnologia, Universidade Federal da Paraíba, Caixa Postal 5009, 58051970 João Pessoa-PB, Brazil.
authors.forename: A F
authors.initials: AF
authors.lastname: Marinho
authors.forename: J M
authors.initials: JM
authors.lastname: Barbosa-Filho
authors.forename: E J
authors.initials: EJ
authors.lastname: Oliveira
mesh_terms: D000470:Alkaloids / Q000302:isolation & purification*; D044182:Benzylisoquinolines / Q000737:chemistry / Q000302:isolation & purification; D015415:Biomarkers / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D031606:Cissampelos / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry; D005638:Fruit / Q000737:chemistry; D016014:Linear Models; D009019:Morphinans / Q000737:chemistry / Q000302:isolation & purification; D010936:Plant Extracts / Q000737:chemistry*; D018515:Plant Leaves / Q000737:chemistry*; D012621:Seasons; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
chemical_list: D000470:Alkaloids; D044182:Benzylisoquinolines; D015415:Biomarkers; D009019:Morphinans; D010936:Plant Extracts; C557713:milonine; C003931:warifteine; D000431:Ethanol