Food Research International 34 (2001) 223227

www.elsevier.com/locate/foodres

Determination of avonols in green and black tea leaves and green

tea infusions by high-performance liquid chromatography
Huafu Wang *, Keith Helliwell
R&D Department, William Ransom and Son plc, Hitchin, Herts SG5 1LY, UK
Received 26 May 2000; accepted 9 August 2000

Abstract
Tea avonols are potent antioxidants and make up 23% of the water-soluble solids from tea leaves. In this paper, the conditions
necessary for hydrolysing and analysing avonols in tea leaves and tea infusions are optimised and an isocratic elution system for
the determination of the hydrolysed avonols by high-performance liquid chromotography is presented. Aqueous ethanol was
selected as the best solution for hydrolysing avonoids in tea leaves. The contents of avonols on a dry weight base in green tea
leaves ranged from 0.831.59, 1.794.05, and 1.563.31 g/kg, and in black tea leaves from 0.240.52, 1.043.03, and 1.722.31 g/kg
for myricetin, quercetin, and kaempferol, respectively. It was observed that the particle size of ground tea leaves signicantly
inuenced the yield of avonols. The contents of avonols in dierent green tea infusions are given. # 2001 Elsevier Science Ltd.
All rights reserved.
Keywords: Tea; Flavonols; Myricetin; Quercetin; Kaempferol; HPLC

1. Introduction
Flavonoids are natural products widely distributed in
the plant kingdom. They are subdivided into six classes:
avones, avanones, isoavones, avonols, avanols
and anthocyanins varying in their structural characteristics around the heterocyclic oxygen ring (Rice-Evans
& Miller, 1997). Many avonoids are predominantly
present as glycosides rather than non-glycosylated
forms (aglycones). For example, avonols occur generally as O-glycosides with a sugar residue mostly at the
C-3 position. The type and number of sugar residues,
together with chain branching, can result in a very large
number of individual glycosides.
The main avonoids found in tea are avanols and avonols. They are of interest because they have a wide range
of pharmaceutical properties including antioxidative,
anticarcinogenic and antiarteriosclerotic (Dreosti, Wargovich, & Yang, 1997; Jankun, Selman, Swiercz, & Skrzypczak-Jankun, 1997; Wiseman, Balentine, & Frei, 1997;
Yamamoto, Juneja, Chu, & Kim, 1997; Yang, 1997). Tea
catechins are structurally, primarily, avanols, and these
form about 20% of the dry weight of green tea, while tea
* Corresponding author. Tel.: +44-1462-437615; fax: +44-1462420528.
E-mail address: hwang@williamransom.com (H. Wang).

avonols make up 23% of the water-soluble solids of tea.

Therefore, the catechins have attracted more attention
from researchers. However, avonols are also regarded to
be potent antioxidants due to the combination of a keto
group conjugated to a double bond in the C ring, together with adjacent hydroxyl groups in the B ring (Fig. 1)
(Bors, Heller, Michel, & Stettmaier, 1996). Flavonols
are structurally more stable than catechins, and it has
been shown that tea is a major dietary source of these
compounds (Hertog, Hollman, & van de Putte, 1993).
Fieschi, Codignola, and Luppi Mosca (1989) measured the total avonoid glycoside and aglycone contents of dierent types of black tea infusions using
paper chromatography followed by spectrophotometric
measurements. Price, Rhodes, and Barnes (1998) investigated avonol glycoside in tea infusions and tea products using high-perfomance liquid chromotogarphy
(HPLC) gradient elution system. However, this
approach was not suitable for routine analysis as most
reference compounds are not commercially available.
Hertog et al. (1993) analysed the content of avonoids
in tea infusions, wines, and fruit juices by hydrolysing
all glycosides to aglycones followed by HPLC determination. This oered a practical method for the quantitative determination of avonoids in foods.
In this paper, the conditions for hydrolysing and
analysing avonols in tea leaves and tea infusions are

