Food Research International 53 (2013)

900–908

Contents lists available at ScienceDirect

Food Research International

journal homepage: www.elsevier.com/locate/foodres

Antioxidant activity of white, green and black tea obtained

from the same tea cultivar
Patricia Carloni a, Luca Tiano b, Lucia Padella b, Tiziana Bacchetti c, Chisomo Customu d,
Alexander Kay d, Elisabetta Damiani c,⁎
a
Dipartimento Scienze Agrarie, Alimentari e Ambientali, Università Politecnica delle Marche, Ancona I‐60131, Italy
b
Dipartimento Scienze Cliniche, Specialistiche e Odontostomatologiche, Università Politecnica delle Marche, Ancona I‐60131, Italy
c
Dipartimento Scienze della Vita e dell'Ambiente, Università Politecnica delle Marche, Ancona I-60131, Italy
d
Satemwa Tea Estates, P.O. Box 6, Thyolo, Malawi

a r t i c l e i
abstract
n f o
The present study explored what effect manufacturing has on the antioxidant properties
Article history:
Received 17 February 2012
of teas coming from a single cultivar but processed differently to give a white, two black
Received in revised form 24 July (Orthodox and CTC) and two green (low-caffeine and non-decaffeinated) teas. Total phenol
2012 Accepted 26 July 2012 (TPC), flavonoids (TFC), theaflavins, individual cate- chins content, and chelating activity
were also investigated. Using the ABTS, ORAC and LDL assays the follow- ing ‘antioxidant
Keywords: profile’ was obtained: green≥low-caffeine green> white≥black Orthodox> black CTC, with
Tea statistically significant correlation between ORAC and LDL assays (r2 = 0.444, p=
Green/black/white tea processing 0.0067), whereas TPC and TFC significantly correlate with the ABTS one (r2 = 0.871, p=
Antioxidant activity 0.000 and r2 = 0.438, p= 0.007, respectively). Metal chelating activity, which was lowest
Chelating activity in the green teas, does not correlate with antioxidant activity but appears to be in fluenced
LDL
by theaflavins content. The results contribute to better understand how the manufacturing
Phenolic composition
process influences the antioxidant activity of tea when variables (geographical region,
envi- ronmental conditions, cultivar type, plucking techniques) are kept to a minimum.
Secondly, we show that novel African green, white and black Orthodox teas, made from
tea varieties typically used in black CTC tea production, may have potential health
benefits comparable with commonly consumed teas.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction dell'Ambiente, Via Brecce Bianche, Università Politecnica delle Marche, I-

60131 Ancona, Italy. Tel.: +39 71 2204135; fax: +39 71 2204398.
E-mail address: e.damiani@univpm.it (E. Damiani).
Tea is the most popular beverage after water, enjoyed
by many people across the globe, and which is receiving
continual interest due to the many beneficial health effects
associated with its regular consumption.
All varieties of tea are produced from the young, tender
leaves of the Camellia sinensis (L.) (family Theaceae), a
perennial, evergreen, leafy crop that enjoys warm and humid
climates with plenty of rainfall. Freshly, plucked leaves are
then processed to give black tea (fermented), oolong tea
(semi-fermented), green and white teas (unfermented).
Fermenta- tion consists in the enzymatic oxidation by
endogenous polyphenol oxidases and peroxidases of tea
polyphenols, namely colorless flavanols, that are partially
converted into theaflavins and thearubigins, respon- sible for
the characteristic aroma and color of black and oolong teas
(Obanda, Owuor,& Mang'oka, 2004). This oxidation process
is accelerat- ed by rupturing the withered tea leaves using
orthodox rollers or ma- chines (CTC: crush-tear-curl), so that
the oxidases released can more readily react with oxygen. In
unfermented teas, fermentation of the withered leaves is
prevented by inactivating the endogenous enzymes

⁎ Corresponding author at: Dipartimento Scienze della Vita e

through steaming or heating using different methods loss (Dufresne & Farnworth, 2001). These health benefits
(pan frying, roasting, baking) before the rolling and are attributed to its high content of catechins which have
drying process. Instead, white tea is the least processed been described as potent antioxidants ameliorating
in that it goes only through sunshine withering and disease states re- lated to reactive oxygen species (ROS)
drying. Furthermore, white teas are characterized by the (Rietveld & Wiseman, 2003). Since tea leaves are
fact that only buds still covered with fine white hair, and processed differently to produce black, green, oolong and
one or two very young leaves are used (Balentine, 1992). white teas, it is of interest to know which tea could poten-
Consumers of black tea far outweigh those of green tially be more beneficial in terms of antioxidant activity.
tea world- wide. However, in recent times green tea Many studies compare the activities of these teas but a
consumption has increased especially in the West, due conclusive answer has yet to be reached. Generally,
to the numerous studies that indicate a wide variety of antioxidant activity decreases in the order: green tea>
health benefits following its regular consumption: oolong tea> black tea (Richelle, Tavazzi, & Offord, 2001;
reduced risk of cardiovascular disease and certain Roginsky, Barsukova, Hsu, & Kilmartin, 2003; von Gadow,
types of cancer, in- flammatory bowel, liver and Joubert, & Hansmann, 1997) although some studies show
neurodegenerative diseases, diabetes, and even weight that black teas are better than green ones (Hoff &
Singleton, 1977; Khokhar &

