International Journal of Food Science and Technology 2010, 45, 2071–2079 2071

Original article
A simple and rapid turbidimetric method for determining catechins
in beverages

Motokazu Nakayama,1* Naofumi Shigemune,1 Takashi Tsugukuni,1 Hajime Tokuda1 & Takahisa Miyamoto2
1 Kao Corporation, Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan
2 Division of Food Biotechnology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University,
6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
(Received 8 March 2010; Accepted in revised form 1 July 2010)

Summary We have developed a simple and rapid turbidimetric method to determine catechins based on the fact that
many polyphenols produce hydrogen peroxide in an alkaline environment and that hydrogen peroxide
oxidises cerium to generate cerium oxide precipitates. Four catechins (epicatechin, epigallocatechin,
epicatechin gallate, epigallocatechin gallate) aggregated with these precipitates to form massive precipitates
with increased turbidity. The catechins solution (0.18 mL) was mixed with 0.02 mL of 1% CeCl3 solution,
and absorbance (650 nm) was measured immediately after agitation for 3 min using a spectrophotometer.
Absorbance was strongly correlated (0.99) with the concentration of each catechin compound. For
commercially bottled green tea, the estimated catechin content determined using this turbidimetric method
showed better correlation with the content determined by high-performance liquid chromatography than
that determined using ferrous tartrate method, which is the official Japanese method for determining the
tannin content of green tea.
Keywords Catechin, CeCl3, hydrogen peroxide, polyphenol measurement, turbidimetric method.

ellagic acid, and curcumin, in addition to their poly-

Practical Application
We present a turbidimetric method for determining
catechins that is simple, rapid, and accurate compared
to conventional methods. This novel method could be
applied for the rapid determination of catechins in the
process control of beverage production.
Introduction
Polyphenols with multiple phenolic hydroxyls as food
components are attracting much attention because of
their various physiological effects, including anticancer
(Buschman, 1998; Ahmad et al., 1999), anti-obesity
(Murase et al., 2002; Maki et al., 2009), anti-atherogenic
(Dilip et al., 2009), and anti-allergic (Yoshino et al.,
2004) effects. Thus, the determination of polyphenols is
important for evaluating the health function of foods.
Polyphenols are characteristic to plants, being biosyn-
thesised via various pathways depending on the plant.
Major classifications of polyphenols include flavonoids,
which are monomers, chlorogenic acid, gallic acid,
*Correspondence: Fax: +81-285-68-7403;
e-mail: nakayama.motokazu@kao.co.jp
merised tannins (Dilip & Arjan, 2009), comprising more
than 1000 varieties of polyphenols (Dilip & Arjan,
2009). Foods made from plants contain mixtures of
several of these varieties. Owing to such variety,
numerous methods to determine polyphenol content
have been proposed based on different principles.
Among them, the colorimetric method using ferrous
tartrate has become the official Japanese method for
determining green tea tannins (Nihal et al., 2006), while
the widely used Folin-Denis method (1915) is the official
method of the AOAC International. These methods, as
well as colorimetric determination utilising spectros-
copy, however, have various problems related to sample
colour effects and time-consuming colour reactions
(approximately 40–60 min). Determination using high-
performance liquid chromatography (HPLC) (Natsume
et al., 2000) also has drawbacks, including prolonged
analysis time, required preparation of reference stan-
dards, and limitations based on the structure of the
components to be determined. Thus, a simple and rapid
determination method is in high demand, especially for
process control during beverage production. We have
been investigating various methods of polyphenol
determination by considering factors, such as analysis

