Abstract
Following optimization of extraction, separation and analytical conditions, a rapid, sensitive and simple reverse-phase high performance liquid chromatography-photo diode array (HPLC-PDA) method has been developed for the identification and quantification of wedelolactone in different extracts of Eclipta alba. The separation of wedelolactone was achieved on a C18 column using the solvent system consisting of a mixture of methanol: water: acetic acid (95: 5: 0.04) as a mobile phase in isocratic elution mode followed by photo diode array detection at 352 nm. The developed method was validated as per the guidelines of the International Conference on Harmonization (ICH). Calibration curve presented good linear regression (r²>0.998) within the test range and the maximum relative standard deviation (RSD, %) values for intra-day assay were found to be 0.15, 1.30 and 1.1 for low (5 µg/mL), medium (20 µg/mL) and high (80 µg/mL) concentrations of wedelolactone. For inter-day assay the maximum RSD (%) values were found to be 2.83, 1.51 and 2.06 for low, medium and high concentrations, respectively. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 2 and 5 µg/mL respectively. Analytical recovery of wedelolactone was greater than 95%. Wedelolactone in different extracts of Eclipta alba was identified and quantified using the developed HPLC method. The validated HPLC method allowed precise quantitative analysis of wedelolactone in Eclipta. alba extracts.
Highlights
IntroductionThe coumestan, wedelolactone (Figure 1), is the bioactive principle of aerial parts of E. alba
Shoot juice of E. alba mixed in equal amount with shoot juice of Oxalis corniculata Linn. is used as drops in injured eyes
Coumestans, polypeptides, polyacetylenes, thiophene derivatives, steroids, triterpenes, and flavonoids have been reported as the main constituents of E. alba (Sarg et al, 1981). β-Amyrin, wedelolactone and luetolin 7-O-glucoside have been isolated from the aerial parts of the plant (Sikoria et al, 1982)
Summary
The coumestan, wedelolactone (Figure 1), is the bioactive principle of aerial parts of E. alba. Various biological activities such as hepatoprotective, antiplasmodial, sedative, anxiolytic, nootropic and anti-stress have been reported for wedelolactone (Saxena et al, 1993; Simonsen et al, 2001; Thakur et al, 2005). Chemical synthesis of wedelolactone has been reported by several groups (Chang et al, 2008), there are only few high performance liquid chromatography method for identification and quantification for wedelolactone in extracts of E. alba. Present investigation was undertaken to develop a rapid, sensitive high performance liquid chromatography method for identification and quantification of wedelolactone in extracts of E. alba
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