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2012.07.057
Magnusdottir, 2002; Venditti et al., 2010) while others report
2.2. Tea samples and preparation
the ab- sence of any significant differences (Hodgson et al.,
1999; Lin, Juan, Chen, Liang,& Lin, 1996). The numerous
Five tea samples processed from hand plucked leaves of
variables that affect tea compo- nents according to cultivar type,
the superior cultivar PC108, bred in Malawi for typical black
growth conditions (season, climate, soil), horticultural practices
CTC tea production by the Tea Research Foundation based
(mechanical- or hand-plucking, age of leaves) and the different
in Mulanje, and grown ona private es- tate, were analyzed.
technologies of the tea companies may account for these
The green leaves had been harvested from the same field at
conflicting reports (Kan, 1980; Wicremasinghe, 1974). Hence, it
three different times: February (typically overcast, wet,
is not strictly correct to compare the antioxidant activities of
warm con- ditions), April (bright sunny days and cool nights)
black, white, and green teas unless these variables are
and September (hot clear days and warm nights) 2008.
minimized.
Fresh leaves were then processed according to the flow-
Therefore, the present study was carried out to explore
chart reported in Fig. 1 to give five different teas: white,
what effect manufacturing has on the antioxidant properties
green, low-caffeine green, black CTC and black Orthodox
of teas coming from a single cultivar (grown and plucked
teas. The withering was done in troughs and ambient or
under the same conditions) but processed differently in the
heated air was blown into a plenum chamber and up
same factory to give two black teas (Orthodox and CTC),
through the leaf. In order to achieve the target moisture
two green teas (low-caffeine and non-decaffeinated) and one
content, the withering time varied depending on the
white tea. Total phenol, flavonoids, theaflavins, individual
prevailing weather conditions and conditions of leaf on
catechins and caffeine content, as well as chelating activity
receipt into the factory. The target moisture content for all
were also investigated.
teas at the end of the drying process carried out in a fluid
bed drier was 3–4%.
Prior to preparation of hot tea infusions, the teas were
2. Materials and methods
ground using a pestle and mortar to obtain a homogeneous
fine powder. The infusions were prepared by pouring 20 ml
2.1. Chemicals and equipment
of mineral water at 90 °C on 0.5 g of tea and brewed for 7
min. They were then filtered through Whatman paper filters
All reagents, standards for HPLC analysis and solvents of
43–48 μm and diluted appropriately with water according to
highest purity available, were purchased from Sigma-Aldrich
each specific assay.
Chemical Co. (Milan, Italy) and used as received. Ultrapure
water was used through- out and obtained from a Milli-Q
2.3. Total phenol content (TPC)
system from Millipore (Milford, MA), except for preparation
of teas where bottled mineral water purchased from local
Total phenol content in the tea infusions was determined
retail shops was used.
using the Folin–Ciocalteu reagent (Singleton, Orthofer,
Spectrophotometric measurements were recorded on a Lamuela-Raventos, & Lester, 1999). To 1.975 ml water, 0.125
UV Kontron 941 spectrophotometer or on a microplate ml of Folin–Ciocalteu reagent followed by 0.025 ml of tea
reader (Synergy HT, Biotek, Winooski, VT, USA). previously diluted 5 fold, or appropriately diluted gallic acid
standard ethanolic solution, or water as blank, were
Fig. 1. Flow chart showing the different stages in the manufacture of the different teas investigated. LTP= Lawrie tea processor; CTC= crush-tear-curl (see
Materials and methods for details).
added and mixed. After 10 min, 0.375 ml of 20% Na2CO3 reacting aqueous stock solutions of ABTS [2,2′-azinobis-(3-
were added, mixed and samples were left for 2 h at room ethylbenzothiazoline-6-sulfonic acid) diammonium salt] and
temperature in the dark. Absorbance was read at 760 nm K2S2O8 to reach final concentrations of 6.3 mM and 2.45 mM
and the results were expressed as mM Gallic Acid respectively, for 12–16 h in the dark at room temperature.
Equivalents (GAE) using the linear regression value Prior to use, the ABTS+• stock solution was diluted~ 80 fold
obtained from the gallic acid calibration curve. with water in order to obtain an ab- sorbance at 734 nm
ranging between 0.6 and 0.8. To 2.475 ml of this ABTS+•
2.4. Total flavonoid content (TFC) solution, 0.025 ml of tea previously diluted 40 fold or appro-
priately diluted Trolox (6-hydroxy-2,5,7,8-
The total flavonoid content in the tea infusions was tetramethylchroman-2- carboxylic acid) standard ethanolic
measured using a colorimetric assay according to the solution, or water as control,
method of Gursoy, Sarikurkcu, Cengiz, and Solak (2009)
with some modifications. Briefly, 0.05 ml of tea infusion or
appropriately diluted (+)-catechin standard ethanolic
solution, or water as blank, were added to 1.35 ml of
distilled water. After mixing, 0.05 ml of 5% NaNO 2
followed by 10% AlCl3 (0.05 ml) were added and mixed
and samples were left for 10 min at room tem- perature in
the dark. Absorbance was read at 415 nm and the results
were expressed as mM Catechin Equivalents (CE) using
the linear re- gression value obtained from the catechin
calibration curve.

2.5. Total theaflavins content (TTC)

The total theaflavins content in the tea infusions was

determined according to the Flavognost method (Hilton,
1972). Tea infusions (3 ml) were mixed with 3 ml of 4-
methylpentan-2-one (IBMK), vortexed for 10 min and then
allowed to stand until the layers separated. One milliliter of
the upper layer was pipetted into a test tube, followed by 2
ml ethanol and 1 ml Flavognost reagent (2% w/v in ethanol).
The contents were mixed and left for 15 min to allow for
color development. Absorbance
(A) at 625 nm was read against an IBMK/ethanol (1:1 v/v)
blank. The TTC of the aqueous solution was calculated from
Beer's law using the equation [TTC] (mmol/l)=1.1496 ×A625
(Spiro & Price, 1986).

