International Journal of Food Science and Technology 2015, 50, 885891

885

Original article
Analysis of tea catechins in vegetable oils by high-performance
liquid chromatography combined with liquidliquid extraction
Xin Zhang,* Zufang Wu, Peifang Weng & Yang Yang
Department of Food Science and Engineering, School of Marine Science, NingBo University, Ningbo 315211, China
(Received 26 August 2014; Accepted in revised form 16 November 2014)

Summary

In this study, a method was developed for the determination of various tea catechins in vegetable oils.
Firstly, vegetable oils including tea seed oil, sunower seed oil and soya bean oil were extracted by methanol/water (40:60, v/v), and then, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of GA, caeine, EGC, EGCG, EC, ECG, GC, GCG, C and
CG. For the compounds detected in tested vegetable oils, LODs were in the range of 0.051.65 ng, both
intraday and interday relative standard deviations (RSDs) were <5.0%, and the recovery rates were in the
range of 96.2100.5% with RSD <3.7%. The results showed in vegetable oils which declared to had
added tea catechins in, the concentrations of tea catechins were less than that showed in package label,
and the content of EGCG was the highest in all samples. Therefore, the advancement made in our study
will facilitate studies of tea catechins in oil industry.

Keywords

high-performance liquid chromatography, liquidliquid extraction, tea catechins, vegetable oil.

Introduction

Tea polyphenols are one of the bioactive ingredients in

tea, mainly tea catechins, avonoids and phenolic
acids (Bolling et al., 2009). As its main ingredient, tea
catechins accounted to 6080% of total tea polyphenols, including (-)-epigallocatechin gallate (EGCG),
(-)-epigallocatechin (EGC), (-)-epicatechin gallate
(ECG) and (-)-epicatechin (EC). The chemical structures of tea catechins are illustrated in Fig. 1. The
major purine alkaloid in tea is caeine. Although caffeine has physiological eects on various body systems
(Bode & Dong, 2007), there are reports about the
adverse eects of excessive intake of caeine (Lodato
et al., 2013). Tea catechins have been involved in
many biological activities and widely used in food processing and medicine (Gramza & Korczak, 2005).
Relative to the current commonly used antioxidant
TBHQ, tea catechins have many advantages such as
natural, anticancer, anti-arteriosclerosis and they have
attracted increasing attention in oils and fats industry
(Chen & Chan, 1996; Wang et al., 2000). A number of
methods have been explored to add tea catechins to
vegetable oil; however, for the characteristic of water
soluble, the solubility of tea catechins in the oil is limited, and the determination is relatively dicult (Chen
*Correspondent: E-mail: zhangxin@nbu.edu.cn

doi:10.1111/ijfs.12726
2014 Institute of Food Science and Technology

et al., 1996; Pirisi et al., 2000). Although some

methods have been developed to determine the total
contents of phenolic compounds and various phenols
in olive oils (G
omez-Alonso et al., 2002; Ocakoglu
et al., 2009), the determination of the presence of various tea catechins in vegetable oils has not been
reported. Consequently, there is a demand for rapid
and eective analytical methods for the analysis of tea
catechins in the oil, which should be suitable across a
wide range of research and practical applications.
For the high sensitivity, high-performance liquid
chromatography (HPLC) is always selected as the
main method to determine tea catechins (Yang et al.,
2007; Hu et al., 2009). However, tea catechins in vegetable oils cannot be determined directly by HPLC, and
it should necessarily be pretreated. Although there are
studies for extraction procedures (solid/liquid SPE and
liquid/liquid LLE) and HPLC separation and quantication methods of phenolic compounds in olive oils
(Pirisi et al., 2000), reports about the application of
extraction for the sample preparation in the analysis
of tea catechins in vegetable oils are not available.
Therefore, we report here in detail liquidliquid extraction combined with HPLC for the determination of
tea catechins in vegetable oils. In this study, using
Zorbax Eclipse XDB-C18 column (Agilent Co. Ltd.,
Santa Clara, CA, USA) and diode array detector
(DAD), we established a rapid analysis of tea

886

Analysis of tea catechins in vegetable oils X. Zhang et al.

