Simultaneous spectrofluorimetric determination of scopoletin and ...

Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of TraditionalMedicines, 2008, 3 ( 6 )OH which shows one exchangeable proton with D 2O.After taking into consideration the melting point andthe analytical data obtained from UV, IR and NMRanalysis, the isolated compound was considered to bescopoletin.Preliminary analysisA preliminary analysis was carried out todetermine the wavelength at which maximum intensityis exhibited by pure scopoletin and mangiferin. Forthis purpose, 100 µg/ml samples of pure scopoletinand mangiferin were prepared in methanol. Theywere scanned spectrofluorimetrically to obtain theexcitation and emission wavelengths. The λ maxshown by scopoletin had an excitation at 430 nmand an emission at 460 nm while the λ maxshownby mangiferin had an excitation at 248 nm and anemission at 520 nm.Preliminary spectrofluorimetric screening of themethanolic extract for testing the wavelengthrangeThe methanolic extract of CD was scanned in thespectrofluorimeter in concentrations of 0.1 µg/ml and0.01 µg/ml. The scanning was performed at excitationand emission wavelengths of 200 nm to 700 nm andabsorption maxima was found at 315 nm and 435 nm.This wavelength corresponded to λ maxof mangiferinand scopoletin, respectively. No other absorptionpeaks were detected in the range screened. Scanningbetween 248 nm and 520 nm did not exhibit any otherpeak, thus the range selected for analysis was expectedto screen only mangiferin and scopoletin in the testsample.Preparation of standard curveStandard curves of scopoletin and mangiferinwere prepared in methanol. First of all, stock solutioncontaining 100 µg/ml of scopoletin and mangiferinwas prepared in methanol. Then, this stock solutionwas used to prepare required dilutions containing5, 10, 15, 20, 25 and 30 µg/ml of scopoletin andmangiferin. The volume of stock solution used was0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 ml, respectively. Thesamples were analyzed in the spectrofluorimeteragainst solvent blank (methanol). The wavelength andintensity of each sample was recorded and a standardcurve was prepared for concentration versus theintensity of fluorescence.Determination of scopoletin and mangiferinconcentration in methanolic extract10 mg of the methanolic extract was weighedaccurately and dissolved in 10 ml of methanol withvigorous shaking. It was then filtered and the volumemade up to 100 ml with methanol. This solution wasanalyzed in the spectrofluorimeter and intensity offluorescence was recorded. The concentration of bothscopoletin and mangiferin in the extract samples wasdetermined from their standard curves.Spectrofluorimetric method development forsimultaneous estimation of scopoletin andmangiferinAfter the success of the spectrofluorimetricanalysis in determining the concentration of scopoletinand mangiferin individually in the methanolic extractof CD, it was thought worthwhile to develop a methodfor the simultaneous estimation of scopoletin andmangiferin concentrations in crude drug samples.For this purpose, 1 g shade dried powdered drug wastaken and subjected to methanolic extraction. Themethanolic extract was transferred to a volumetricflask and the volume made up to 100 ml withmethanol. The fluorescence intensity of this dilutedsample was determined by spectrofluorimetry. Thewhole procedure was repeated three times to obtaintriplicate readings. The concentration of scopoletinand mangiferin in all the samples was obtained byextrapolating from the standard curves. The meanconcentration of scopoletin and mangiferin presentin 1 g crude drug was determined. After determiningthe concentration of scopoletin and mangiferin perml of the methanolic extract, the mean concentration226