Gallic acid metabolites are markers of black tea intake in humans

Article abstract of DOI:10.1021/jf000089s

Gallic acid is one of the main phenolic components of black tea. The objective of this study was to identify urinary gallic acid metabolites with potential for use as markers of black tea intake. In an initial study, nine compounds, assessed by using gas chromatography-mass spectrometry, were found to increase in concentration in urine after 3 cups of black tea over 3 h. A subsequent study employed a controlled crossover design in which 10 subjects consumed 5 cups per day of black tea or water for 4 weeks in random order. Twenty-four hour urine samples were collected at the end of each period. Of the 9 candidate compounds identified in the initial study, only 3 were present at higher concentrations in urine of all 10 subjects during tea- drinking in comparison to water-drinking periods. These compounds were identified as 4-O-methylgallic acid, 3-O-methylgallic acid, and 3,4-O- dimethylgallic acid, all methyl ether derivatives of gallic acid. It is suggested that these compounds have the potential to be used as markers of black tea intake.

Full text of DOI:10.1021/jf000089s

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2276
J. Agric. Food Chem. 2000, 48, 2276−2280
Ga llic Acid Meta bolites Ar e Ma r k er s of Bla ck Tea In ta k e in Hu m a n s
J onathan M. Hodgson,* Lincoln W. Morton, Ian B. Puddey, Lawrence J . Beilin, and
Kevin D. Croft
Department of Medicine, University of Western Australia, and Heartsearch, Royal Perth Hospital,
Perth, Western Australia, Australia
Gallic acid is one of the main phenolic components of black tea. The objective of this study was to
identify urinary gallic acid metabolites with potential for use as markers of black tea intake. In an
initial study, nine compounds, assessed by using gas chromatography-mass spectrometry, were
found to increase in concentration in urine after 3 cups of black tea over 3 h. A subsequent study
employed a controlled crossover design in which 10 subjects consumed 5 cups per day of black tea
or water for 4 weeks in random order. Twenty-four hour urine samples were collected at the end of
each period. Of the 9 candidate compounds identified in the initial study, only 3 were present at
higher concentrations in urine of all 10 subjects during tea-drinking in comparison to water-drinking
periods. These compounds were identified as 4-O-methylgallic acid, 3-O-methylgallic acid, and 3,4-
O-dimethylgallic acid, all methyl ether derivatives of gallic acid. It is suggested that these compounds
have the potential to be used as markers of black tea intake.
Keyw or d s: Black tea; gallic acid; 4-O-methylgallic acid; 3-O-methylgallic acid; 3,4-O-dimethylgallic
acid
INTRODUCTION
as markers of black tea intake. The methyl ether
derivative of gallic acid, 4-O-methylgallic acid (4OMGA),
has previously been identified as a major metabolite of
gallic acid in humans (Shahrzad and Bitsch, 1998). The
possibility that gallic acid is metabolized to other methyl
ether derivatives and/or other phenolic acids has been
investigated in the present study.
The major objectives of this study were therefore (1)
to identify phenolic acid metabolites of gallic acid that
increase in concentration in human urine following
black tea ingestion and (2) to establish whether con-
centrations of these compounds are consistently raised
during regular black tea ingestion in comparison to
regular water ingestion. Compounds present at consis-
tently higher concentrations in urine of volunteers
taking their usual diet during regular black tea inges-
tion can be considered to be of possible use as markers
of black tea ingestion.
The potential health benefits of drinking tea are
suggested to be chiefly due to polyphenolic components
of tea (Hertog et al., 1993; J ick et al., 1973; Keli et al.,
1996). Results of epidemiological studies show that
black tea (Hertog et al., 1993; J ick et al., 1973; Keli et
al., 1996) and polyphenols derived from black tea
(Hertog et al., 1993, 1995; Keli et al., 1996; Knekt et
al., 1996) are associated with reduced risk of cardiovas-
cular disease. Tea can be a major dietary source of
polyphenolic compounds: polyphenols make up ∼30-
40% of the weight of the tea leaf (Harbowy and Ballen-
tine, 1997).