2.6. Analysis of phenolic compounds and caffeine

HPLC for the analysis of phenolic compounds and caffeine

was conducted on a Gilson liquid chromatograph equipped
with a 321 solvent pump, a 234 Autoinjector and a Dionex
AD20 Absorbance Detec- tor set at 280 nm. A C18 guard
column and a Waters μ-Bondapack C18 pre-packed column (3.9
×300 mm) were used for separation. The solvent
compositions used were water:acetic acid, 93:3 v/v (solvent A)
and HPLC grade methanol (solvent B), with a flow rate of 0.8
ml min−1. The conditions for HPLC analysis were modified
from those described by Zuo, Chen, and Deng (2002). Briefly,
the elution was performed with a gradient starting at 100%
A to reach 80% at 15 min and 100% at 40 min. The freshly
prepared tea infusions from samples harvested in April were
filtered through a 0.45 μm membrane filter and 0.02 ml of
each sample were injected after a 1:10 dilution. Quantification
of the compounds at 280 nm was based on peak area, obtained
with valley- to-valley integrations, and retention times, using
external standards. A dilution series of each standard (0.01
mg/ml–0.1 mg/ml) had been previ- ously prepared for
quantification.

2.7. ABTS assay

For measuring the antioxidant activity of the different

teas, the ABTS assay was performed according to the
method of Pellegrini, Ke, Yang, Rice-Evans, and Lester
(1999). The radical cation (ABTS+•) was gener- ated by
were added and mixed. The samples were left for 2 h of the iron (II)-ferrozine complex (Viollier, Inglett, Hunter,
in the dark at room temperature, and absorbance was Roychoudhury, & Van Cappellen, 2000) with modifications.
read at 734 nm against water. Inhibition percentage To 0.1 ml of a 0.8 mM FeSO4 solution in
values were calculated according to the equation: 0.1 M HCl, 0.2 ml of tea previously diluted 5 fold, or of
appropriately diluted EDTA (ethylenediaminetetraacetic
Inhibition of A734 ð%Þ ¼ ð1−Ac =A0 Þ × 100 acid) standard solution, or water as control, were added
followed by 0.1 ml of 5 mM ferrozine [4,4′-[3-(2-pyridinyl)-
where Ac = absorbance of the samples, A0 = 1,2,4-triazine-5,6-diyl]bisbenzenesulfonic acid] solution in 0.1
absorbance of the con- trol. Antioxidant activity was M PBS. Finally, 1.6 ml of 0.1 M PBS was added to neutral-
expressed as mM Trolox Equivalents (TE) using the ize the solution. After standing for 10 min in the dark at
linear regression value obtained from the Trolox cal- room temper- ature, absorbance was read at 562 nm
ibration curve. against a blank containing water instead of tea and
lacking FeSO4. Antioxidant activity was expressed as
2.8. ORAC assay

Antioxidant activity of the different teas was also

assessed with the ORAC (Oxygen Radical Absorbance
Capacity) assay according to the method of Gillespie,
Chae, and Ainsworth (2007). Briefly, in each well of
a solid black 96-well microplate, 0.15 ml of 0.08 μM
fluorescein (3′,6′-dihydrosyspiro[isobenzofuran-1[3H],9′[9H]-
xanthen]- 3-one) dissolved in 75 mM PBS (phosphate
buffered saline) was added followed by 0.025 ml of tea
previously diluted 1000 fold. After 10 min incubation in
the dark at 37 °C, 0.025 ml of 147 mM AAPH [2,2′-
azobis(2-methylpropionamidine)dihydrochloride] were
rapidly added to each well and fluorescence recorded
from the top every 120 s for 3 h, using an excitation
wavelength of 485/20 nm and an emission filter of
528/20 nm. The kinetics showed a classic fluorescence
decay due to decomposition of fluorescein that was
delayed in the presence of tea samples or of Trolox
standard ethanolic solution diluted with PBS. AUC (area
under the fluorescence decay curve) was automatically
calculated by the analytical software KC4 (Biotek,
Winooski, VT, USA) connected to the Synergy HT reader.
The net AUC for each standard/compound was obtained
by subtracting the area of the blank sample (PBS).
Antioxidant activity was expressed as mM Trolox
Equivalents using the linear regres- sion value obtained
from the Trolox calibration curve.

2.9. LDL isolation and oxidation

LDL (low density lipoprotein) particles were isolated

from pooled, fresh, human blood obtained from
volunteers after overnight fasting according to the
method described by Venditti et al. (2010).
To determine the effects of tea samples on the
inhibition of LDL oxidation, conjugated dienes (a
measure of lipid oxidation) were monitored in LDL
samples incubated in the presence and absence of
different teas in a 96-well microplate reader. The
volume of each well was adjusted with deionized
water to reach a final volume of
0.2 ml. Briefly, to 0.01 ml of LDL (100 μg protein/ml), 0.01
ml of tea
previously diluted 400 fold was added. The oxidation
reaction was initiated by adding 0.1 ml of 0.01 mM
CuSO4 to each well and conju- gated dienes were
monitored at 234 nm for 6 h at 37 °C with read- ings
taken every 120 s. The control sample lacked tea. The
lag time was then determined graphically and reported
as such.