OH

OH
OH

OH

OH

OH

O
OH

OH

OH
OH

OH

(-)-Epicatechin (EC)

(-)-Epigallocatechin (EGC)

OH

OH
OH

OH

OH

OH
OH

OH

OH

OH

OH

OH

OH
O

(-)-Epicatechin gallate (ECG)

OH

OH

(-)-Epigallocatechin gallate (EGCG)

catechins in vegetable oils by HPLC method and laid

foundation for the determination of tea catechins in
vegetable oil. In addition, the contents of tea catechins
in dierent vegetable oils have also been analysed
using this developed method.
Materials and methods

Materials

Tea seed oils, sunower seed oils and soya bean oils
were obtained locally (Ningbo, Zhejiang province),
and their production dates were all within half a
month before analysis. Standards of EGCG (>98%),
ECG (>98%), EGC (>98%) and EC (>98%) were purchased from Funakoshi (Tokyo, Japan). Standards of
GA (gallic acid) (>98%) and caeine (>98%) were
obtained from Sigma-Aldrich (St. Louis, MO, USA).
Toyopearl HW-40S resin was purchased from Tosoh
(Tokyo, Japan). Folin-Ciocalteu reagent was obtained
from Fluka (Buchs, Switzerland). HPLC grade of
methanol was obtained from Hanbon Science and
Technology (Jiangsu, China). All other chemicals were
of analytical grade.

Figure 1 Chemical structures of tea catechins.

modications (Hu et al., 2009). Briey, the solution

of EC, ECG, EGC and EGCG was autoclaved and
then loaded onto a column of Toyopearl HW-40S
column pre-equilibrated with 80% ethanol. The column was eluted with 80% ethanol, and the elution
was monitored by measuring the absorbance at
280 nm and auto-collected. The eluted fractions were
analysed by HPLC, and the desired fractions were
collected, concentrated, loaded onto a column of
Toyopearl HW-40S, respectively, and treated as
described above. As a result, the fractions containing
C, CG, GC and GCG were concentrated and freezedried by a Freeze-Dry System (Labconco, Kansas
City, MO, USA), respectively.
The structures of prepared tea catechin monomers
were conrmed by electrospray ionisation time-ofight mass spectrometry (ESI-TOF-MS) and 1H
NMR. 1H NMR spectrum was recorded in D2O as
solvent with a Bruker DRX-500 spectrometer (Bruker
Co. Ltd., Rheinstetten, Germany) operated at 300 K.
Chemical shifts (d) are given in ppm, and J values are
given in Hz. ESI-TOF-MS spectra were recorded on
an Applied Biosystems mass spectrometer.
Treatment of vegetable oil by liquidliquid extraction

Preparation of tea catechins

Standards of EC, ECG, EGC and EGCG were dissolved in ddH2O directly and then ltered through a
0.45-lm cellulose lter before HPLC analysis. Epimers of C, CG, GC and GCG were prepared by heat
epimerisation of EC, ECG, EGC and EGCG, respectively, according to the reported method with some

International Journal of Food Science and Technology 2015

The extractions of tea catechins from vegetable oil

were prepared according to the reported methods
with some modications (Chen & Chan, 1996).
Before analysis, all vegetable oils were stored in the
dark. Firstly, a sample of vegetable oil (1 mL) was
extracted by 3 mL of methanol/water (40:60, v/v).
The mixture was sonicated for 10 min and then

2014 Institute of Food Science and Technology

Analysis of tea catechins in vegetable oils X. Zhang et al.

centrifuged at 4000 g at room temperature for

15 min. After the centrifugation, the methanol/water
layer was taken out and washed by 1 mL of n-hexane. The evaporation of solvent was done using a
series of evaporation methods (nitrogen gas, aspirator and rotary evaporator, respectively). After the
evaporation, the residue was dissolved in 1 mL
ddH2O. Final extract was ltered through a 0.45-lm
cellulose lter membrane and injected to HPLCDAD immediately.
Tea polyphenols content analysis

The content of tea polyphenols in extract was determined by Folin-Ciocalteu method according to the
reported procedure (Liu et al., 2009). A calibration
curve of GA (ranging from 0.02 to 0.10 mg mL 1)
was prepared, and the content of tea polyphenols was
standardised against gallic acid and expressed as mg
GA equivalent per gram of sample on a dry weight
(DW) basis.
Tea catechins content analysis