Compounds that provide an indication of tea polyphe-
nol intake and exposure may be useful as markers of
tea intake. Markers of tea intake will be useful in
epidemiological and intervention studies focusing on tea
and disease-related endpoints. Identification of such
compounds has proved to be troublesome, due largely
to the complex nature of many of the polyphenols
present in black tea and lack of understanding of the
metabolism of these compounds. Methods for the mea-
surement of a particular class of polyphenolic com-
pounds (van het Hof et al., 1998) or total polyphenolic
concentrations (He and Kies, 1994) may be useful in
providing an indication of black tea intake, but these
methods are nonspecific.
MATERIALS AND METHODS
Two studies were performed to identify and describe urinary
gallic acid metabolites with potential for use as markers of
black tea intake. In the first study phenolic acids (lower
molecular weight compounds in the acid fraction of urine) that
increased significantly in concentration after 3 cups of tea over
3 h in comparison to a baseline fasting urine sample were
assessed. The methyl ether derivatives of gallic acid were
specifically targeted as likely metabolites of gallic acid.
Compounds identified as having increased acutely were then
assessed in 24-h urine samples during 4 weeks of regular black
tea or water (5 cups per day) consumption.
Su bjects. Ten healthy subjects (eight men, two women)
were recruited from the general population in response to
newspaper advertisements. Volunteers were excluded after
initial screening if they reported the use of any medication or
dietary supplements; a history of major illness including heart
disease, diabetes, liver disease, renal disease, and gastrointes-
tinal conditions; current smoking or smoking cessation for <6
months; body mass index > 35 kg/m2; an alcohol intake
Gallic acid is one of the main phenolic components of
tea. Gallic acid occurs in black tea in free and esterified
forms and is estimated to be present at ∼5% of the
weight of the tea leaf (Harbowy and Ballentine, 1997).
We propose that gallic acid metabolites may be useful
* Address correspondence to this author at the University
Department of Medicine, GPO Box X2213, Perth, WA 6001,
Australia (telephone 61 8 9224 0267; fax 61 8 9224 0246; e-mail
jonathan@cyllene.uwa.edu.au).
10.1021/jf000089s CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/27/2000

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Markers of Black Tea Intake
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2277
averaging >40 g per day; or regular tea intake averaging <1
cup per day. The Royal Perth Hospital Ethics Committee
approved the project, and all participants gave informed
written consent.
4-O-methylgallic acid, which was purified by preparative thin-
layer chromatography on silica gel.
3-O-Methylgallic Acid. The method of J urd (1959) was
modified for the synthesis of 3-O-methylgallic acid (3OMGA).
Methyl gallate was first prepared according to a standard
Fischer-Speier esterification using gallic acid and 1% metha-
nolic HCl. Methyl gallate (0.5 g, 2.72 mmol) was mixed with
dichlorodiphenylmethane (0.695 g, 2.93 mmol) and potassium
carbonate (1.62 g, 11.7 mmol) and heated at 170-180 °C for
10 min until it turned brown and no further gas (HCl) was
evolved. The mixture was extracted with ethyl acetate (3 ×
20 mL), and the combined extracts were filtered, washed with
water (2 × 20 mL), and dried (MgSO4); the solvent was
removed under reduced pressure and with warming to yield a
brown oil, which formed brown amorphous crystals with
cooling. The solid was dissolved in the minimum of ether, and
an equal volume of hexane was added. Colorless crystals of
methyl 3-hydroxy-4,5-diphenylmethylenedioxybenzoate formed
(660 mg, 70%, mp 162-164 °C, lit. 164 °C; J urd, 1959).