2.10. Metal chelating assay

The ferrous ion chelating activity of the tea samples

was measured by the decrease in absorbance at 562 nm
mM EDTA Equivalents (EE) using the inhibition percentage with TPC and TFC data from the April harvest (data not
value obtained from the EDTA calibration curve as described shown), a statistically significant correlation was observed
for the ABTS assay. in both cases (r2=0.8091, p= 0.0377; r2=0.9018,
p=0.0135, respectively).
2.11.Data analysis The caffeine levels were similar in all the teas, except for
the black
Appropriate controls were carried out throughout all the CTC which had the lowest. This result is not surprising
experiments described above. The data reported represent since fermen- tation results in a slight loss of extractable
average values from at least three independent experiments caffeine (Cloughley, 1983) and the black CTC tea, which
each performed in duplicate. Statistical significance of undergoes severe leaf disruption leading to a greater
correlations was evaluated using a two-tailed probability test. surface area for enzymatic oxidation of the catechins, is the
Statistical significance was performed using one-way ANOVA most fermented one. However, surprising is the fact that
and Tukey's multiple comparison test. A value of pb 0.05 was the tea
considered statistically significant.

3. Results

3.1. Levels of total tea polyphenols, flavonoids and

theaflavins

The distribution of the data of total phenols (TPC) and

flavonoids (TFC) content measured in the tea infusions is
reported in Fig. 2A and B. TPC was found to be higher in
green and low-caffeine green teas (23.6 ± 5.4 and 22.6 ±
5.0 mM GAE) followed by the white (18.1 ± 5.5 mM GAE)
and black CTC and Orthodox teas (10.7 ± 4.0 and 14.9 ±
5.9 mM GAE). No significant differences were observed
amongst the green teas, whilst differences can be
observed amongst the two black teas processed
differently: the black Orthodox tea has statistically
significant (pb 0.05) higher levels of polyphenols than the
black CTC tea. The trend of TFC is similar to that of TPC,
except for white tea (4.75 ± 0.65 mM CE) whose value is
lower than black Orthodox tea (6.60 ± 1.52 mM CE).
Total theaflavins content (TTC) was also determined, since
it is considered to be an important criteri- on of black tea
quality. The TTC (Fig. 2C) was very low in the green teas
as expected (b 0.01 mM theaflavins), while higher levels
were found in the remaining teas, with black CTC
exhibiting the highest levels (0.172 ± 0.019 mM
theaflavins) being the most oxidized tea. White tea which
is not usually considered a fermented tea, also showed a
signifi- cant amount of theaflavins (0.093 ± 0.018 mM
theaflavins).

3.2. Individual phenolics and caffeine content

Individual phenolic compounds and caffeine content in tea

infusions were determined in the teas harvested in April only
and are reported in Table 1. Epigallocatechin (EGC) is the
major flavanol in all the tested in- fusions, especially in the
two green teas, whereas in black CTC, catechin
(C) is the major one. The levels of epigallocatechin-3-
gallate (EGCG) are similar for the two green teas and
black Orthodox tea, lower in the white tea and very low in
the black CTC one. Epicatechin-3-gallate (ECG) levels are
instead similar in all teas except in the black CTC which
has the lowest level of this flavanol. The levels of C were
found to be low in the black Orthodox and in the white tea
while gallic acid (GA) was found to be highest in the black
Orthodox tea. The distribution of the data of the total
catechins content is reported in the box-plot of Fig. 3.
Total catechin levels are significantly higher in the low-
caffeine green tea than in the black CTC and white teas
but not with respect to the green and black Orthodox teas.
Furthermore, black CTC has signifi- cantly lower total
catechin levels than the non-decaffeinated green tea. By
analyzing the total catechins content reported in Table 1
which was treated to reduce caffeine levels (low-caffeine from the same culti- var, grown in the same conditions, and
green tea) had only slightly lower, non-significant levels processed differently in the same factory to make white,
of caffeine than the green tea. This unexpected result green and black tea. Since no single mea- surement of
may be due to the fact that the tea leaves had been antioxidant status is sufficient, a “battery” of different assays,
treated in boiling water for only 15 s (Fig. 1) which may which work on different principles, was employed (Prior &
have been too short a time for sufficient extraction of Cao, 1999; Roginsky et al., 2003). The ABTS assay is an
water- soluble caffeine. In fact, although a common indirect meth- od that simply measures the capacity of the
technique of discarding a short steep (30–60 s) is ABTS radical cation to ab- stract an hydrogen atom or an
believed to reduce caffeine content in a subse- quent electron from the compound/s under study (Prior, Wu, &
brew by 80–90%, research suggests that a 5 min steep Schaich, 2005). The simplicity of the method has
yields up to 70% of the caffeine, and a second steep has a
third of the caffeine of the first (about 23% of the total
caffeine in the leaves) (Hicks, Hseih, & Bell, 1996).