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H. Wang, K. Helliwell / Food Research International 34 (2001) 223227

optimised and an isocratic elution system for the determination of the hydrolysed avonols by HPLC is
presented. The content of avonols in dierent tea
leaves and green tea infusions, brewed in the way people
normally make this beverage, are detailed.
2. Materials and methods
2.1. Samples
Zhejiang roasted green tea and Keemun black tea
were obtained from the Tea Research Institute, Chinese
Academy of Agricultural Sciences. Gunpowder tea and
Ceylon black tea were purchased from a local teashop
in Hitchin, Hertfordshire, UK. Sencha tea was a present
from Japan.
2.2. Chemicals
Myricetin, quercetin, and kaempferol were purchased
from Sigma Chemical Co. Acetonitrile, methanol, ethanol and acetone were of HPLC quality and purchased
from Fisher Scientic (Essex, UK). HCl and KH2PO4
were of analytical grade, and also purchased from Fisher
Scientic (Essex, UK). The water used in HPLC and
sampling was prepared with a Super Purity Water System
(Purite Ltd, England) with a resistivity over 17.5 M
cm.
2.3. Method for the hydrolysis of avonoids
One gram of tea leaves was mixed with 40 ml of 60%
aqueous ethanol and 5 ml of 6 M HCl. After reuxing
at 95 C for 2 h, the hydrolysed solution was ltered into
a 50-ml volumetric ask and subsequently made up to
the volume with 60% aqueous ethanol. Approximately
1ml of the nal solution was allowed to cool under
running water and ltered through a 0.45 m lter (Nylon

Acrodisc 13, Gelman) prior to injection for HPLC analysis. For tea infusion, according to the conventional tea
brewing method, 1 g of gunpowder tea leaves was infused
with 100 ml of boiling distilled water for 5 min, 70 ml liquid
were ltered o and cooled to room temperature under
running water; a second infusion was made by adding a
further 70 ml boiling distilled water to the tea leaves for
5 min, and ltering o 70 ml which was cooled to room
temperature under running water; a third infusion was
made using the same procedure. Sixteen millilitres of
each infusion were mixed with 24 ml absolute ethanol
(99.99%) to produce an ethanol concentration of 60%,
5 ml 6 M HCl were added for hydrolysis. Other procedures were the same as those for tea leaves.
2.4. Analytical determinations
The analysis of avonols in tea was carried out by the
following HPLC method. A HP 1100 series liquid
chromatograph system, comprising vacuum degasser,
quaternary pump, auto-sampler, thermostated column
compartment, and diode array detector, was used. The
column was a C18 reversed phase Kingsorb 5 m C18
(1504.6 mm) (phenomenex1, UK) with a Kingsorb 5
m C18 (304.6 mm) guard column. Mobile phase consisted of 30% acetonitrile in 0.025 M KH2PO4 buer
solution (v/v); the pH of the mobile phase was adjusted
with 6 M HCl to 2.5. The ow rate was 1.0 ml/min. The
column was operated at 30 C. The sample injection
volume was 20 ml. UV spectra were recorded from 200
to 400 nm, and peak areas were measured at 370 nm.
The UV spectra obtained for each peak, after subtraction of the corresponding UV base spectrum, were
computer normalised and the plots were superimposed.
Peaks were considered to be chromatographically pure
when there was exact coincidence to their corresponding
UV spectra. Chromatographic peaks in the samples
were identied by comparing their retention time and
UV spectra with those of the reference standards.
Working standard solutions (580 ml) were injected into
the HPLC, and peak area responses were obtained. A
standard graph for each component was prepared by
plotting concentration versus area. Quantication was
carried out from integrated peak areas of the sample
against the corresponding standard graph. Each sample
was analysed in triplicate.
3. Results and discussion
3.1. Hydrolysis of avonoid glycosides

Fig.1. Chemical structures of avonols in tea.