doi:10.1111/j.1365-2621.2010.02372.x
 2010 The Authors. International Journal of Food Science and Technology  2010 Institute of Food Science and Technology
2072 Rapid catechins determination method M. Nakayama et al.
time and effects of sample colour. Our results led us to
develop a simple and rapid turbidimetric method for
determining catechins, which have structure of 3-hy-
droxyflavan, based on the fact that many polyphenols
produce hydrogen peroxide in an alkaline environment
(Arakawa et al., 2002) and that hydrogen peroxide
oxidises cerium to produce cerium oxide precipitates
(3H2O2 + 2Ce3+ + 6OH) fi 2Ce(OH)3OOHfl +
2H2O) (Briggs et al., 1975).
Materials and methods
Reagents
The following polyphenol compounds were used in this
study: epigallocatechin gallate (EGCg; Teavigo; DSM
Nutrition Japan, Tokyo, Japan); epigallocatechin (Sig-
ma-Aldrich Chemical Co., St Louis, MO, USA);
epicatechin gallate (Sigma-Aldrich); epicatechin
(Sigma-Aldrich); gallic acid monohydrate (Wako Pure
Chemical Industries, Ltd., Osaka, Japan); ethyl gallate
(Wako Pure Chemical Industries, Ltd., Osaka, Japan);
and chlorogenic acid (Cayman Chemical Co. Ann
Arbor, MI, USA). The chemical structure of each
compound is shown in Fig. 1.
Measurement of hydrogen peroxide produced by catechins
The effects of pH on hydrogen peroxide production of
catechins were investigated using 500 ppm EGCg
dissolved in 10 mm 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid (HEPES) buffer (Irvine Scientific
Figure 1 Structure of some polyphenols.
International Journal of Food Science and Technology 2010
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Co. Santa Ana, CA, USA) at various pH levels with
NaOH or HCl. The solutions were incubated for
30 min before measuring the hydrogen peroxide con-
centration using the colorimetric hydrogen peroxide
kit (Assay Designs Co. Ann Arbor, MI, USA).
The catechin concentration-dependent productivity of
hydrogen peroxide and the time course of hydrogen
peroxide production were investigated for EGCg at
concentrations of 250, 500, or 1000 ppm in 10 mm
HEPES buffer at pH 8.0. Each solution was incubated
for 0, 5, 10, 20, 30, and 60 min before measurement of
the hydrogen peroxide concentration using the same
hydrogen peroxide kit.
Measurement of UV ⁄ VS absorption spectrum
Each of the four catechins (500 ppm), or purified water
as a control, was dissolved in 10 mm HEPES buffer (pH
8.0), and then the UV ⁄ VS absorption spectrum of each
solution was measured using a spectrophotometer
(Spectra Max 190; Molecular Devices Co. Sunnyvale,
CA, USA). The UV ⁄ VS absorption spectrum of each
compound (500 ppm; and purified water) in 10 mm
HEPES buffer (pH 8.0) was also measured immediately
after the addition of 1% CeCl3 solution at 1 ⁄ 10 (v ⁄ v) the
total volume of the solution.
Aggregate production from hydrogen peroxide or EGCg
following the cerium reaction
In a 1.5-mL test tube, 0.05 mL of 2% CeCl3 solution
was added to 0.95 mL of 10 mm HEPES buffer (pH 8.0)
 2010 The Authors

