TOTAL PHENOL ANALYSIS PROTOCOL (Zielinski et al.

, 2014)

Materials
 Water
 Folin-Ciolcateu reagent
 Sodium carbonate
 Gallic acid monohydrates
 Dried tea leaves

Exraction
1. Mix 30 gram dried materials and homogenized
2. Extract 2 gram sample with 100 ml distilled water at 80oC in a glass flask
covered with a lid
3. Stir for 7.5 minutes using magneting stirring
4. Filter and transfer the mixture to falcon tube
5. Frozen at -20oC until further analysis

Total phenolic content

1. Add 8.4 ml distilled water to the test tube
2. Add 0.1 ml of diluted tea (1:7)
3. Add 0.5 FC reagent to the mixture
4. After 3 minutes, add 1 ml sodium carbonate
5. Centrifuge for 10 second in a vortex
6. Let stand the mixture in the dark for 1 hour

Measurement procedure
1. Measure absorbance at 725 nm using spectrophotometer
2. Transfer all absorbance values to a central phenol sheet where estimates of
secondary metabolites is expressed as gallic acid equivalent (GAE mg/L)
relative to standard curve