3.1.1. Eect of HCl concentration and hydrolysis time

The inuence of three HCl concentrations (1.0, 3.0
and 6.0 M) on the hydrolysis of avonoids in the
samples has been investigated. Fig. 2 shows the time

H. Wang, K. Helliwell / Food Research International 34 (2001) 223227

225

Fig. 2. The time course of hydrolysis of avonols in tea. Values

represent means of triplicate analyses with their standard deviations
less than 0.1.

course for the hydrolysis of avonols in tea with the

addition of 5 ml 6 M HCl. As can be seen, both myricetin and quercetin reached their highest yield within 2
h, but for kaempferol, it continued to increase throughout the hydrolysis period of 3 h. Using lower concentrations of HCl, a much longer time was needed to
achieve the same result. For eciency of analysis, a
concentration of 6 M HCl and hydrolysis time of 2 h
were selected. Other acids were not considered because a
comparison of HCl with other acids was made by Hertog, Hollman and Venema (1992) who demonstrated
that HCl was the most eective.
3.1.2. Eect of dierent solvents and solvent
concentrations
Three extraction solvents were tested: ethanol,
methanol, and acetone, with concentrations varying
from 090% of the solvent in water. Fig. 3AC show
the eect of the solvents in dierent concentrations on
the hydrolysis of myricetin, quercetin and kaempferol,
respectively. The highest yield for the three avonols
was found by using 60% ethanol in aqueous solution,
and at concentrations of 6090% ethanol in water, the
yield was unchanged. Using methanol, the yield of the
three avonols continued to increase with increasing
concentration of methanol, but was still lower than that
using ethanol. Using acetone, the highest yield was
achieved at a concentration of 30%, after that the yield
deteriorated by 38 and 17% for myricetin and quercetin,
respectively. Hydrolysis of avonoids in some vegetables and fruits with HCl in aqueous methanol has
been described by Hertog et al. (1992). However, as our
results show, aqueous ethanol was superior to aqueous
methanol, and therefore aqueous ethanol (60%) was
selected as the extraction solvent.

Fig. 3. Eect of the solvents on the hydrolysis of the avonoids. A,

Myricetin; B, Quercetin; C, Kaempferol. Values represent means of
triplicate analyses with their standard deviations less than 0.1.

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H. Wang, K. Helliwell / Food Research International 34 (2001) 223227

Fig. 4. A typical chromatogram of avonols in tea leaves. The chromatographic conditions are described Section 2. 1, Myricetin; 2, quercetin; 3,
kaempferol.

3.2. Validation of the method

In order to verify the accuracy and precision of the
analytical procedure, recovery studies were carried out
by spiking selected samples of tea with the avonol
standard solution. The recovery for myricetin, quercetin, and kaempferol was 93.6, 102, and 106%, respectively. All the analytes exhibited very good linearity
over the range tested with correlation coecients (r) of
1.000.
To test the precision of the assay method, the standard solution of avonols and one of the samples to be
analysed were injected seven times under the chromatographic conditions described above. The experiment
showed that the variation coecients were quite low:
for the standard solution, the coecients of all analytes
were within 0.24%, while for an actual sample, within
0.58%.
3.3. Quantitative measurement of dierent tea samples
3.3.1. Content of avonols in tea leaves
Fig. 4 shows a typical chromatogram of avonols in
tea leaves. Dierent kinds of green tea, namely, gunpowder which is popular in the UK, Zhejiang roasted
green tea which is popular in China, and Sencha which
is popular in Japan, and two kinds of black tea, Keemun black tea and Sri Lanka black tea were prepared
for analysis. The content of avonols has been calculated as milligrams per kilograms of tea leaves on dry
weight base (Table 1).
It has been reported that avonol glycosides are not
aected by polyphenol oxidase (Finger, Engelhardt, &
Wray, 1991), therefore, their variations in content in the
dierent types of tea are thought to be due to the different tea varieties, geographic location or agricultural
conditions. However, it can be seen that the content of
myricetin is proportionally lower in black teas than in
green teas. This is possibly because myricetin and myr-