containing 0, 250, or 500 ppm EGCg. In addition,
20 mm HEPES buffer (pH 9.0) was mixed with 30%
hydrogen peroxide (special grade; Wako Pure Chemical
Industries, Ltd.) at a 1:1 ratio, adjusted to pH 8.0 by
adding 1 N NaOH. The pH-adjusted solution was
diluted with HEPES buffer (pH 8.0) to 0, 0.003, 0.3,
3.0, 30.0, or 300.0 ppm hydrogen peroxide, and 0.95 mL
of each dilution was mixed with 0.05 mL of 2% CeCl3
solution. Immediately after the agitation of each aggre-
gation solution, 200 lL of reaction solution was trans-
ferred to the well of a microtitre plate and absorbance
(650 nm) was measured and compared between reac-
tions.
FT-IR analysis
Samples of EGCg and CeCl3 were fine powders and
their reaction products were obtained for analysis by
adding 50 lL of 2.0% CeCl3 solution to 950 lL of
1000 ppm solution of EGCg in pH 8.0 HEPES buffer.
Precipitates were collected by centrifugation at 20 000 g,
washing with deionised water, and centrifugation again.
Samples of EGCg, CeCl3 and their reaction products
were analysed using an Fourier transform infrared
spectrophotometer (FT-IR) spectrometer (Spectru-
mOne, PerkinElmer Co. Waltham, MA, USA) equipped
with a single reflection diamond ATR accessory (Uni-
versalATR, PerkinElmer Co.) with a 4-cm)1 wavenum-
ber resolution four consecutive times (accumulation:
four times).
Turbidimetric method using cerium for polyphenol
determination and turbidity stability
Each of the four catechins was dissolved at 1000 ppm in
10 mm HEPES buffer adjusted to pH 6.0, 7.0, or 8.0.
Each of the catechin solutions was diluted with HEPES
buffer to attain a final concentration ranging from 0 to
1000 ppm. Ethyl gallate and chlorogenic acid were also
dissolved in 10 mm HEPES buffer adjusted to pH 8.0.
Each solution (0.18 mL) was dispensed into a well of a
96-well microtitre plate (Becton and Dickinson Co.
Franklin Lakes, NJ, USA), followed by the addition of
0.02 mL of 1% CeCl3 solution. Immediately after
agitating the plate, absorbance (650 nm) was measured
using the spectrophotometer. Calibration curves were
prepared by plotting concentrations of the catechin
compound, ethyl gallate, and chlorogenic acid against
absorbance.
The stability of the reaction products was also anal-
ysed for EGCg at concentrations of 0, 62.5, 125, 250, and
500 ppm in 10 mm HEPES buffer (pH 8.0). Each
solution (0.18 mL) was dispensed in a well of a 96-well
microtitre plate, followed by the addition of 0.02 mL of
1% CeCl3 solution. Absorbance (650 nm) was measured
at 0, 10, 20, 40, and 60 min after agitating the plate.
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International Journal of Food Science and Technology  2010 Institute of Food Science and Technology
Rapid catechins determination method M. Nakayama et al. 2073
Comparison of catechin concentration determined by the
present turbidimetric method using cerium and by HPLC
To prepare an EGCg solution of unknown concentra-
tion, an appropriate amount of EGCg was dissolved in
10 mm of HEPES buffer (pH 5.0). Determination was
performed both by the turbidimetric method using
cerium and by HPLC, and the results were compared.
For the turbidimetric method using cerium, turbidity
was measured immediately after adding NaOH to adjust
the solution to pH 8.0, followed by the addition of 1%
CeCl3 solution. The calibration curves prepared as
