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

Spectrofluorimetric determination of scopoletin and mangiferin in Canscora decussata / Asian Journal of TraditionalMedicines, 2008, 3 ( 6 )per gram of crude drug (mg/g) was calculated. Thescopoletin and mangiferin content in crude drugpowder of CD was found to be 4 mg/g and 7 mg/g,respectively.Thus, a simple spectrofluorimetric method foranalysis of scopoletin and mangiferin in CD wasdeveloped. Further recovery studies were performedto validate this novel analytical method.Analytical method validation- LinearityStandard solutions (5 µg/ml to 30 µg/ml) wereprepared in methanol and the intensity of fluorescencewas recorded in the spectrofluorimeter. The standardcurve was prepared by plotting the concentrationas the abscissa versus the intensity of fluorescenceas the ordinate. A linear dependence of intensityon concentration was observed over the entireconcentration range tested.- Precision and accuracyThe precision of the method was checkedusing standard solutions of the methanolic extract at aconcentration of 0.1, 0.2, 0.5 and 1.0 µg/ml, prepared byappropriate dilutions with methanol. The solutionswere analyzed in a spectrofluorimeter at 430 nm and 460nm (excitation and emission wavelengths) for scopoletinand 248 nm and 520 nm (excitation and emissionwavelengths) for mangiferin and the intensities wererecorded. The corresponding concentrations wereextrapolated from the standard curve.The entire procedure was repeated three times foreach dilution and the readings were expressed as Mean± S.D. (n=3). Then, 0.1 µg/ml solutions of scopoletinand mangiferin were prepared by appropriatedilutions and analyzed spectrofluorimetrically. Theconcentrations of scopoletin and mangiferin werecalculated for the sample.This sample of known concentration wasadded in an equal volume (1 ml) to all theprevious dilutions, and analyzed to see whetherthe observed concentrations corresponded to thetheoretical concentrations from the standard curve.The % recoveries were calculated on the basisof determination of the analyte added to a samplecontaining a known amount of scopoletin and mangiferin(Table 1).Results and discussionStandard curves for scopoletin and mangiferinwere prepared at excitation and emission wavelengthsof 430 nm and 460 nm and 248 nm and 520 nm,respectively, using a spectrofluorimeter. In both cases,the plots of concentration versus intensity exhibited alinear relationship. The equation of the straight linefor scopoletin was y=34.642x and that for mangiferinwas y=5.157x-114.06. A methanolic extract of CDwas also analyzed at the same excitation and emissionwavelengths. The scopoletin and mangiferin contentcalculated from the standard curve was found to be 4mg/g and 7 mg/g, respectively.Thus, a simple analytical method was developedfor determining concentrations of scopoletin andmangiferin simultaneously in CD. The developedmethod was validated for linearity, reproducibility andaccuracy. The linearity was found to be in the rangeof 5-30 µg/ml. The correlation coefficients (r) were0.9919 and 0.9937 for scopoletin and mangiferin,respectively, indicating good linearity between thefluorescence intensity and concentration. Scanningof the samples three times allowed the precision ofthe method to be checked. The reproducibility andaccuracy of the method was checked by carryingout recovery studies. A known concentration ofscopoletin and mangiferin was added to differentconcentrations of the methanolic extract i.e. 0.1, 0.2,0.5 and 1.0 µg/ml. A sample of known concentrationwas added in equal volume to the various dilutionsof the extract and analyzed spectrofluorimetricallyto see whether the observed concentration obtainedcorresponded to the theoretical concentrationobtained from the standard curve. The percentage227