Dimethyl sulfate (180 mg, 1.4 mmol) was dissolved in ether
(2 mL) and added to a solution of methyl 3-hydroxy-4,5-
diphenylmethylenedioxybenzoate (395 mg, 1.1 mmol) in ether
(10 mL) and a slurry of potassium carbonate (160 mg, 1.2
mmol) in acetone (3 mL). The mixture was stirred at room
temperature overnight. Water (5 mL) and saturated sodium
chloride solution/hydrochloric acid (20 mL, pH 3) were added,
the organic phase was separated, and the aqueous phase was
extracted with ethyl acetate (2 × 20 mL). The combined
organic phase was dried (MgSO4), and the solvent was removed
under reduced pressure with warming to yield a white solid.
This was recrystallized from ether/hexane to yield methyl
3-methoxy-4,5-diphenylmethylenedioxybenzoate [378 mg, 1.0
mmol, 92%, mp 135 °C (sharp), lit. 135 °C; J urd, 1959].
Methyl 3-methoxy-4,5-diphenylmethylenedioxybenzoate was
treated with 80% acetic acid (108 °C, 5 h), diluted with 4
volumes of water, washed with hexane (2 × 10 mL, discarded),
and extracted with ethyl acetate (2 × 20 mL). The organic
extracts were combined, and the solvent was evaporated under
reduced pressure to yield a white solid, which was dissolved
in equal volumes of methanolic potassium hydroxide (1 M) and
water and heated at 55 °C for 36 h under nitrogen. The
solution was diluted with water, acidified (pH 1.5, 5 M HCl),
extracted with ethyl acetate (5 × 10 mL), and purified by silica
preparative thin-layer chromatography plates. The plates were
developed with ethyl acetate/hexane/glacial acetic acid )
50:50:1. The lowest band was removed, extracted with ethyl
acetate, and shown to be 3OMGA by a combination of 200 MHz
1H NMR and GC-MS.
Exp er im en ta l Design : Stu d y 1. Urinary phenolic acids
were measured in two volunteers before and after consumption
of 3 cups of black tea. Subjects had fasted for at least 12 h
and had not consumed tea for at least 2 weeks prior to
measurements. Black tea was prepared by allowing 2 g of tea
leaves to infuse in boiled water for 1 min. The tea was ingested
without additives such as milk and sugar. A spot urine sample
was taken for baseline measurements. The subjects then
consumed 3 cups of black tea at hourly intervals over the
following 3 h (at 1, 2, and 3 h after the baseline sample). A
spot urine sample was taken again 1 h after the last drink of
tea. Urine samples were frozen at -80 °C until analysis.
Exp er im en ta l Design : Stu d y 2. Compounds identified as
having increased acutely were measured in 24-h urine samples
collected during 4-week periods of regular black tea or water
consumption. During the study subjects avoided all coffee,
chocolate drinks, herbal infusions, and all teassapart from
that supplied. After a 4-week wash-out period during which
subjects consumed 5 cups per day of hot water, subjects were
randomly assigned to drink either black tea or hot water for
4 weeks. Subjects then crossed over to the alternate drink for
a further 4 weeks. Black tea or hot water intake was 5 cups
per day without additives such as milk and sugar. The tea
was prepared by allowing 2 g of tea leaves to infuse in 250
mL of boiled water for 1 min. Twenty-four-hour urine samples
were collected during the last week of each of the 4-week
intervention periods. The volume of urine was recorded, and
then an aliquot was frozen at -80 °C for subsequent analysis.
Assessm en t of P h en olic Com p ou n d s by Ga s Ch r om a -
togr a p h y)Ma ss Sp ectr om etr y (GC-MS). Urine (2 mL) and
1-hydroxy-2-naphthoic acid (1 µg, internal standard) were
acidified to pH 4.8 with 1 M HCl. â-Glucuronidase (30 µL, with
3000 units of activity) (Sigma catalog no. G707) was added,
mixed, and incubated at 37 °C for 24 h with occasional mixing.