described previously were then used for the determina-
tion. For determination by HPLC, sample solutions
were filtered (0.45-lm pore size; Millipore Co. Billerica,
MA, USA) and analysed using the gradient method with
an HPLC system (SCL-10AVP; Shimadzu Corp. Kyoto,
Japan) and an octadecyl silane column (L-column ODS;
u 4.6 · 250 mm; Chemical Evaluation and Research
Institute, Tokyo, Japan). The column conditions were as
follows: mobile phase A, distilled water containing 0.1 m
acetic acid; mobile phase B, acetonitrile containing
0.1 m acetic acid; flow rate, 1 mL per min; injection
volume, 10 lL; UV detector (at 280 nm); and column
temperature, 35 C. The linear gradient conditions used
for analysis are shown in Table 1. To prepare the EGCg
calibration curve, 50 mg EGCg was weighed out and
mixed with a 50% aqueous solution of methanol (v ⁄ v,
containing 4 mm HCl) and then made up to 50 mL in a
volumetric flask. This solution was serially diluted
twofold up to 32-fold and used to measure the calibra-
tion curve. EGCg concentration was calculated on the
basis of this curve.
Effect of the presence of ascorbic acid (reducing agent)
EGCg solutions in 10 mm HEPES buffer (pH 8.0) were
prepared at concentrations of 0, 125, 250, 500, and
1000 ppm without and with the addition of 0.006, 0.010,
0.030, 0.050, 0.100% ascorbic acid. Each solution
(0.18 mL) was dispensed in a well of a 96-well microtitre
Table 1 Gradient conditions used for analysis
Concentration of Concentration
solution A* of solution B†
Time (min) (% by volume) (% by volume)
0.0 97 3
37.0 80 20
43.0 80 20
43.5 0 100
48.5 0 100
49.0 97 3
60.0 97 3
*
Solution A is distilled water containing 0.1 M acetic acid.
†
Solution B is acetonitrile containing 0.1 M acetic acid.
International Journal of Food Science and Technology 2010
2074 Rapid catechins determination method M. Nakayama et al.
plate (Becton and Dickinson Co.), followed by the
addition of 0.02 mL of 1% CeCl3 solution. Immediately
after agitating the plate, absorbance (650 nm) was
measured. A calibration curve was prepared by plotting
catechin concentration against absorbance.
Analysis of catechins in green tea beverages
Two different beverages, labelled drinks A and B, were
each mixed at a 1:1 ratio with 20 mm HEPES buffer (pH
8.0). The catechins content in each beverage was then
determined using the turbidimetric method with cerium
as described previously. In the same manner as the
ferrous tartrate method, a calibration curve was prepared
for ethyl gallate and the catechins composition in the
beverages was analysed. First, 100 mg ferrous sulphate
(ferrous sulphate heptahydrate, Wako Pure Chemical
Industries, Ltd.) and 500 mg potassium sodium tartrate
(potassium sodium(+)-tartrate tetrahydrate, Wako Pure
Chemical Industries, Ltd.) were dissolved in water to
prepare 10 mL of ferrous tartrate reagent. A 5-level
calibration curve was prepared using 5–25 mg ⁄ 100 mL
ethyl gallate (Tokyo Chemical Industry Co., Ltd.) in
5-mg increments, in accordance with the official Japanese
method for the determination of tannins in tea beverages.
After combining a 5-mL sample with 5 mL ferrous
tartrate reagent in a 25-mL volumetric flask, 67 mm
phosphate buffer (pH 7.5) was added to the fixed volume
(25 mL). The amount of ethyl gallate was calculated