3.3. Antioxidant activity

The antioxidant activity of the tea infusions was

assessed using three independent assays, ABTS, ORAC,
and inhibition of LDL oxidation. The results obtained from
the ABTS assay reported in the box-plot of Fig. 4A show
that the green tea has significantly higher antioxidant ac-
tivity (55.0 ±12.7 mM TE) compared to all the rest, while
similar activ- ities are found for the low-caffeine green
(44.0 ± 10.2 mM TE), white (38.4 ± 9.9 mM TE) and
black Orthodox (34.0 ± 8.4 mM TE) teas; the antioxidant
activity of the black CTC tea is significantly lower than all
the others (25.5 ± 7.5 mM TE). Furthermore, a
statistically significant correlation was found between the
results obtained with the ABTS assay and TPC and TFC
reported in Fig. 2A and B (r2=0.871, p=0.000 and
r2=0.438, p=0.007, respectively).
The results reported in Fig. 4B from the ORAC assay,
follow in gen- eral a similar trend to the ABTS one,
although not in total accordance with it. In fact, the high
antioxidant activity of the green teas is re- versed: the
low-caffeine tea has a higher (70 ± 9 mM TE) but not sig-
nificant antioxidant activity than the non-decaffeinated
one (61 ± 10 mM TE). Furthermore, there appears to be
a remarkable antioxi- dant activity in the black Orthodox
tea (62 ± 8 mM TE) which is sig- nificantly greater than
the white tea (47 ± 8 mM TE) and parallels that of the
green teas. Once again, the black CTC tea shows the
lowest antioxidant activity (32 ± 4 mM TE).
The lag times reported in Fig. 4C concerning the
ability of the teas to suppress copper-induced LDL
oxidation, are in significant agreement with the trend of
antioxidant activity using the ORAC assay reported in
Fig. 4B (r2=0.444, p=0.0067). Furthermore, the black
CTC tea (123 ±9 min) did not appreciably prolong the
lag time compared to the control without tea (111± 19
min).

3.4. Metal chelating activity

The chelating activity of the tea infusions measures

how effective the compounds in them can compete with
ferrozine for ferrous ions. Fig. 5 shows that the two
green teas have the lowest chelating activity (0.178 ±
0.090 and 0.147 ± 0.066 mM EE) with no differences
among each other, while the white tea displays the
highest (0.477± 0.136 mM EE). Among the black teas,
the Orthodox one shows a significantly higher chelating
power (0.449 ± 0.110 mM EE) compared to the CTC one
(0.249 ±0.056 mM EE).

4. Discussion

The present study was carried out to determine how

antioxidant activity varies in infusions of tea leaves plucked
 A

Fig. 2. Box-plot diagrams of the levels of total tea polyphenols, flavonoids and theaflavins in the tested teas. The cross represents the median value, 50% of
the data are contained within the box and the lines indicate the upper and lower quartiles, each one corresponding to 25% of the data. Panel A: Total
polyphenols content (expressed as mM Gallic Acid Equivalents) determined using the Folin–Ciocalteu reagent; Panel B: Total flavonoid content (expressed
as mM Catechin Equivalents) determined using the aluminum chloride colorimetric method; Panel C: Total theaflavin content (expressed as mM
theaflavines) determined using the Flavognost reagent (see Materials and methods for details). Statistical significance of one-way ANOVA was pb 0.0001.
p for Tukey's least significant difference is summarized below each box-plot; ns= non-significant.
Table 1
Content of phenolic compounds and caffeine in tea infusions expressed in mg/ml.

GA C EGC EGCG ECG Total Cathechins CAFF

content
White 0.018 ± 0.004 0.078 ± 0.022 0.331 ± 0.213 ± 0.120 ± 0.80 ± 0.36 0.547 ±
0.071 0.052 0.096 0.044
Green 0.007 ± 0.008 0.283 ± 0.064 1.214 ± 0.544 ± 0.178 ± 1.94 ± 0.57 0.604 ±
0.151 0.091 0.052 0.043
Green low-caffeine 0.017 ± 0.183 ± 0.110 1.333 ± 0.506 ± 0.135 ± 2.17 ± 0.55 0.514 ±
0.003 0.163 0.145 0.116 0.039
Black CTC 0.045 ± 0.004 0.163 ± 0.026 0.029 ± 0.011 ± 0.009 0.038 ± 0.24 ± 0.06 0.361 ±
0.007 0.010 0.047
Black Orthodox 0.081 ± 0.006 0.0209 ± 0.051 0.848 ± 0.461 ± 0.230 ± 1.75 ± 0.37 0.617 ±
0.118 0.103 0.066 0.070
GA= gallic acid, C=(+)-catechin, EGC=(−)-epigallocatechin, EGCG=(−)-epigallocatechingallate, ECG=(−)‐epicatechingallate, CAFF= caffeine.
Values are mean of duplicate determinations (n= 6)± standard deviation.

