Estimation of Scopoletin from Roots of Argyreia nervosa (Burm. fill.) Bojer Using a Validated HPLC-Fluorescence Method Optimized Using the Design of Experiment Approach.

Abstract

Argyreia nervosa (Burm. Fil.) Bojer., a woody climber, is indicated in Ayurveda to treat debilities of the male reproductive system, diseases of the nervous system, and chronic ulcers. A sensitive analytical method was developed to estimate bioactive scopoletin from methanolic extract prepared from the medicinally active dried roots of Argyreia nervosa (Burm. fil.) Bojer using HPLC equipped with a fluorescence detector. Chromatographic separation was achieved using a LunaTM (C18, 250 × 4.6 mm, id: 5 μm) column using an isocratic mobile phase comprising phosphate buffer (pH 3.5)-acetonitrile (80 + 20, by volume) at a flow rate of 1.0 mL/min. The excitation wavelength was 345 nm, and the emission wavelength was 444 nm. The chromatographic parameters were optimized using the design of the experiment approach after determining the combined effects of selected independent variables on area, retention time, and tailing factor (TF) for the peak corresponding to scopoletin, and the experimental design was validated by navigating through the design space. The developed method was found linear in the range 10-140 ng/mL. The results of the studies confirmed the accuracy, precision, and robustness of the developed analytical method. The plant material was found to contain 0.0125 ± 0.0001% w/w scopoletin on a dried weight basis when estimated using the developed method. The method was developed using the HPLC-fluoresence detection by adopting the design of experiment approach and simple sample preparation for the estimation of scopoletin from roots of A. nervosa. This extremely sensitive analytical method with one-step sample preparation has the potential to be adapted for routine QC procedures.