using the calibration curve prepared by measuring
absorbance (540 nm) of water as the blank, and the
amount of tannins was calculated by multiplying this
value by 1.5. The amounts were then compared to those
of catechins calculated by HPLC. The HPLC catechin
calibration curve was prepared by weighing out 10 mg
catechin (Mitsui Norin Co. Ltd. Shizuoka, Japan), 25 mg
gallocatechin (Mitsui Norin Co. Ltd.), 10 mg catechin
gallate (Mitsui Norin Co. Ltd.), 25 mg gallocatechin
gallate (Mitsui Norin Co. Ltd.), 15 mg epicatechin,
50 mg epigallocatechin, 15 mg epicatechin gallate, and
50 mg EGCg; these were mixed with a 50% aqueous
solution of methanol (v ⁄ v, containing 4 mm HCl) and
made up to 50 mL in a volumetric flask. The solutions
were serially diluted twofold up to 32-fold, and the
resulting solutions were measured for the calibration
curve.
Results and discussion
Amount of hydrogen peroxide produced by catechins
After dissolving 500 ppm EGCg in solutions of 10 mm
HEPES buffer adjusted to various pHs, solutions were
incubated for 30 min before analysing the hydrogen
peroxide concentration in each solution. The hydrogen
peroxide concentration increased in a pH-dependent
International Journal of Food Science and Technology 2010
International Journal of Food Science and Technology  2010 Institute of Food Science and Technology
Figure 2 Time course of hydrogen peroxide generation from EGCg at
250 (m), 500 (n), and 1000 (¤) ppm in 10 mm HEPES buffer (pH 8.0).
manner in solutions at pH 7.0 and 8.0; hydrogen
peroxide concentrations in solutions at pH 5.0 and 6.0
were below the limit of detection.
The time course change in the hydrogen peroxide
concentration in each solution was measured for various
concentrations of EGCg (250, 500, and 1000 ppm)
dissolved in 10 mm HEPES buffer (pH 8.0) (Fig. 2).
The hydrogen peroxide concentration increased rapidly
immediately after adding EGCg. The rate of hydrogen
peroxide production decreased after 5 (1000 ppm), 10
(500 ppm), and 20 (250 ppm) min. Thus, unless other-
wise stated, measurements were taken within the 5-min
period in which linearity was observed between a wide
range of EGCg concentrations and hydrogen peroxide
production. In addition, for all subsequent data, a
calibration curve was produced every time a measure-
ment was taken.
Conditions for the turbidimetric method
To confirm the absorbance wavelength that is most
suitable for determining the reaction products which
result from the addition of CeCl3 to catechins (10 mm
HEPES buffer, pH 8.0), we measured the absorbance
spectra in the UV ⁄ Vis range (200–760 nm; Fig. 3).
Solutions with and without CeCl3 showed high levels
of absorption at UV wavelengths specific to catechins.
No absorption was detected for the CeCl3 solution alone
(data not shown). Absorption also occurred at wave-
lengths above 400 nm, but only in solutions in which
CeCl3 reacted with a catechin, most likely corresponding
to aggregates produced during the reaction. Based on
these results, aggregate was analysed by turbidimetry at
650 nm, a wavelength frequently used in turbidimetry.
Comparison of turbidity between catechin and hydrogen
peroxide after the CeCl3 reaction and FT-IR analysis
After a 10-min incubation, approximately 5 ppm hydro-
gen peroxide was produced from 500 ppm EGCg at
 2010 The Authors

