New usage of fluorometric method to assay antioxidant activity in plant extract

A new fluorometric method was developed for measuring antioxidant activity of natural antioxidant. In this method, oxidative conversion of non –fluorescent dichlorofluorecin (LDCF) to fluorescent dichlorofluorocein (DCF) was occurred. The rate of the reaction was followed by monitoring the formation of DCF as a function of time [1]. The antioxidant assay was carried out for different concentrations of gallic acid, Salvia mirzayani crude extract and its fractions obtained by thin layer chromatography (TLC) [2]. In this assay, hematin was added to H₂O₂ followed by the addition of tris buffer. This mixture was used as base reaction. Then for measuring antioxidant activity, antioxidant compounds (plant extract or gallic acid) were added. The data were analyzed for statistical significance using one way ANOVA. P values less than 0.05 were considered significant. Fluorometric method was very sensitive, thus we were able to determine the antioxidant activity of eight fractions obtained in TLC of Salvia mirzayani. Subfractions 6,7, and 8 represented the highest antioxidant activities (Fig 1). The lowest concentration of gallic acid which can be detected by this method was 0.001µg/ml (Fig 2). The comparisons of existing methods with fluorometric assay showed that the fluorescent method could be used to detect lower concentrations of antioxidants.

Fig 1: Antioxidant activity of eight fractions obtained from TLC of Salvia mirzayani compared with gallic acid

Fig 2: The use of different concentration of gallic acid to detect the limit of concentration in fluorometric method. The most steep line is the base reaction that is lowered by the effect of several concentrations of gallic acid .0.001µg/ml of gallic acid is shown to be detectable.

References: 1: Cathcart, R., Shwiers, E., et al. (1983) Anal Biochem. 134:111–116. 2: Moein, S., Farzami, B.,et al. (2007) Pharm Biol. 45:458–463.

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