The determination of tea catechins in the solution was

determined by HPLC-DAD using a model G1379A
degasser, a model G1311A pump with a low-pressure
gradient mixer (G1311-69701), a model G1316A column oven and a model G1315B DAD system. The
separation was achieved on a Zorbax Eclipse XDBC18 column (150 mm 9 4.6 mm, 5 lm; Agilent). The
temperature of column oven was set at 40 C. The
mobile phase consisted of formic acid solution (pH
2.5, A) and methanol (B). Elution was performed with
a linear gradient by decreasing A from 80% to 40%
within a period of 15 min (Hu et al., 2009). System
ow rate was 1.0 mL min 1. Chromatographic data
were collected and integrated using Agilent Chemstation software. Calibration plots were constructed with
authentic standards by plotting peak areas from the
DAD absorbance signal at 280 nm vs. standard concentrations. The sample was ltered through 0.45-lm
cellulose lter prior to injection, and the injection
volume was 20 lL.
HPLC-DAD method validation

The selectivity criterion for an assay method is that

the analytic peaks will have a chromatographic baseline with a suitable resolution from all of the other
sample components. Mean peak areas from triplicate
HPLC-DAD analyses of a range of solution concentrations were used to determine calibration curves for
each standard, which was dened as the least-squares
regression line relating absorbance peak area at 280
nm to sample concentration. Calibration curves for all

2014 Institute of Food Science and Technology

authentic standards were prepared by performing

HPLC-DAD analysis in triplicate on ve incremental
dilutions of each standard. LOD was determined by
performing HPLC-DAD analysis in triplicate on incrementally diluted solutions of each standard until the
ratio of signal (peak height) to noise at 280 nm was
reduced to below 3:1.
The accuracy of the HPLC-DAD method was
assessed by recovery experiments. In each case,
known amounts of standards of tea catechins were
added to dierent vegetable oil samples, and then,
each sample was subjected to the preparing procedure as described in the treatment of vegetable oil
by liquidliquid extraction. The aliquots of infusion
were analysed by HPLC-DAD. Therefore, the recovery was calculated by comparing the amount of the
standards measured to that of added. Intraday variation (repeatability) was evaluated by performing
HPLC-DAD analysis on aliquots of each infusion
ve times on the same day. Interday variation
(reproducibility) was assessed by performing HPLCDAD analysis on aliquots of each infusion in triplicate on three consecutive days.
Liquidliquid extraction method validation

To demonstrate that liquidliquid extraction is an

ecient way to extract tea catechins from vegetable
oils, special experiments were designed. Based on our
previous experiments, the extracting eciency of
methanol was signicantly higher (P < 0.05) than
other solvents, including water and ethanol. Therefore, extracting verify experiment was carried out. A
known quantity of standard tea catechins solution
(1.0 mg mL 1) was added to vegetable oil sample
(10.0 mL), and then, the sample was extracted by
water, 20%, 40% and 60% methanol, respectively,
and the corresponding extractions were collected as
fraction 1 (F1), fraction 2 (F2), fraction 3 (F3) and
fraction 4 (F4), respectively. The extractions of vegetable oils (1.0 mL) by methanol/water were washed
by n-hexane, and the obtained solutions were collected. All fractions were evaporated, and the residue
was dissolved in a known volume (1.0 mL) of ddH2O
and then ltered through a 0.45-lm cellulose lter
membrane. The contents of tea catechins (including
EC, EGC, C, EGCG, EC, GCG, ECG and CG) and
tea polyphenols in fraction 14 were analysed by
HPLC-DAD and Folin-Ciocalteu method according
to our previously reported method.
Statistical analysis

The results obtained were analysed using SPSS version

16.0 (SPSS Inc., Chicago, IL, USA). Any signicant
dierence was determined by one-way analysis of vari-

International Journal of Food Science and Technology 2015

887

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Analysis of tea catechins in vegetable oils X. Zhang et al.

ance (ANOVA) followed by the Tukey test for multiple

comparisons considering dierence statistically at
P < 0.05.