made it very popular and suitable for assessing the ability of

tea in disease states related to reactive oxygen species
compounds to act as hydrogen/electron donors and to evaluate
(ROS), devel- opment of cancer, cardiovascular diseases
their antioxidant ac- tivity (Re et al., 1999). The ORAC assay is
and other pathologies (Dufresne & Farnworth, 2001;
instead a direct method that measures antioxidant inhibition of
Rietveld & Wiseman, 2003). From our results, the green
peroxyl radical-induced oxidations and thus reflects classical,
teas have the highest content of catechins, especial- ly
radical chain-breaking antioxidant activity by hydrogen atom
EGC and EGCG, and they display the highest antioxidant
transfer; its popularity is due to the fact that it can measure
activity. The remarkable antioxidant effect of these two
both antioxidant capacity (duration of antioxidative action)
catechins has been at- tributed to the higher degree of
and reactivity (Prior et al., 2005). The suppression of the
hydroxylation of the B ring and/or hy- droxyl group at C-3
oxidation of LDL, i.e. a test that directly analyzes the effect of
position of the basic catechin structure (Amic, Davidovic-
a food containing antiox- idants on the oxidative degradation of
Amic, Beslo, & Trinajstic, 2003) which increases its
a model system, was also evaluat- ed (Sanchez-Moreno,
radical scavenging ability. The high antioxidant activity of
Jimenez-Escrig, & Saura-Calixto, 2000). From the results
catechins is fur- ther demonstrated when comparing the
obtained using the different tests, a significant correlation was
two black teas manufactured with the Orthodox method
found between the ORAC and LDL assays which are both based
(for large leaf grades) and with the CTC method (small
on the same principle, i.e. inhibition of an oxidative process in
particles suitable for tea bags), where significant dif-
which several oxidative species are involved; hence a similar
ferences were always observed. The CTC processing
trend in results should be expected. Instead, the ABTS assay
method involves a more rapid and severe leaf disruption
gives an indication of the scaveng- ing efficiency of the
producing smaller fragments and a greater surface area
different teas towards only one radical species,
for enzymatic oxidation and this leads to a higher degree
i.e. ABTS radical cation, which reacts readily with
of fermentation which translates into lower catechin levels,
hydrogen/electron donors such as polyphenols. As expected, a
as confirmed by the HPLC analysis (Table 1). Astill et al.
strong correlation was found between the results obtained
also found lower total catechin contents in CTC-
from the ABTS and TPC assays, but both do not correlate with
manufactured black teas compared with Orthodox-
those of LDL and ORAC ones.
manufactured ones determined on a batch of leaves from
Although some small differences in the results using
the same area of an Assam estate (Astill, Birch, Dacombe,
the ABTS, ORAC and LDL assays were found, a general
Humphrey, & Martin, 2001). Orthodox teas have an
trend in antioxidant ac- tivity can be delineated, i.e. green
enhanced yellow color and higher aroma than the poorer
tea≥low-caffeine green tea> white tea≥black Orthodox
flavor of CTC teas (Saikia & Mahanta, 2002), and now from
tea> black CTC tea, which clearly demonstrates that the
the present study one can add that they also have a more
manufacturing process influences the properties of tea. It
potent antioxidant activity. From Fig. 2A and B, one can also
is well known that green tea is higher in total catechins
observe that TPC and TFC generally decrease from green to
than black tea, since the fermentation process reduces
white to black tea, in good agreement with the antioxidant
these levels in the latter one as they are converted to
theaflavins and thearubigins. It is also known that high activities observed, and also with the TPC trend generally
catechins levels positively correlate with antioxidant reported in the literature (Lin, Tsai, Tsay, & Lin, 2003;
activity (Gramza-Michalowska & Korczak, 2007; Karori, Roginsky et al., 2003).
Wachira, Wanyoko, & Ngure, 2007), which contribute to The trend in metal ion chelating activity observed in Fig. 5
the protective role of deserves a separate discussion. This activity is among one of
the several factors which account for the antioxidant activity of
polyphenols that includes
Fig. 3. Box-plot diagram of the total catechins content (mg/ml) in hot tea infusions from tea leaves harvested in April. The cross represents the median
value, 50% of the data are contained within the box and the lines indicate the upper and lower quartiles, each one corresponding to 25% of the data. The
graph was constructed from the total catechins content reported in Table 1. Statistical significance of one-way ANOVA was pb 0.0001. p for Tukey's least
significant difference is summarized below each box-plot; ns=non-significant.
 A

Fig. 4. Box-plot diagrams of antioxidant activity of the tested teas. The cross represents the median value, 50% of the data are contained within the box
and the lines indicate the upper and lower quartiles, each one corresponding to 25% of the data. Panel A: ABTS assay results expressed as mM Trolox
Equivalents; Panel B: ORAC assay expressed as mM Trolox Equivalents; Panel C: LDL assay shown as the lag time for formation of conjugated dienes.
Human LDL (100 μg protein/ml) oxidation was initiated by addition of 5 μM Cu2+ and formation of conjugated dienes at 234 nm was determined in the
presence of tea infusions. The control sample lacked tea. The lag time was calculated from the sigmoidal kinetic curves of LDL oxidation obtained for
each sample. See Materials and methods for details. Statistical significance of one-way ANOVA was pb 0.0001. p for Tukey's least significant difference is
summarized below each box-plot; ns= non-significant.
Fig. 5. Box-plot diagram of metal chelating activity (expressed as mM EDTA Equivalents) of the tested teas determined using the Ferrozine assay (see
Materials and methods for details). The cross represents the median value, 50% of the data are contained within the box and the lines indicate the upper
and lower quartiles, each one corresponding to 25% of the data. Statistical significance of one-way ANOVA was pb 0.0001. p for Tukey's least
significant difference is summarized below each box-plot; ns= non-significant.

