0.2 n folin ciocalteu reagent

Total phenolic content of the plant extracts was determined by the Folin-Ciocalteu (FC) reagents using gallic acid as a standard (Hazra et al., 2008 (link); Mishra et al., 2015 (link)). Different concentrations of the plant extracts (50–1000 μg ml^-1) were mixed with 2.5 ml 0.2 N Folin–Ciocalteu reagent (Sigma-Aldrich, USA) and were incubated for 5 min, followed by the addition of 2 ml sodium carbonate (Na₂CO_3; 75 g l^-1). The reaction mixtures were incubated for a further 90 min at room temperature. The absorbance was measured at 760 nm and the total phenolic content was calculated as mg ml^-1 gallic acid per 100 mg extract from a standard curve.

The total amount of phenolic and flavonoid compounds of each extract was determined according to the methods of Tsai et al. [29 (link)]. For the determination of total polyphenols content, an aliquot of 0.15 mL extract sample was mixed with 0.75 mL of a 0.2 N Folin-Ciocalteu reagent (Sigma-Aldrich) and 0.6 mL of a 7.5% sodium carbonate solution. The mixture was kept at room temperature for 30 min and then measured at 765 nm with a spectrophotometer (Ultrospec 2100 pro, GE Health-care, Amersham Place, UK). The total polyphenols content was estimated from the calibration curve that was built using gallic acid as a standard and is expressed as gallic acid equivalents of dry extract.
To determine the total flavonoids content, an aliquot of 0.3 mL of the sample, 1.2 mL of deionized water, and 0.075 mL of 15% Na₂CO₃ was added to a test tube. After 5 min, 0.15 mL of 10% AlCl₃ was added and kept at room temperature for 6 min. A solution containing 0.5 mL of 1 M NaOH and 0.275 mL of deionized water were added. After mixing, the absorbance of the solution was measured at 510 nm. The total flavonoids content was estimated from the calibration curve built using catechin as a standard and is expressed as catechin equivalents of dry extract.

Total phenolic content was determined with the Folin-Ciocalteu method [13 ]. Kombucha sample (5 mL) was diluted to 50 mL with Milli-Q water and filtered through Whatman No. 1 paper. Solution (0.5 mL) was mixed with 2.5 mL of 0.2 N Folin-Ciocalteu reagent (Sigma-Aldrich, North Ryde, Australia) for 5 min and 2 mL of 75 g/L sodium carbonate was then added. Mixture was incubated at ambient temperature for 2 h before reading with a Lambda 35 UV–vis spectrophotometer. Absorbance was measured at 760 nm against an ethanol blank. Gallic acid was used to produce a standard curve from 0 to 100 mg/L. All analyses were carried out in triplicate and the mean was expressed in mg of gallic acid equivalents (GAE)/100 mL kombucha.