(a)
Figure 3 UV ⁄ VIS absorption spectra of cate-
chin solutions before and after reaction with
CeCl3. UV ⁄ VIS absorption spectra of epi-
catechin (n), epigallocatechin ()), epicatechin
gallate (•), or EGCg (4) were measured at
500 ppm in 10 mm HEPES buffer (pH 8.0),
before (a) and after (b) adding 0.1% CeCl3
(final concentration).
Figure 4 Relationship between hydrogen peroxide concentration and
turbidity of the reaction mixture. EGCg or hydrogen peroxide was
reacted with cerium chloride. EGCg (m) and hydrogen peroxide (¤)
were dissolved in 10 mm HEPES buffer (pH 8.0), and CeCl3 was added
to a final concentration of 0.1%. EGCg concentration was 250 and
500 ppm, producing 8.5 and 13.0 ppm hydrogen peroxide, respectively
at pH8.0.
pH 8 (Fig. 4). Mean turbidity of 0.64 resulted from the
reaction of 500 ppm EGCg with CeCl3, while it was
much lower (0.23) in the reaction of 15% hydrogen
peroxide (data not shown). Comparing the IR spectral
patterns of the three samples of EGCg, CeCl3, and their
aggregates, peaks specific to EGCg were confirmed in
the aggregates, indicating that aggregates contained
both Ce(OH)3OOH, produced by the reaction between
CeCl3 and hydrogen peroxide, and EGCg. Turbidity
resulting from the CeCl3 reaction with 500 ppm cate-
chins was high when extremely low levels of hydrogen
peroxide were produced compared to the turbidity of
15% hydrogen peroxide. Thus, upon reaction of cerium
with hydrogen peroxide, cerium oxide is produced,
which subsequently reacts with EGCg to form com-
plexes (Japanese Patent Publication; P2000-203834).
This results in further reaction of the catechins, which
are oxides, with cerium oxide to form complexes, which
 2010 The Authors
International Journal of Food Science and Technology  2010 Institute of Food Science and Technology
Rapid catechins determination method M. Nakayama et al. 2075
(b)
Figure 5 FT-IR spectra of EGCg, CeCl3, and products generated by
EGCg and CeCl3.
we observed as large aggregates. Our FT-IR results
showing EGCg within the aggregates (Fig. 5) confirmed
this observation. The FT-IR spectra also showed
broadening of the peaks derived from catechins in the
aggregate, compared to those for catechin alone, which
is usual considering that EGCg gives sharper IR
absorbance peaks, in a dry state. It appears, however,
that these peaks show broadening and some peak shift
with hydration. Changes of absorption wavelength were
also observed as a result of aggregation. Major absorp-
tion peak patterns accorded when spectra were com-
pared before and after aggregation, indicating that these
broad peaks correspond to those specific to EGCg.
Thus, the results show that catechin aggregates with the
cerium oxide precipitate to form complex which subse-
quently grows more massive, causing a further increase
in turbidity.
Trace amounts of hydrogen peroxide produced by
catechins might consequently result in large changes
in turbidity in this system. Preliminary electron micro-
scopic observations of the aggregates support this
conclusion; that is, aggregates resulting from the
International Journal of Food Science and Technology 2010
2076 Rapid catechins determination method M. Nakayama et al.
Figure 6 Effect of reaction time of CeCl3 and EGCg on turbidity of the
reaction mixture. Concentration of EGCg: 500 (¤), 250 (n), 125 (m),
63 (·), and 0 (*) ppm.
reaction of CeCl3 and 500 ppm EGCg were larger than
those produced by the reaction of CeCl3 and hydrogen
peroxide with a 30 000-fold higher concentration than
that produced by 500 ppm EGCg (data not shown). We
also analysed the stability of the resulting aggregates
(Fig. 6). After the reaction of EGCg and CeCl3 for up to
20 min, the EGCg concentration in the mixtures was
strongly correlated with absorbance, although turbidity
slightly decreased over time. Conversely, at 62.5 ppm
EGCg, however, the post-reaction absorbance tended to
increase over time; the reason is unclear for this gradual
decrease in turbidity over time with increasing EGCg
concentration, when at 62.5 ppm the turbidity rises.
However, as highly quantitative values appear to be
obtained at a reaction time 10 min or less, measure-
ments were made at 3 min in this study, unless otherwise
stated.
(a) (c)
(b) (d)
International Journal of Food Science and Technology 2010
International Journal of Food Science and Technology  2010 Institute of Food Science and Technology
Determination of polyphenols and stability of turbidity
The four catechins were reacted with CeCl3 to produce
aggregates that were analysed by measuring absorbance
at 650 nm (Fig. 7). Calibration curves showing the
catechin concentration relative to absorbance showed
that at pH 6, none of the catechins produced aggregates
and there was no correlation between the catechin
concentration and absorbance. Regression analysis per-
formed using the least squares method and correlation
coefficients to calculate regression equations, however,
showed at pH of 7 or higher, a strong correlation
between absorbance and the catechin concentration for
each catechin, despite differences in the rate of change of
absorbance at each pH and the range of concentrations
in which the linear increase in absorbance occurred. For
EGCg, epigallocatechin, epicatechin gallate, and epi-
catechin, the correlation coefficients were high (>0.99)
and their calibration curves were confirmed to be linear.
There was no difference between epi-type and non-epi-
type catechins in the slope of the calibration curve or
where it intercepts the y-axis (data not shown). As
already indicated, catechins produce large quantities of