Table 1
Content of avonols in dierent tea leaves (g/kg dry leaves)a
Tea leaves

Myricetin

Quercetin

Kaempferol

Green tea
Gunpowder
Zhejiang
RGT
Sencha
Longjing

1.590.04 (22.1)b
0.930.04 (15.1)

4.050.02 (56.3)
2.840.03 (46.2)

1.560.04 (21.6)
2.380.03 (38.7)

1.320.03 (15.8)
0.830.02 (16.5)

3.750.04 (44.7)
1.790.02 (35.6)

3.310.04 (39.5)
2.410.01 (47.9)

Black tea
Qimen
Ceylon

0.240.02 (6.7)
0.520.04 (9.9)

1.040.02 (29.0)
3.030.02 (57.5)

2.310.01 (64.3)
1.720.02 (32.6)

a
All the data for content of avonols are average values of triplicate analyses, and are given with standard deviations.
b
Data in the parentheses are expressed as percentage of the compound in three avonols.

Table 2
Content of avonols in dierent infusions of gunpowder tea (mg/L)a
Infusion

Myricetin

Quercetin

Kaempferol

First
Second
Third

6.400.02
4.200.03
2.700.02

23.870.06
15.900.10
10.010.03

9.010.02
6.500.03
3.490.02

13.30

49.78

19.00

Total

a
The tea infusions were made according to the method described in
Section 2. Values represent means of triplicate analyses, and are given
with standard deviations.

icetin glycosides can still be aected by tea producing

processing to some extent. McDowell, Bailey, and
Howard (1990) also found that myricetin and myricetin
glycosides were the most likely of the three avonols to
be oxidised. Quercetin is generally regarded the most
important avonol, followed by kaempferol. However, it is noticeable that the content of myricetin is
much higher than in many other herbal plants analysed

H. Wang, K. Helliwell / Food Research International 34 (2001) 223227

by us (data not shown). Therefore myricetin should

not be ignored when discussing tea leaves and their
extracts.
The eect of the particle size of ground tea leaves on
the yield of avonols during extraction was also
investigated. The result showed that the detected content of myricetin, quercetin and kaempferol in ground
samples was approximately 36% higher than that in
unground ones. It is expected that the ner the size of
ground tea leaves the more eciently they are extracted.
Therefore, it is recommended that ground tea samples
be used for the determination.
3.3.2. Content of avonols in dierent tea infusions
Although there is a signicant interest in the use of tea
extracts, making a cup of tea by brewing of tea leaves is
still the most common way to consume tea. In China
and some other southeast Asian countries people are
accustomed to making tea infusions two or three times
with the same tea leaves. So far, little attention has been
paid to the quantitative determination of avonols in
tea infusions. However, it is important to measure the
content of avonols in the dierent tea infusions when
the intake of avonols from consuming tea is evaluated.
Table 2 shows the content of avonols in dierent tea
infusions brewed with gunpowder tea.
It was observed that the highest content of avonols
was in the rst infusion, and then the content gradually
decreased with later infusions. Because this data resulted from the normal tea brewing process it can be used
to evaluate the likely intake of avonols from drinking
tea. For example, if 5 g of gunpowder tea are consumed
per day, over 40 mg of combined avonols may be
ingested. Fujiki et al. (1992) proposed drinking green
tea in large amounts, such as ten cups per day, as a
possible method of cancer prevention for the general
population. If so, the intake of avonols would be more
than the quantity calculated from our results. Kuhnau
(1976) estimated that the total intake of avonoids in
the United States was 1 g/day expressed as glycosides or
170 mg/day expressed as aglycones. However, Hertog et
al. (1993) found that the average intake of avonols and
avones in the Netherlands was 23 mg/day (expressed
as aglycones), with tea being the major source (48% of
total intake). Our results appear to be comparable to
the data given by Hertog et al. (1993).

227

Acknowledgements
The authors would like to thank Mr. Clive Welham and
Miss. Catherine Marshall for their kind help in this work.
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