mAU
140

120

Results and discussion

100

Isolation, purification and identification of tea catechin

monomers

In the present study, EC, ECG, EGC and EGCG were

used for the preparation of C, CG, GC and GCG
using heat epimerisation according to the reported
method. In addition, the structures of C, GC, CG and
GCG were conrmed by 1H NMR and ESI-TOF-MS.
ESI-MS data were m/z 313.6, 329.1, 465.6 and 481.1
for [M+Na]+, respectively. And 1H NMR data of C,
GC, CG and GCG were all identical to those of C,
GC, CG and GCG, respectively (Seto et al., 1997).
Development and validation of HPLC-DAD analytical
method

The representative chromatogram of standard EC,

EGC, C, EGCG, EC, GCG, ECG, CG, GA and caffeine is shown in Fig. 2. For vegetable oils treated by
liquidliquid extraction, elution proles of tea catechins, GA and caeine were similar to those observed
for their corresponding standards as shown in Fig. 3.
Therefore, peak identities were assigned based on
comparison of peak retention times and online DAD
spectra of the authentic standards.

mAU
9

350
300

5
6

250

10

200
150
100

80
60
40
7

20

4
1

0
0

10
10

10

12

14

tR (min)

Figure 3 A representative chromatograms of tea catechin extracts

from soya bean oil prepared by liquidliquid extraction.

It can be seen in Table 1 that the coecients of

determination (R2) were above 0.999 for calibration
curves of all standards. For calculating the LOD, it
can be found that LODs were in the range of 0.05
1.65 ng, EGC had the highest LOD at 1.65 ng, following were EC and ECG at 1.45 ng, while GA had the
lowest LOD at 0.05 ng. Linear range represents the
concentration range over which the least-squares
regression line relating peak area to standard concentration exhibited R2 > 0.999, and that of each tea catechins, GA and caeine are shown in Table 1.
Both intraday and interday RSDs were generally
<5.0% for the compounds detected in tested vegetable
oils (Table 2). Intraday RSDs were generally smaller
than interday RSDs for all the tested samples. The relatively low RSDs for GA, caeine and tea catechins in
both intraday and interday experiments indicate this
method is both repeatable and reproducible. The recovery rates were investigated with vegetable oil samples
prepared by the method described in HPLC-DAD
method validation. As a result, the recovery rates were
within the range of 96.2100.5% with RSD <3.7%.
Liquidliquid extraction treatment of vegetable oil

50

0
0

10

12

14

tR (min)

Figure 2 A typical HPLC-DAD chromatogram of GA, caeine and

tea catechins. 1, GA; 2, GC; 3, EGC; 4, C; 5, EGCG; 6, Caeine; 7,
EC; 8, GCG; 9, ECG; and 10, CG.

International Journal of Food Science and Technology 2015

During the treatment of vegetable oils by methanol/

water extraction, dierent fractions were collected as
mentioned in the experimental section, and the content
of tea catechins and tea polyphenols was determined.
As shown in Table 3, there was an increasing trend in
the concentration of methanol from 0% to 60%. Statistical analysis showed that signicant dierences were
existing among 0%, 20% and 40%, and 0%, 20%,

2014 Institute of Food Science and Technology

Analysis of tea catechins in vegetable oils X. Zhang et al.

Table 1 Summary of data used to identify and quantify major components of tea infusions eluted by the HPLC-DAD methods: HPLC-DAD
observed retention time (tR), calibration curve regression equation, calibration curve coecient of determination (R2), limit of detection (LOD)
and linear range (LR)
Identification

Calibration

Peak

Compound*

tR (min)

Regression equation

R2

LOD(ng)

LR(lg mL 1)

1
2
3
4
5
6
7
8
9
10

GA
GC
EGC
C
EGCG
Caffeine
EC
GCG
ECG
CG

3.2
3.4
4.9
5.6
6.9
7.2
7.5
8.4
9.8
10.6

y
y
y
y
y
y
y
y
y
y

0.9999
0.9998
0.9999
0.9998
0.9997
0.9999
0.9998
1
0.9998
0.9999

0.05
1.1
1.6
0.7
0.65
0.15
1.4
0.57
0.36
0.2

0.05100
0.05100
1.25100
0.05100
0.05100
0.05100
0.05100
0.05100
0.05100
0.05100

=
=
=
=
=
=
=
=
=
=

9.429x 27.523
5.553x 1.042
7.745x 6.325
36.463x 12.548
38.367x 37.103
55.783x 5.922
25.389x+13.021
18.446x 37.882
38.696x 17.526
75.119x 23.021

Identification and calibration data for GA, tea catechins and purine alkaloids were obtained using authentic standards.
y, peak area; x, the amount of each analytical injected (mg L 1).