hydrogen peroxide in an alkaline environment, although
epicatechin has been shown to produce little hydrogen
peroxide, particularly at pH 8.0, under the same
experimental conditions in this study (Akagawa et al.,
2003). While the plot for epicatechin had only a gentle
slope, the plots for the three other catechins showed
steep slopes. Thus, there appears to be a strong
correlation between hydrogen peroxide produced by
the catechins and absorbance. The amount of hydrogen
peroxide produced in catechin solutions increased in a
pH-dependent manner as the solution became alkaline.
Figure 7 Relationship between polyphenol
concentration and turbidity determined by
the present rapid method on various catechins
in 10 mm HEPES buffer at pH 8.0 (¤), pH 7.0
(n), pH 6.0 (·).
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(a)
Figure 8 Relationship between polyphenol
concentration and turbidity determined
by the present rapid method on ethyl gal-
late and chlorogenic acid in 10 mm HEPES
buffer (pH 8.0).
On the other hand, turbidity as a result of aggregates
formation by the addition of CeCl3 also increased in a
pH-dependent manner, suggesting that aggregates
formed upon adding CeCl3 to the four catechins; that
is, these products were formed by the reaction of cerium
ions and the hydrogen peroxide produced by the
catechins.
Regression analysis by the least squares method was
similarly performed for ethyl gallate and chlorogenic
acid at pH 8.0 (Fig. 8) and resulted in a high correlation
coefficient (‡0.99), indicating a very strong correlation
between absorbance and the ethyl gallate and chloro-
genic acid concentrations.
As the production of hydrogen peroxide by polyphe-
nols requires disassociation of their –OH group, the
conditions for the reaction between cerium and catechins
must be alkaline. The disassociation state of each
catechin compound can be predicted using molecular
orbital calculation software (Herrero-Martı́nez et al.,
2005). Using such software, the pH at which –OH
disassociation initially occurs (Nakayama et al., 2009) is
roughly equivalent to the pH at which the correlation
between turbidity and catechin concentration occurs
(data not shown). Thus, pH during the reaction is an
important factor in the determination of catechins.
Aggregation of catechins with cerium oxide precipitates
occurs rapidly at pH ‡8.0, leading to unstable determi-
nation results. Moreover, at pH <7.0, hydrogen perox-
ide production is low. Although pH 8.0 was thought to
be optimal for analysis, problems arose because of
detection limitations. In addition, when the slopes of the
calibration curves created with our turbidimetric method
(Fig. 7) were compared at the same pH for each catechin
compound, the steepness of the slope increased relative
to the amount of hydrogen peroxide produced by the
catechin compound; that is, EGCg > epigallocate-
chin = epicatechin gallate  epicatechin (Akagawa
et al., 2003). Similarly, turbidity was strongly correlated
with the ethyl gallate and chlorogenic acid concentra-
tions (Fig. 8). These results clearly demonstrate that our
turbidimetric method using cerium has excellent sensi-
tivity for pyrogallol containing polyphenol determina-
tion.
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Rapid catechins determination method M. Nakayama et al. 2077
(b)
Determination capability compared to HPLC
To investigate the correlation between the results of our
turbidimetric method using cerium and the conventional
quantitative method by HPLC, samples (n = 3) were
prepared with unknown concentrations of EGCg (around
400 ppm). There was a strong correlation between the
samples using the two methods (data not shown). That is,
the quantitative value of the sample by our method was
92.6–94.7% of the value obtained by HPLC.
Effect of ascorbic acid (reducing agent)
Ascorbic acid is usually added to beverages containing
polyphenols, such as tea catechins, to prevent aggregation
and precipitation as a result of oxidative degradation and
oxidative polymerisation of polyphenols. Using our
turbidimetric method with cerium for polyphenol deter-
mination in commercial tea beverages, reducing agents
like ascorbic acid that remove hydrogen peroxide are
considered to interfere with measurement. Accordingly,
we analysed EGCg at various concentrations with our
method both with and without ascorbic acid (Fig. 9).
Although the addition of ascorbic acid decreased the
slope of the calibration curve, turbidity after the reaction
with CeCl3 increased with the amount of EGCg. More-
over, the correlation between the EGCg concentration
and turbidity was high, with a correlation coefficient of
‡0.99, and the calibration curve was confirmed to be
linear. In our turbidimetric method, the amount of
polyphenol is basically calculated from the amount
of hydrogen peroxide produced. When an agent that
eliminates hydrogen peroxide, such as ascorbic acid, is
present, however, the results are still correlated with those
of the HPLC analysis, although the slope of the calibra-
tion curve is decreased. Although ascorbic acid did not
alter the slope of the calibration curve for EGCg at
0.006%, it inhibited the reaction for cerium oxide
formation at 0.01% or above. Commercial bottled green
tea products commonly contain about 0.03% ascorbic
acid. It seems that 10-fold diluted sample would be
suitable for the determination of catechins in the bottled
green tea by our method.
International Journal of Food Science and Technology 2010
2078 Rapid catechins determination method M. Nakayama et al.

