Antioxidant activity
Total phenolic content
The TPC of each herbal tea infusion was measured using the
method described by K¨ahk¨onen et al.14 The herbal tea infusion
(3 mL) was added to 1.5 mL of Folin–Ciocalteu’s phenol reagent
(10% v/v) and was allowed to react for 5 min. Next, 1.2 mL of 7.5%
w/v sodium carbonate was added to the reaction mixture and
incubated for 30 min. The resulting blue complex was measured
at765 nm,andTPCwasexpressed asmg g−1 gallic acid equivalents
(GAE). Measurements were performed in triplicate.
Total flavonoid content
The total flavonoid content (TFC) was assayed as described by
Zhishen et al.15 In brief, 0.5 mL of herbal tea infusion was mixed
with 2 mL of distilled water and 0.15 mL of 20% w/v sodium
nitrite and was left to stand for 5 min. Then 0.3 mL of 10% w/v
aluminium chloride was added to this mixture. After 6 min, 2 mL
of 1 mol L−1 sodium hydroxide and 0.2 mL of distilled water were
added. The absorbance was read at 510 nm. The results were
expressed as mg g−1 quercetin equivalents (QE). Measurements
were performed in triplicate.
Total proanthocyanidin content
The total proanthocyanidin content (TAC) of each of the herbal
tea infusions was measured using the method described by Sun
et al.16 First, 2.5 mL of 1% (w/v) vanillin in methanol and 2.5 mL
of 9.0 mol L−1 hydrochloric acid in methanol were added to 1 mL
of herbal tea infusion. After incubation at 30 ◦C for 20 min, the
absorbance was measured at 500 nm using a spectrophotometer
(Shimadzu UV 1240; Shimadzu,) and expressed as mg g−1
catechine equivalents (CE). Measurements were performed in
quintuplicate.
1,1-Diphenyl-2-picrylhydrazyl free radical scavenging activity
The DPPH free radical scavenging activity of each herbal tea
infusion was determined according to the method described by
Leong and Shui.17 A 0.1 mmol L−1 solution of DPPH in methanol
was prepared. An aliquot of 0.1 mL of herbal tea infusion was
added to 2.9 mL of methanolic DPPH solution and kept in the
dark for 30 min. Absorbance was assayed at 517 nm using a
spectrophotometer (Shimadzu UV 1240). Trolox solution was used
to perform the calibration curves. The results were expressed
as μmol L−1 g−1 Trolox equivalents (TE). The measurement was
performed in triplicate.
Ferric-reducing antioxidant potential assay
The ability of the herbal teas to reduce ferric ions was measured
according to the method described by Benzie and Strain.18 The
FRAP reagent was prepared using 300 mmol L−1 sodium acetate
buffer at pH 3.6, 20 mmol L−1 iron chloride and 10 mmol L−1
2,4,6-tripyridyl-s-triazine dissolved in 40 mmol L−1 hydrochloric
acid at a ratio of 10 : 1:1 (v : v : v). The reagent was incubated in
a water bath at 37 ◦C for 5 min before use. The initial reading of
the reagent was measured at 593 nm using a Shimadzu UV 1240
spectrophotometer. An aliquot of 0.1 mL of herbal tea infusion
was then added to 2.9 mL of FRAP reagent and kept in the dark for
30 min. Trolox solution was used to create the calibration curves.
The results were expressed as μmol L−1 g−1 of Trolox equivalents
(TE). Measurements were performed in triplicate.