Folin ciocalteu reagent

Total polyphenolics from ginger ENPs were purified by methanol extraction. Briefly, 20 µl of ENPs were mixed with 100 µl of absolute methanol, vortexed and incubated at room temperature for 10 min. After centrifugation at 10,000 × g for 5 minutes, supernatant fraction was utilized for TPC estimation using a modified protocol described by Alhakmani et al.^{26 (link)}. In brief, the supernatant fraction was mixed with 400 µl of Folin-Ciocalteu reagent (HiMedia laboratories) (diluted tenfold with water) and vortexed. After the addition of 800 µl of 7.5% sodium carbonate, samples were incubated at room temperature for 30 mins. Samples were transferred to 96 well colorimetric plates and the blue color developed was measured using an ELISA plate reader at 765 nm wavelength. Gallic acid was used to generate standard curve and TPC values are represented as gallic acid equivalents per gram of ginger.

Total phenolic content in the extracts was determined using modified Folin-Ciocalteu method [35 (link)]. 0.5 mL of the extract was mixed with 5 mL Folin-Ciocalteu reagent (Himedia India) (previously diluted with water at 1 : 10 v/v) and 4 mL (75 g/L) of sodium carbonate (Himedia India). The tubes were vortexed for 15 sec and incubated at 40°C for 30 min in water-bath. Absorbance was measured at 765 nm using the Hewlett Packard UV-VS spectrophotometer. Samples of extracts were evaluated at a final concentration of 10 mg/mL. Total phenolic content was expressed as mg/g tannic acid equivalent, using the following equation based on the calibration curve [36 ]:
$\begin{array}{c}y=\mathrm{0.1216}x,\\ R^{\mathrm{2}}=\mathrm{0.9365},\end{array}$
where x = absorbance and y = tannic acid equivalent (mg/g).