LOD represents total ng on-column (calculated by multiplying the solution concentration by the injection volume, 20 lL) and represents HPLC-DAD
detection values.

LR represents the concentration range over which the least-squares regression line relating peak area to standard concentration exhibited
R2 0.999.

Table 2 Intraday* and interday of repeatability of peak areas of vegetable oils and recoveries
Tea seed oil

Sunflower seed oil

Soya bean oil

Peak

Compounds

Intraday
RSD (%)

Interday
RSD (%)

Recovery
(%)

Recovery
RSD (%)

Intraday
RSD (%)

Interday
RSD (%)

Recovery
(%)

Recovery
RSD (%)

Intraday
RSD (%)

Interday
RSD (%)

Recovery
(%)

Recovery
RSD (%)

1
2
3
4
5
6
7
8
9
10

GA
GC
EGC
C
EGCG
Caffeine
EC
GCG
ECG
CG

2.4
2.3
2.0
2.4
2.1
1.8
1.9
1.7
2.6
2.3

3.7
3.5
2.9
3.4
3.2
2.8
2.6
3.0
3.3
3.2

100.5
98.9
97.4
98.7
99.2
96.2
97.8
96.3
98.6
96.6

1.3
1.6
2.0
2.5
2.6
1.9
3.7
2.2
1.5
2.6

2.7
2.4
2.5
1.7
3.0
1.3
2.0
1.6
2.4
2.3

3.9
3.1
3.2
2.8
3.5
3.0
2.9
2.6
3.7
3.4

100.1
98.7
96.4
96.9
97.9
96.3
96.4
99.2
98.8
98.9

1.1
2.8
1.7
2.6
2.5
2.4
3.3
2.7
2.3
2.3

1.9
2.1
1.6
2.0
2.4
1.6
1.3
2.1
2.2
2.1

2.8
3.2
2.4
2.8
3.4
2.5
2.3
3.0
2.9
3.1

99.9
99.4
97.5
98.7
99.4
96.8
98.3
99.4
98.8
99.9

1.7
1.9
2.3
1.5
2.7
2.2
2.4
2.3
1.8
2.3

Intraday RSDs represent data from five analyses of each infusion within the same day.
Interday RSDs represent data from triplicate analyses of each infusion performed on three consecutive days; triplicate analyses on the same day
were averaged to give single data points used to assess variation among days.

Average of recoveries at three spiked levels.

Table 3 The content of tea catechins and tea polyphenols in F1F4

Components

F1 (mg)

F2 (mg)

Tea catechins
Tea polyphenols

24.56  1.24
54.53  1.77a
a

F3 (mg)

33.32  1.36
73.29  2.43b
b

F4 (mg)

35.03  2.03
78.94  1.25c
c

35.58  3.12c
79.21  2.86c

Different lowercase alphabet letters are significantly different at the level of P < 0.05.

and 60% (P < 0.05), but there was no signicant difference between 40% and 60% (P > 0.05). It indicated
that there is a positively signicant eect of methanol

2014 Institute of Food Science and Technology

concentration on tea catechins content when it is

<40%, while the eect is not signicant when it is
higher than 40%. Therefore, 40% was selected as the

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889

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Analysis of tea catechins in vegetable oils X. Zhang et al.

concentration of methanol in the experiment as higher

methanol concentration will bring about reagent waste
and increasing cost for extraction process.
Determination of tea catechins and tea polyphenols in
vegetable oils