radical scavenging, stability of the resulting phenoxyl of all the other analy-
radical, solubility in the lipophilic phase, redox potential ses were similar to those of this season.
(Cao, Sofic, & Prior, 1997; Morel, Lescoat, Cillard, & In conclusion, this paper contains key information on how
Cillard, 1994), hence the reason for investigating this the manufacturing process of tea affects antioxidant activity,
activity. In this study, the green teas showed the lowest TPC, TFC, TTC and individual catechins content when
metal chelating activity while the white tea and the black variables are kept to a mini- mum: tea from a single specialty
Orthodox displayed the highest. This trend is unusual as cultivar grown under the same condi- tions and processed in
one would expect that teas with higher, overall catechins the same factory as a green, white and black tea. This is the
content and hence higher antioxidant activity would also first study of its kind as most literature reports investigating
display higher metal chelating activity. However, this does
not ap- pear to be the case, as also observed by Lin, Liu,
and Mau (2008). It seems that components other than
catechins might contribute most to the chelating ability; the
presence in a compound of a specific group bearing
adjacent hydroxyl and carbonyl functions has been
suggested to play a role in metal chelation (Lin et al.,
2008). This group for example, can be found in theaflavins
which are present in fermented teas and interestingly, in
our study, the teas that show the highest chelating activ- ity
(white and black teas) are also those that have the highest
content of theaflavins (Fig. 2C). White tea is commonly
considered a non- fermented tea although this is not strictly
correct: in fact, white teas do not undergo the inactivation
of enzymes before withering, and in this stage, polyphenol
oxidases and peroxidases, essential for color develop- ment
of this tea, remain active (Jiang, 2009). Therefore white tea
poly- phenols do undergo slight and slow oxidation, as
confirmed by the presence of significant amounts of
theaflavins found in the white tea in- vestigated (Fig. 2C).
The slight oxidation of white tea polyphenols might also
explain why the antioxidant activity observed for this tea is
always slightly lower than the green teas and similar to the
black Orthodox one. Tea products are manufactured with
tea leaves harvested at differ- ent seasons which is known
to influence the flavor and aroma of tea products (Kan,
1980). In our study, seasonal variations were observed but
it was not possible to predict which season produced
teas with higher antioxidant activities since there were no
remarkable differ- ences and an overall trend could not be
deduced. Hence the data obtained from the three seasons
were all pooled and analyzed togeth- er. The only
exception was the HPLC analysis of individual catechin
and caffeine contents reported in Table 1 and Fig. 3 which
were rela- tive to the April harvest, since the average data
the antioxidant properties of teas compare black, green
and white teas from different geographical regions.
Those that have studied teas from the same geographical
region, have either used teas processed in differ- ent tea
factories and from different cultivars (Karori et al.,
2007), or compared teas processed in the same way, i.e.
just green teas (Saito et al., 2007) or compared different
cultivars (Baruah & Mahanta, 2003). Secondly, since
there are very few non-CTC tea producers in Africa, this
study shows that black Orthodox, white and green teas,
can be made from tea varieties bred as quick fermenting,
astringent types, typ- ically used in black CTC tea
production. This initial study on the antiox- idant
properties of the novel African green, white and black
Orthodox teas, indicate that they may have comparable
or superior potential health benefits that could rival
commonly consumed teas present on the market.

Acknowledgments

The authors thank the Polytechnic University of the

Marche for financial support. The authors declare that
they have no conflict of interest.

