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*
Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan and
Institute of Nutritional Sciences, Justus Liebig University Giessen, D-35392 Giessen, Germany
2To whom correspondence should be addressed. E-mail: shahrzad{at}md.tsukuba.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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KEY WORDS: • tea • gallic acid • bioavailability • humans • HPLC
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2
3)
, and in
free or bound forms, it is found in large amounts in tea leaves
(3)
, from which it is extracted in hot water infusions.
GA is a strong antioxidant that possesses antimutagenic and
anticarcinogenic activities (4
5
6
7
8
9
10)
. Its derivative,
4-O-methylgallic acid (4OMGA), has been reported as the main
metabolite of GA in rats, rabbits and chickens (1
,11
12
13
14
15)
and humans (16)
, but despite the special activities of GA,
there are no data available about the extent of its absorption,
elimination or relative bioavailability from foodstuffs like tea, which
is one of the most common drinks for humans. To estimate such data, our
study was performed in healthy adult humans.
As a source of pure GA, acidum gallicum tablets were used that
contained 10% GA and 90% glucose. Acidum gallicum tablets have been
in use for more than four decades as therapy for patients with
bronchitis (17)
. Here, we compare the bioavailability and
some pharmacokinetic parameters of GA from a single oral dose of acidum
gallicum tablets with that of tea.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Each trial was performed with 10 healthy volunteers (5 men and 5 women)
with ages ranging from 23 to 36 y (mean: 28 y), with body
weights of 47–82 kg (mean: 63 kg) and heights of 152–190 cm (mean:
171 cm). They did not use any medication. The study was approved by the
Ethics Commission of Justus Liebig University Giessen, and the subjects
were fully informed of the protocol and intent of the study. The acute
toxicity of GA is very low; 24–29 mmol/kg subcutaneous or
intraperitoneal is required for a 50% lethal dose to mice or rats.
Prolonged consumption in amounts well above normal food levels is not
known to produce untoward effects (1)
.
Study design.
For a bioavailability study involving tea GA, we determined free and
bound GA in different kinds of tea. To avoid the possible interference
of conjugated forms of GA such as theogallin
(5-O-galloyl-quinic acid),
(-)-epigallocatechin-3-gallate or (-)-epicatechin-gallate in this
study, we chose and used Assam black tea extract, which contains
93% of its GA in free form.
Tea or acidum gallicum was administered at 0800 h after an overnight fasting. On the day before and during the study, subjects were asked to consume a GA-free diet, which consisted of white bread, cheese and water. In the first trial, 2 acidum gallicum tablets [0.15 mmol (25 mg) GA in a single tablet] were swallowed with 200 mL water. In the second trial, the tea brews were prepared immediately before administration. Each time, 125 mL Assam black tea brew containing 0.3 mmol (50 mg) free GA was adjusted with distilled water to 200 mL and ingested orally.
Blood samples (10 mL) were collected into heparinized blood containers (KABE Labortechnik, Nuembrecht-Elsenroth, Germany) before the administration and at different time intervals (i.e., 0.67, 1.33, 2, 3, 4, 5.5, 7.75 and 12 h) after the dose. The samples were centrifuged, and the harvested plasma was frozen immediately and stored at -18°C until analyzed. Urine was collected before dosing and quantitatively during the 24 h after dosing. Total urine volume was measured for each collection period, and 1-mL aliquots were stored frozen until assayed for GA and 4OMGA.
Plasma and urine samples were analyzed for GA and its metabolite,
4OMGA, using a previously reported HPLC method (16)
. The
preparation procedure for the samples was the same as described for
analysis of the tea brews. The plasma samples were centrifuged at 1800
x g for 10 min after extraction with ethyl acetate
and before separation of the organic fraction. The limit of
quantification was 0.30 µmol/L, and a limit of detection
(signal-to-noise ratio of 3) of 0.15 µmol/L was obtained in both
predose human plasma and human urine samples.
Tea brews.
Commercial samples (25 g) of Darjeeling, Assam (CTC Numalighur), Sri Lanka and Chinese green tea were put into a pot, extracted with 500 mL hot water and brewed on a hot plate for 5 min (the procedure was conducted with a coffee machine). The brew was filtered, and 10 mL of every tea brew was used for determination of its free and bound GA and 4OMGA concentrations.
To extract GA and 4OMGA, 2 mL of 1 mol sulfuric acid/L was added to 2 mL tea brew in glass tubes. The tubes were put in a boiling water bath, and the samples were hydrolyzed for 30 min and then cooled in a cold-water bath. These samples and two nonhydrolyzed samples were subjected to extraction twice with a 4-fold volume of ethyl acetate. The organic phase was pipetted in round-bottom flasks, and the combined ethyl acetate extracts were evaporated to dryness under vacuum with a rotary evaporator, with the path temperature maintained at <35°C. Each extract was redissolved in an appropriate amount of HPLC mobile phase before HPLC analysis.
To validate the extraction method, 0.03, 0.3, 0.5, 3 and 6 µmol GA and 4OMGA were added to 10-mL tea brews. Then, 2 mL of each sample before hydrolysis and 2 mL after hydrolysis were extracted and analyzed. This procedure was repeated twice for each tea.
Chromatographic condition.
