To estimate total phenolic compounds (TPC), 1 g of fresh leaves
was extracted with methanol: water (10 ml, 1:1 v/v) twice and the
supernatant after evaporation was dissolved in 3 ml methanol. To
this, 0.5 ml of Folin-Ciocalteau’s reagent was added followed by
incubation at room temperature and 2 ml of 20% sodium carbonate
was again added with thorough mixing. The appeared colour was
analyzed using UVeVIS spectrophotometer (Shimadzu UV-1700) at
650 nm as described by Sadasivam and Manickam (1996). Total
phenolics were expressed in terms of gallic acid equivalents per
gram of fresh leaves. Analytical grade reagents were used
throughout the experiment.