The Rapid Method of Estimating Antioxidant Activity and Total Polyphenols Content of Sample Plant Extracts

Estimation of total polyphenols content

Figure1. The appearance of bluish-black colour after the addition of reagents on sample plant extracts during the estimation of total polyphenols content. 

We estimated total polyphenol content by a standard protocol (Singleton et al. 1999 and Stankovic 2011) with slight modification. Briefly, we separately mixed each plant extract (0.5 ml; 1 mg/ml)  with the Folin-Ciocalteu phenol reagent (2.5 ml; 10%) and aqueous sodium bicarbonate solution (2.5 ml; 7.5%). Then, the reaction mixture was allowed to stand for about 45 minutes and the absorbance was measured at 765 nm using the UV- spectrophotometer (Thermo Fisher Scientific, Genesystem-10.5).The calibration curve was plotted using gallic acid as standard in ethanol (absolute) using the concentration series of 25, 50, 75, and 100 µg/ml. Here, the reagent mixture with the same volume of ethanol instead of plant extract solution was used as a blank. On the basis of this calibration curve, the concentration of the individual samples was calculated. The total polyphenol content was expressed in terms of the milligrams of the gallic acid equivalent per gram of the dry mass (mg GAE/ g). The test was done in triplicates. 

Figure 2. The flow-chart for the estimation of total polyphenols content. 

Estimation of antioxidant activity

We performed the antioxidant activity of orchid extracts by following standard protocol according to Zhao et al. (2006) with slight modification. Briefly, we made a series of the solution with concentration 50 µg,  100 µg, 200 µg, 400 µg, 800 µg/ml for each plant extracts. Then, the  sample solution was made by mixing plant extract solution (1.5 ml of each concentration of a series) and DPPH free radical solution (1.5 ml; 0.25 mM in ethanol); blank solution by plant extract solution (1.5 ml of each concentration) and absolute ethanol (1.5 ml); control solution by DPPH free radical solution (1.5 ml; 0.25 mM) and absolute ethanol (1.5 ml). The quercetin was taken as a reference compound. The solutions of reference compound were made as it was done in case of plant extract solution. These solutions were vigorously shaken and allowed to stand for 30 minutes under dark immediately after preparation. Then, the absorbance of each solution was taken at 517 nm using UV- visible spectrophotometer. The experiment was done in triplicate.

Finally, we  applied the following equation to calculate scavenging activity of plant extracts and reference compound:

Scavenging rate = [1-(A₁-A₂)/A₀] ×100 %

Here, A₀ = Absorbance of control solution; A₁ = Absorbance in sample solution (i.e., in presence of plant extract); A₂ = Absorbance of blank solution (i.e., without DPPH). Finally, the IC₅₀ value of each plant extract was determined by nonlinear regression equation of plot of percentage antioxidant activity (AA %) against concentration.

Figure 3. Flow-chart for the estimation of antioxidant activity (DPPH radical scavenging activity) by sample plant extracts.