The samples were acidified (pH 2, 1 M HCl) and extracted with
ethyl acetate (1 × 2 mL), and the aqueous phase was
discarded. The organic phase was extracted with sodium
bicarbonate (5% w/w, 1 × 2 mL), and the organic phase was
discarded. The aqueous phase was acidified (pH 2, 5 M HCl)
and extracted with ethyl acetate. The organic phase was
retained, and the solvent was removed under a stream of
nitrogen to yield the acid fraction of the urine samples. The
trimethylsilyl derivative was made using BSTFA (50% v/v) in
pyridine.
Phenolic acids and other compounds in the acid fraction
were analyzed on a Hewlett-Packard HP 5890 gas chromato-
graph coupled to an HP 5970 mass spectrometer. Larger
molecular weight polyphenols such as flavonoids were not
specifically targeted. An HP-5MS column (25 m × 0.20 mm,
0.33 µm film thickness, Hewlett-Packard) was used with
helium as the carrier gas and an inlet pressure of 30 kPa.
Injections were made in a splitless mode. The initial column
temperature of 120 °C was held for 0.5 min and then increased
at 15 °C/min to 280 °C, at which it was held for 5 min. The
mass spectrometer was operated in the electron impact mode
(70 eV). All ions were monitored in the scan mode. Analyte
abundances were compared using extracted ion chromato-
grams.
3,4-O-Dimethylgallic Acid. The authentic standard of 3,4-
O-dimethylgallic acid (3,4OdiMGA) was purchased from Sigma
(catalog no. H-7009, St. Louis, MO).
For quantification, response factors were established by
measuring peak areas versus response in comparison to the
internal standard.
Sta tistics. All statistical analyses were performed using
SPSS (Chicago, IL). Results are presented as means ( SEM,
and P < 0.05 was used as the level of significance. The paired
samples t test was used to compare urinary concentrations of
compounds during the water-drinking period with the urinary
concentrations during the tea-drinking period.
RESULTS
Com p ou n d Id en tifica tion . Those compounds that were
present at a higher concentration during regular black tea
drinking, compared to the water period, in all 10 subjects were
identified. Identification of the compounds was based on
retention times and mass spectra compared with authentic
standards.
4-O-Methylgallic Acid. The authentic standard of 4-O-
methylgallic acid was prepared according to literature proce-
dures (Luz Cardona et al., 1986). Briefly, gallic acid (1 mol
equiv) was methylated with methyl sulfate (2 mol equiv) and
potassium carbonate (2 mol equiv) in acetone at room tem-
perature for 12 h. The methyl ester was hydrolyzed with 1
mol/L methanolic potassium hydroxide under nitrogen to give
The subjects recruited for this study were between
43 and 75 years with a mean age of 61.8 ( 2.7 years.
They were healthy nonsmokers with a body mass index
at baseline of between 23.5 and 35.7 kg/m2 (mean ) 28.0
( 1.2 kg/m2).
Following 3 cups of black tea (study 1), gallic acid was
not detected in urine. A total of nine compounds was
found, using GC-MS, to have increased in concentration
following black tea ingestion. The retention times, major
ions, and compound names (if known) of these nine
compounds are presented in Table 1. Each of these

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2278 J. Agric. Food Chem., Vol. 48, No. 6, 2000
Hodgson et al.
F igu r e 1. Structures and mass spectra for gallic acid and its major methylated metabolites identified in human urine (spectra
presented are trimethylsilyl derivatives).
Ta ble 1. Reten tion Tim es, Ma jor Ion s, a n d Com p ou n d
Na m es (If Kn ow n ) of th e Nin e Com p ou n d s F ou n d To
Ha ve In cr ea sed Acu tely in Con cen tr a tion follow in g
Bla ck Tea In gestion
Ta ble 2. 24-h Ur in a r y Excr etion of 4OMGA, 3OMGA, a n d
3,4-Od iMGA for Ea ch Su bject a n d Gr ou p Mea n s d u r in g
th e Wa ter - a n d Tea -Dr in k in g P er iod sa
4OMGA, µg/day
subject water tea
414
3OMGA, µg/day 3,4OdiMGA, µg/day
compound
no.