Using this liquidliquid extraction analytical method,

the content of tea catechins, GA and caeine in dierent vegetable oils were determined. It can be seen in
Table 4, in vegetable oils that declared to had added
tea polyphenols in, the content of tea polyphenols was
less than that declared in package label, which were all
shown more than 100 mg L 1. For the content of tea
catechin, GA and caeine, in sunower seed oils, no
tea catechins were detected, which was inconsistent
with the label; in tea seed oils, only trace esteried tea
catechins (EGCG and GCG) and caeine could be
detected; and in soya bean oil, the content of tea catechins was relatively higher. Notably, in soya bean oil,
esteried catechins were the most abundant tea catechins and accounted for more than 50% of tea catechins. In addition, EGCG was of the highest content.
It can also be seen that the content of caeine
increased with tea catechins, which may be added to
vegetable oils with the addition of tea catechins. Scientic evidence supports that, when consumed in moderation, caeine has no adverse health eects (Heckman
et al., 2010). The extract amount of caeine may produce an adverse eect which varies from person to
person, depending on their weight and sensitivity to
caeine (Higdon & Frei, 2006).
The measured tea catechin contents that were lower
than labelled values of the oils may be attributed to the
following two factors: one is oxidation or decomposition
during transportation and storage, and the other is poor
solubility of catechins in oils. Edible fats, oils and prodTable 4 Concentrations of tea catechins, GA, caeine and tea polyphenols of vegetable oils measured by HPLC-DAD analysis

ucts including fats undergo the processes of oxidation,

both during production and storage. This process in food
causes a sequence of unfavourable changes, mainly deterioration in sensory properties of the product and
decrease in nutritional value (Gramza & Korczak, 2005).
Due to the special structures, which comprises several
compounds sharing a common phenylchromane skeleton
(Fig. 1), tea catechins were prone to be oxidised, and the
processing, transportation and storage conditions also
had great eect on tea catechin content. In our study, the
solubility results of tea catechins in vegetable oils were
low as shown in previous reports (Nwuha et al., 1999).
Meanwhile, the poor solubility of tea catechins in oils is
another important reason for their low solubility results.
Tea polyphenols have good water solubility; nevertheless, they are poorly absorbed because of their large size,
incompatible with a process of passive diusion and/or
their poor miscibility with oils and other lipids (Manach
et al., 2004). Phospholipids are small lipid molecules
where glycerol is bonded to two fatty acids, with the
third hydroxyl, normally one of the two primary methylenes, bearing a phosphate group. Phospholipids from
soya, mainly phosphatidylcholine, are lipophilic substances and readily complex polyphenolics. In recent
studies, phosphatidylcholine, the major molecular building block of cell membranes and a compound miscible in
both water and oil/lipid environments, is well absorbed
orally and has the potential to act as a chaperon for polyphenolics, accompanying them through biological membranes (Semalty et al., 2010).
For good bioavailability, tea polyphenols must
have a good balance between hydrophilicity (for dissolving in the gastrointestinal uids) and lipophilicity (to cross lipidic biomembranes). The preparation of
tea polyphenolsphospholipids complex may eectively improve the solubility of tea polyphenols in vegetable oils and overall absorption and bioavailability
as well.
Conclusions

Vegetable oil

Components
GA
GC
EGC
C
EGCG
Caffeine
EC
GCG
ECG
CG
Tea
polyphenols

Tea seed
oil (mg L 1)

Sunflower seed
oil (mg L 1)

Soya bean
oil (mg L 1)

0.52
0.66
3.07
1.12
9.89
8.93
2.63
0.43
6.76
0.82
61.38

3.96  0.18
2.14  0.12

1.39  0.08

11.73  0.75

International Journal of Food Science and Technology 2015













0.02
0.03
0.33
0.42
0.74
0.22
0.11
0.02
0.43
0.08
2.87

In this study, an eective method to determine the

contents of tea catechins in vegetable oils has been
successfully developed using HPLC-DAD combined
with liquidliquid extraction. All data of calibration,
LOD, repeatability, reproducibility, and recovery rate
demonstrate that this rapid HPLC-DAD method is
repeatable, reproducible, sensitive and practical. The
results showed that the solubility of tea catechins in
vegetable oils was low as shown in previous reports,
and their contents were far less than the label declared,
which may due to their special structures. Finally, our
results suggest that HPLC combined with liquidliquid
extraction is suitable for determination of tea catechins
in vegetable oil, and provide theoretical basis for quality assessment of vegetable oil during the storage.

2014 Institute of Food Science and Technology

Analysis of tea catechins in vegetable oils X. Zhang et al.

Acknowledgments

This work is sponsored by K.C. Wong Magna Fund

in Ningbo University.
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