Chlorophyll a and b and total carotenoids were determined spectrophotometrically
in acetone extract of samples by the procedure
described by Singh et al. (2011). For chlorophyll a and b, the absorbance
was recorded at 645 nm and 663 nm and its total chlorophyll
content was calculated using Arnon’s equation (Arnon, 1949), total
chlorophyll (mg/100 g) = Chla + Chlb. Where, Chla = [(12.7 A663)
(2.6 A645) ml acetone/mg sample] and Chlb = [(22.9 A645)
(4.68 A663) ml acetone/mg sample]. Total carotenoids in sample
extracts were estimated using the equation described by
Lichtenthaler and Buschmann (2001) i.e. C(x + c) (mg/ml of sample
extract) = (1000A470 1.90Ca 63.14Cb)/214, where Ca – chlorophyll
a; Cb – chlorophyll b; C – carotenoid and x-xanthophyll.
Tannin content was determined using Folin–Denis reagent as
described by Sadasivam and Manikam (1995) with tannic acid as
standard and expressed as mg tannic acid/100 g. Flavonoid content
in the samples was determined by spectrophotometer using standard
protocol described by Sadasivam and Manikam (1995).
Briefly, 0.5 ml of 2% solution of AlCl3 in ethanol was mixed with
the same volume of sample extract (500 lg/ml) and absorbance
was taken at 415 nm after 1 h against a blank. Total flavonoid content
was determined using a standard curve of rutin standard (0–
50 mg/lt) and expressed at mg rutin equivalent (mg/100 g fresh
weight).