References
Amic, D., Davidovic-Amic, D., Beslo, D., & Trinajstic, N. (2003). Structure–
radical scav- enging activity relationships of flavonoids. Croatica
Chemica Acta, 76, 55–61.
Astill, C., Birch, M. R., Dacombe, C., Humphrey, P. G., & Martin, P. T.
(2001). Factors affect- ing the caffeine and polyphenol contents of black
and green tea infusions. Journal of Agricultural and Food Chemistry,
49(11), 5340–5347.
Balentine, D. (1992). Manufacturing and chemistry of tea. In C. -T. Ho, T.
Osawa, M. -T. Huang, & R. T. Rosen (Eds.), Phenolic compounds in food
and their effects on health (pp. 102–117). Washington, DC: American
Chemical Society.
Baruah, A. M., & Mahanta, P. K. (2003). Fermentation characteristics of
some assamica clones and process optimization of black tea
manufacturing. Journal of Agricultural and Food Chemistry, 51(22),
6578–6588.
Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior
of flavo- noids: Structure–activity relationships. Free Radical Biology &
Medicine, 22(5), 749–760.
Cloughley, J. B. (1983). Factors influencing the caffeine content of black tea:
Part 2 —
The effect of production variables. Food Chemistry, 10, 25–34.
Dufresne, C. J., & Farnworth, E. R. (2001). A review of latest research
findings on the health promotion properties of tea. The Journal of
Nutritional Biochemistry, 12(7), 404–421.
Gillespie, K. M., Chae, J. M., & Ainsworth, E. A. (2007). Rapid measurement
of total antioxidant capacity in plants. Nature Protocols, 2(4), 867–870.
Gramza-Michalowska, A., & Korczak, J. (2007). Polyphenols—Potential food
improve- ment factor. American Journal of Food Technology, 2, 662–670.
Gursoy, N., Sarikurkcu, C., Cengiz, M., & Solak, M. H. (2009). Antioxidant
activities, metal contents, total phenolics and flavonoids of seven
Morchella species. Food and Chemical Toxicology, 47(9), 2381–2388.
Hicks, M. B., Hseih, P., & Bell, L. N. (1996). Tea preparation and its influence
on methyl- xanthine concentration. Food Research International, 29(325– Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C.
330). (1999). Antioxidant activity applying an improved ABTS radical cation
Hilton, P. J. (1972). In vitro oxidation of flavanols from tea leaf. decolorization assay. Free Radical Biology & Medicine, 26, 1231–1237.
Phytochemistry, 18, 1243–1248. Richelle, M., Tavazzi, I., & Offord, E. (2001). Comparison of the antioxidant
Hodgson, J. M., Proudfoot, J. M., Croft, K. D., Puddey, I. B., Mori, T. A., & Beilin, activity of commonly consumed polyphenolic beverages (coffee, cocoa,
L. J. (1999). Comparison of the effects of black and green tea on in vitro and tea) prepared per cup serving. Journal of Agricultural and Food
lipoprotein oxidation in human serum. Journal of the Science of Food and Chemistry, 49, 3438–3442.
Agriculture, 79, 561–566. Rietveld, A., & Wiseman, S. (2003). Antioxidant effects of tea: Evidence from
Hoff, J. F., & Singleton, K. I. (1977). A method for determination of tannin in human clinical trials. Journal of Nutrition, 133(10), 3285S–3292S.
foods by means of immobilized enzymes. Journal of Food Science, 42, Roginsky, V., Barsukova, T., Hsu, C. F., & Kilmartin, P. A. (2003). Chain-
1566–1568. breaking anti- oxidant activity and cyclic voltammetry characterization
Jiang, H. Y. (2009). White tea. Its manufacture, chemistry and health effects. of polyphenols in a range of green, oolong, and black teas. Journal of
In C. T. Ho, Agricultural and Food Chemistry, 51, 5798–5802.
J. K. Lin, & F. Shahidi (Eds.), Tea and tea products. Chemistry and health- Saikia, P., & Mahanta, P. K. (2002). Specific fluctuations in the composition of
promoting properties. (pp. 17–30) Boca Raton: CRC Press. lipoxygenase- and glycosidase-generated flavors in some cultivated teas of
Kan, N. (1980). The aroma constituents of various teas. Food Industry, 12, Assam. Journal of Agricultural and Food Chemistry, 50, 7691–7699.
19–22. Saito, S. T., Gosmann, G., Saffi, J., Presser, M., Richter, M. F., & Bergold, A.
Karori, S. M., Wachira, F. N., Wanyoko, J. K., & Ngure, R. M. (2007). Antioxidant M. (2007). Characterization of the constituents and antioxidant activity
capacity of different types of tea products. African Journal of Biotechnology, of Brazilian green tea (Camellia sinensis var. assamica IAC-259 cultivar)
6(19), 2287–2296. extracts. Journal of Agricultural and Food Chemistry, 55, 9409–9414.
Khokhar, S., & Magnusdottir, S. G. (2002). Total phenol, catechin, and Sanchez-Moreno, C., Jimenez-Escrig, A., & Saura-Calixto, F. (2000). Study
caffeine contents of teas commonly consumed in the United kingdom. of low-density lipoprotein oxidizability indexes to measure the
Journal of Agricultural and Food Chemistry, 50(3), 565–570. antioxidant activity of dietary polyphenols. Nutrition Research, 20,
Lin, Y. -L., Juan, I. -M., Chen, Y. -L., Liang, Y. -C., & Lin, J. -K. (1996). Composition 941–953.
of poly- phenols in fresh tea leaves and associations of their oxygen- Singleton, V. L., Orthofer, R., Lamuela-Raventos, R. M., & Lester, P. (1999).
radical-absorbing ca- pacity with antiproliferative actions in fibroblast cells. Analysis of total phenols and other oxidation substrates and antioxidants
Journal of Agricultural and Food Chemistry, 44, 1387–1394. by means of Folin– Ciocalteu reagent. Methods in Enzymology, 299, 152–
Lin, S. -D., Liu, E. -H., & Mau, J. -L. (2008). Effect of brewing methods on 178.
antioxidant properties of steaming green tea. LWT—Food Science and Spiro, M., & Price, W. E. (1986). Determination of theaflavins in tea
Technology, 41, 1616–1623. solution using the Flavognost complexation method. Analyst, 111,
Lin, Y. S., Tsai, Y. J., Tsay, J. S., & Lin, J. K. (2003). Factors affecting the levels of 331–333.
tea poly- phenols and caffeine in tea leaves. Journal of Agricultural and Food Venditti, E., Bacchetti, T., Tiano, L., Carloni, P., Greci, L., & Damiani, E.
Chemistry, 51, 1864–1873. (2010). Hot vs. cold water steeping of different teas: Do they affect
Morel, I., Lescoat, G., Cillard, P., & Cillard, J. (1994). Role of flavonoids and antioxidant activity? Food Chemistry, 119, 1597–1604.
iron chelation in antioxidant action. Methods in Enzymology, 234, 437– Viollier, E., Inglett, P. W., Hunter, K., Roychoudhury, A. N., & Van Cappellen,
443. P. (2000). The ferrozine method revisited: Fe(II)/Fe(III) determination in
Obanda, M., Owuor, P. O., & Mang'oka, R. (2004). Changes in thearubigen natural waters. Applied Geochemistry, 15, 785–790.
fractions and theaflavins levels due to variations in processing von Gadow, A., Joubert, B., & Hansmann, C. F. (1997). Comparison of the
conditions and their effects on black tea liquor brightness and total antioxidant ac- tivity of rooibos tea (Aspalathus linearis) with green,
colour. Food Chemistry, 85, 163–173. oolong, and black teas. Food Chemistry, 60, 73–77.
Pellegrini, N., Ke, R., Yang, M., Rice-Evans, C., & Lester, P. (1999). Screening Wicremasinghe, R. L. (1974). The mechanism of operation of climatic
of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant factors in the bio- genesis of tea flavor. Phytochemistry, 13, 2057–
activities applying 2,2′-azinobis(3-ethylenebenzothiazoline-6-sulfonic acid) 2061.
radical cation decolori- zation assay. Methods in Enzymology, 299, 379– Zuo, Y., Chen, H., & Deng, Y. (2002). Simultaneous determination of
389. catechins, caffeine and gallic acids in green, Oolong, black and pu-erh
Prior, R. L., & Cao, G. (1999). In vivo total antioxidant capacity: Comparison teas using HPLC with a photo- diode array detector. Talanta, 57, 307–
of different analytical methods. Free Radical Biology & Medicine, 27, 316.
1173–1181.
Prior, R. L., Wu, X., & Schaich, K. (2005). Standardized methods for the
determination of antioxidant capacity and phenolics in foods and dietary
supplements. Journal of Agricultural and Food Chemistry, 53, 4290–4302.