retention
time (min) major ions
water
tea
water
tea
compound name
1
0
0
0
13
13
16
25
24
0
155
5
25
67
1
2
3
4
5
6
7
8
9
8.43
8.61
8.64
9.06
9.40
10.00
10.06
10.08
10.72
342, 312
400, 370
331, 215
400, 223
317
372, 156
378, 338
338, 308
391, 274
3,4-O-dimethylgallic acid
4-O-methylgallic acid
2
3
503
706
124
122
351
183
83
381
212
305
297
107
174
151
140
125
183
129
177
171
33
20
32
81
0
57
17
41
77
0
4
934
3-O-methylgallic acid
5
220
6
7
1618
2580
1303
1376
1404
8
0
0
0
ferulic acida
9
10
263
118
92
53
75
0
55 ( 26 1106 ( 222b 24 ( 9 221 ( 33b 30 ( 10 142 ( 11b
a
mean
Tentative identification.
a
b
Mean values are presented with SEM, n ) 10. P < 0.001 for
comparison with water period.
compounds was considered to be a candidate for inves-
tigation during periods of regular black tea and water
consumption in study 2.
of these compounds during the water- and tea-drinking
periods for each subject is presented in Table 2. The
mean total excretions of the methyl ether metabolites
of gallic acid were 108 ( 41 µg/day during the water-
drinking period and 1469 ( 249 µg/day during the tea-
drinking period (P < 0.001 for difference). Examples of
extracted ion chromatograms showing urinary gallic
acid metabolites from an individual drinking tea and
water for 4 weeks each are presented in Figure 2.
We have also investigated whether other methyl ether
derivatives of gallic acid were present in human urine
following black tea ingestion. Other possible methyl
ether derivatives of gallic acid, including 3,5-O-dimeth-
ylgallic acid (also known as syringic acid) and 3,4,5-O-
trimethylgallic acid, were not present at increased
concentrations following black tea ingestion.
Concentrations of the nine candidate compounds were
assessed in 24-h urine samples taken during 4-week
periods of regular black tea or water consumption (study
2). Compounds that were present at higher concentra-
tion during the tea-drinking period in comparison to the
water-drinking period in all 10 subjects were considered
to be potentially useful as markers of black tea intake.
Compounds present at higher concentrations during the
water-drinking period were deemed to be derived from
dietary sources other than tea. Of the nine candidate
compounds only three fulfilled these criteria.
The three compounds were identified on the basis of
retention time and mass spectral data in comparison
with authentic standards as 4OMGA, 3OMGA, and
3,4OdiMGA. All compounds were methyl ether deriva-
tives of gallic acid. The chemical structures and mass
spectra of gallic acid and these methyl ether derivatives
are presented in Figure 1. The 24-h urinary excretion
DISCUSSION
We have identified three compounds with potential
for use as markers of black tea polyphenol intake. These

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Markers of Black Tea Intake
J. Agric. Food Chem., Vol. 48, No. 6, 2000 2279
take and metabolism. The total gallic acid intake may
therefore be as high as 20-50 mg per cup of black tea,
assuming 2 g of tea leaves infused in hot water gives
20-50% extraction.
The methyl ether derivatives of gallic acid identified
in urine may have resulted from different sources in the
black tea. They may have been derived from free gallic
acid, hydrolysis of gallate esters, and/or metabolism of
flavonoids after ring opening. More complex polyphenols
can be metabolized to phenolic acids (Choudhury et al.,
1999). It is therefore difficult to estimate bioavailablility.
What is clear is that the total excretion of the gallic acid
metabolites measured was ∼1.5 mg/day when subjects
were drinking 5 cups per day of black tea, which
represents a small percentage of gallic acid intake. For
example, assuming 20% extraction into hot water, the
1.5 mg/day of measured gallic acid metabolites would
correspond to 7.5% of ingested free gallic acid, but only
1.5% of ingested total gallic acid.
