Validated Method for the Simultaneous Quantitation of Bioactive Alkaloid Markers in the Leaf Ethanolic Extract of Cissampelos sympodialis Eichl.: a Phenological Variation Study

INTRODUCTION: 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. METHODOLOGY: 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. CONCLUSION: 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.