method indicated that tannin levels of drinks A and B

Figure 9 Effects of ascorbic acid concentration on the present turbi-
dimetric method for determining EGCg. Symbols and calibration
curve: (s) 0.000%, y = 0.0012x + 0.0265; (h) 0.006%,
y = 0.0012x + 0.0204; (·) 0.010%, y = 0.0010x + 0.0084; (m,
y = 0.0009x + 0.0057; (h) 0.050%, y = 0.0008x + 0.0212; (¤)
0.100%, y = 0.0008x + 0.0358.
Analysis of polyphenols in actual green tea beverages
To confirm the use of our turbidimetric method using
cerium for the analysis of actual beverages, the tannin
level in two drinks (A and B) was analysed by the
ferrous tartrate method, HPLC, and our method
(Tables 2 and 3). Results using the ferrous tartrate
were 132.3% and 111.5% those of the values determined
by HPLC, respectively. Large discrepancies were also
found between the levels in the beverages, possibly
because of effects from other components. When
approximately 30 ppm ascorbic acid was included in
both beverages, the values obtained by our method
using calibration curves created with the EGCg solution
containing no ascorbic acid were much lower than those
measured by HPLC: 23.1% and 30.0% of the values
determined by HPLC in drinks A and B, respectively. In
contrast, when the EGCg content of the beverages was
estimated by our method using the calibration curves
created with the EGCg solution containing 30 ppm
ascorbic acid, the respective polyphenol level in drinks A
and B was 83.3% and 78.6% of those determined by
HPLC. The higher values from the ferrous tartrate
method compared with those from HPLC, and the
considerable variation in values between beverages from
the ferrous tartrate method, appear to be as a result of
the effects of components other than polyphenols as
chlorogenic acid and gallic acid. However, while the
values obtained from our method are somewhat lower
than those from HPLC, they vary little from those
obtained by HPLC, indicating that our method yields
satisfactory results. Considering the presence of reduc-
ing agents, including ascorbic acid in foods, our
turbidimetric method showed less interference from
components in the beverage compared to the ferrous
tartrate method, an advantage of our method for
polyphenol determination in food.

Table 2 Measurement of concentration of catechins in drinks by HPLC

Concentration (ppm)

Drink GC EGC C EC EGCg GCg ECg Cg Total

A 89.8 (26.4) 51.9 (15.2) 21.0 (6.2) 15.5 (4.6) 66.2 (19.4) 66.4 (19.5) 18.7 (5.5) 11.3 (3.3) 340.7 (100.0)
B 167.6 (32.0) 93.4 (19.7) 39.2 (7.5) 21.5 (4.1) 77.1 (14.7) 88.3 (16.9) 20.5 (3.9) 15.7 (3.0) 523.2 (100.0)

Values in parentheses are the percentage of catechins in total amount of catechins.

GC, Gallocatechin gallate; EGC, Epigallocatechin gallate; C, Catechin; EC, Epicatechin; EGCg; Epigallocatechin gallate, GCg, gallocatechin gallate; ECg,
Epicatechin gallate; Cg, Catechin gallate.

Table 3 Comparison of polyphenol concentration in drinks measured by different methods

Rapid turbidimetric method

Ferrous tartrate Without ascorbic With ascorbic

Drink HPLC method acid (0.00%) acid (0.05%)

A 340.7 (100.0) 451.1 (132.4) 78.8 (23.1) 284.3 (83.4)

B 523.2 (100.0) 582.8 (111.4) 157.0 (30.0) 410.8 (78.5)

Values in parentheses are the values relative to those obtained by high-performance liquid chromatography (HPLC), where values for HPLC are 100%.

International Journal of Food Science and Technology 2010  2010 The Authors
International Journal of Food Science and Technology  2010 Institute of Food Science and Technology

Conclusion
We developed a simple and rapid turbidimetric method
for catechins determination based on the fact that many
polyphenols produce hydrogen peroxide in an alkaline
environment and that hydrogen peroxide oxidises cerium
to generate cerium oxide precipitates. Our turbidimetric
method can determine catechins simply by preparing a
buffer at a pH that allows the CeCl3 reaction to occur.
Determination can be performed by simply mixing cerium
and catechins at pH 7.0, at which the –OH group
dissociates in catechins, or preferably at pH 8.0, at which
polymerisation is suppressed to a certain extent. This
method has good stability for up to a 20-min reaction
time, at which point the aggregates, which serve as an
indicator, are produced. Although interference by reduc-
ing agents like ascorbic acid should be avoided by
preparing a calibration curve in the presence of reducing
agents, it is a very simple and accurate method for
determining catechins compared with conventional meth-
ods. This method could be used for the rapid determina-
tion of catechins in the process control of beverage
production. This method has already been shown to be
effective in measuring gallic acid and chlorogenic acid,
indicating its applicability to polyphenols other than
catechins.
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