The 4OMGA methyl ether derivative of gallic acid has
previously been identified as a urinary metabolite of
gallic acid in humans (Shahrzad and Bitsch, 1998) and
rats (Zong et al., 1999). We have shown here that
4OMGA is the major metabolite of gallic acid, derived
from black tea, in human urine. In addition, two other
methyl ether derivatives have been identified as minor
gallic acid metabolites. These compounds, 3OMGA and
3,4OdiMGA, have not previously been described as
gallic acid metabolites.
The results of this study demonstrate that tea was
the major dietary source of the gallic acid metabolites
in 10 volunteers consuming their usual diet. However,
it remains uncertain if any particular foods or beverages
other than tea might contribute significantly to urinary
excretion of these compounds and therefore potentially
limit the use of these compounds as markers of tea
ingestion. Contributions of other foods to urinary excre-
tion of the methyl ether derivatives did not appear to
be great in this study. However, the large variability in
4OMGA concentrations is suggestive of differences in
uptake and/or metabolism between individuals. This
may be a limiting factor in the usefulness of 4OMGA
as a marker of black tea intake.
F igu r e 2. Extracted ion chromatograms (m/z 223, 317, 342,
and 400) showing urinary gallic acid metabolites, 3,4-O-
dimethylgallicacid (3,4OdiMGA),4-O-methylgallicacid (4OMGA),
and 3-O-methylgallic acid (3OMGA), from an individual drink-
ing tea or water for 4 weeks each. The unidentified peak at
8.9 min was not consistently elevated during tea drinking.
4OMGA has been measured in 24 h urine at ∼1 mg/
day following 375 mL per day of red wine ingestion
(Abu-Amsha Caccetta, personal communication). This
excretion is similar to the mean 1.1 mg/day following
ingestion of 5 cups per day of black tea found in the
present study. The 4OMGA present in urine following
red wine ingestion may derive from gallic acid and/or
tannic acid (Zhu et al., 1992), which are both present
in red wine. We have not yet established whether
3OMGA and 3,4OdiMGA are also present in human
urine following red wine ingestion and what the relative
contribution of tea and red wine might be to these
metabolites.
Another factor that may influence the value of gallic
acid metabolites as markers of black tea is the kinetics
of these compounds after ingestion. The location of gallic
acid uptake and metabolism will have some bearing on
the kinetics. The importance of the liver and of bacterial
metabolism in the gut is not clear. To date, we have no
data on the kinetics of gallic acid metabolites, except
that plasma levels of 4OMGA peak at 2-4 h after red
wine ingestion (Abu-Amsha Caccetta et al., 2000).
compounds, all methyl ether derivatives of gallic acid,
were 4OMGA, 3OMGA, and 3,4OdiMGA (Figure 1). The
three compounds were present at increased concentra-
tions following ingestion of 3 cups of black tea and
present at significantly higher concentrations in all 10
subjects during regular black tea consumption in com-
parison to a water-drinking control period.
Gallic acid is a major phenolic component of tea. Most
of the gallic acid in black tea will have derived originally
from two flavonoids present at high concentrations
(∼15% of dry weight) in the green tea leaf. These
compounds are epicatechin gallate (ECG) and epigal-
locatechin gallate (EGCG). About 35% of the mass of
these compounds is gallic acid, present as gallate esters
(Harbowy and Ballentine, 1997). It can therefore be
estimated that ∼5% of the weight of the green tea leaf
will be gallic acid. The estimate is similar for black tea,
but the form in which the gallic acid is present will differ
from that in green tea given oxidative changes to the
composition of catechins during black tea production.
It is likely that free gallic acid is higher in the black
teasestimated at 1% of dry weight (Harbowy and
Ballentine, 1997)sand possibly more available for up-
In conclusion, we have identified three metabolites
of gallic acid (4OMGA, 3OMGA, and 3,4OdiMGA) in