The chromatographic condition (16)
was the same for all
samples (tea, plasma and urine samples). A Merck and Hitachi
high-precision pump (model L-6000; Darmstadt, Germany) equipped
with a 100-µL loop was used. The substances were detected using a
Gynkotek spectrophotometer (SP-4; Germering, Germany) at 220 and 270
nm. Separation was carried out using a LiChrospher 100 RP-18 column (5
µm, 120 x 4 mm I.D.; Merck) with a guard column (RP-18, 4
x 4 mm; Merck). Chromatographic data were recorded using a Merck
and Hitachi D-2000 chromatointegrator. The chromatographic separation
of GA and 4OMGA was achieved by reverse phase HPLC using isocratic
elution. Mobile phase was water/acetonitrile (97.5:2.5, v/v) modified
with 2.5 mol phosphoric acid/L. Retention times and absorbance ratios
(at two different wavelengths: 220 and 270 nm) against those of
standards were used to identify the separated peaks and to check their
purity. Quantitative determinations were carried out with the external
standard method.
Data analysis and statistics.
Noncompartmental pharmacokinetic parameters were calculated from plasma
concentration-time data using established methods (with computer
software package TopFit 2) (18
,19)
. The maximum plasma
concentration was determined through visual inspection of the data.
Area under the plasma concentration-time curve (AUC) was calculated
using the trapezoidal rule. Beyond 12 h, the area was in all cases
insignificant. If the concentration after 12 h (C12)
was below the quantification limit, C12 was determined with
the equation C12 = Clast x e-kt, where t is
12 - tlast, and Clast and
tlast are the last measurable concentration
and the respective time. The elimination rate constant
(k) and half-life were calculated from
log-linear regression of the plasma concentration-time profile
in the terminal portion of the curve. Total urinary recovery was
determined by multiplying the concentration of parent GA or its
metabolite (4OMGA) in urine by the volume of the urine sample in each
collection interval and then calculating the sum for all intervals
after dosing. Renal clearance was calculated by dividing the total
amount of parent GA or 4OMGA collected in urine by the respective
plasma AUC during the 12-h interval. Relative bioavailability was
assessed by the tea-to-acidum gallicum ratios of the plasma
AUC(0–12 h).
Results are means ± SD. The statistical significance of differences was tested using the nonparametric Wilcoxon test, and testing for sequence effects was performed with ANOVA with the Bonferroni correction. The differences were considered statistically significant when the calculated P-value was <0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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A study of the repeatability of the analytical method and its reproducibility between days was performed. The repeatability (n = 7) showed a relative standard deviation of 2%. The reproducibility (n = 10) between days was 3.5%, and good response linearity was obtained between 0.1 and 300 µmol/L (in water). Recoveries of both GA and 4OMGA from tea samples exceeded 93%. The results of comparisons of different tea samples showed that Assam black tea brew contained the lowest amount of conjugated GA; 93% of its GA was in free form. (The brew of 25 g tea leaves extracted with 500 mL hot water contained 2.35 ± 0.08 and 0.18 ± 0.03 mmol free and conjugated GA/L, respectively; n = 4.) Therefore, it was used for pharmacokinetic and bioavailability studies. In the Chinese green tea, 97% of GA was in its conjugated forms. We did not find any 4OMGA in the tea samples.
Pharmacokinetics and bioavailability.
The results of the noncompartmental pharmacokinetic analysis are listed
in Table 1
. Figure 1
shows the chromatograms of plasma before and after the administration
of acidum gallicum and Assam black tea brew. The mean plasma
concentration-time profiles of GA and 4OMGA after the
administration of acidum gallicum and tea are shown in Figure 2
.
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The effects of tea consumption on health have recently received a great
deal of attention. Many laboratory studies have demonstrated clearly
and repeatedly the inhibition of tumorigenesis in different animal
models by tea and tea polyphenols (10
,20
21
22
23
24)
.
The molecular mechanisms for these inhibitory actions are not fully
understood. A major problem in investigating the relationship between
tea and cancer is the lack of quantitative data. Even in studies with
animals, mechanistic understanding of the inhibitory effect of tea
against tumorigenesis is hampered by a lack of information on the
bioavailability of the effective components of tea. It has been assumed
that most of the cancer-inhibitory activity of tea is due to the
polyphenols present in the tea. GA has been suggested as a biomarker
for tea consumption. It was found that the GA moiety of theaflavins is
essential for their potent antioxidative and antimutagenic activities
(24)
.
In this study, we obtained some detailed information about the bioavailability of GA of tea in humans. We prepared 200 mL tea brew of 6.24 g black tea leaves (Assam), which was about three times more concentrated than normal tea brews. GA concentration in the stomach could achieve a maximum of 1.5 mmol/L (there was 0.3 mmol GA in 200 mL tea brew). GA was rapidly absorbed, but the highest GA concentration observed in plasma was only 1.83 ± 0.16 µmol/L for the tablets and 2.09 ± 0.22 µmol/L for the tea after 1.3 ± 0.2 and 1.4 ± 0.2 h, respectively. In addition, the highest concentration of its metabolite in plasma was not higher than 2.83 ± 0.25 µmol/L for the tablets and 2.64 ± 0.28 µmol/L for the tea (tmax = 1.5 ± 0.3 h). Whether these low plasma concentrations of GA can have pharmacological activity in the body needs to be investigated. Although with respect to the total amount collected in the urine, >60% of GA excreted was metabolized to 4OMGA, there is no information about whether this metabolite can contribute to the pharmacological effects. Therefore, a greater understanding of pharmacological activities of 4OMGA is highly desirable.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: AUC, area under the plasma concentration-time curve; GA, gallic acid; 4OMGA, 4-O-methylgallic acid.
Manuscript received September 26, 2000. Revision accepted December 19, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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