Mitigation of Oxidative Damage by Green Tea
Polyphenols and Tai Chi Exercise in Postmenopausal
Women with Osteopenia
Guoqing Qian1, Kathy Xue1, Lili Tang1, Franklin Wang1, Xiao Song2, Ming-Chien Chyu3,
Barbara C. Pence4, Chwan-Li Shen4,5,6, Jia-Sheng Wang1*
1Department of Environmental Health Science, University of Georgia, Athens, Georgia, United States of America, 2Department of Epidemiology and Biostatistics,
University of Georgia, Athens, Georgia, United States of America, 3Department of Mechanical Engineering, Texas Tech University, Lubbock, Texas, United States of
America, 4Laboratory Science and Primary Care, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America, 5Department of Pathology,
Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America, 6Laura W. Bush Institute for Women’s Health, Texas Tech University Health
Sciences Center, Lubbock, Texas, United States of America
Background: Osteoporosis is a degenerative bone disease predominantly in postmenopausal women. Green tea
polyphenols (GTP) and Tai Chi (TC) have been shown to be beneficial on human bone health. This study examined the
efficacy of GTP and TC on mitigation of oxidative damage in postmenopausal women with osteopenia.
Methods: A 6-month randomized and placebo-controlled clinical trial was conducted in 171 postmenopausal women with
osteopenia, who were recruited from Lubbock County, Texas. These participants were treated with placebo, GTP (500 mg
daily), placebo + TC (60-minute group exercise, 3 times/week), or GTP (500 mg daily) + TC (60-minute group exercise, 3
times/week), respectively. Their blood and urine samples were collected at the baseline, 1-, 3- and 6-months during
intervention for assessing levels of 8-hydroxy-29-deoxyguanosine (8-OHdG), an oxidative DNA damage biomarker, and
concentrations of serum and urine GTP components.
Results: The elevated concentrations of serum and urinary GTP components demonstrated a good adherence for the trial. A
significant reduction of urinary 8-OHdG concentrations was found in all three treated groups during 3-month (P,0.001) and
6-month (P,0.001) intervention, as compared to the placebo group. The significant time- and dose-effects on mitigation of
the oxidative damage biomarker were also found for GTP, TC, and GTP+TC intervened groups.
Conclusion: Our study demonstrated that GTP and TC interventions were effective strategies of reducing the levels of
oxidative stress, a putative mechanism for osteoporosis in postmenopausal women, and more importantly, working in an
additive manner, which holds the potential as alternative tools to improve bone health in this population.
Trial Registration: ClinicalTrials.gov NCT00625391
Citation: Qian G, Xue K, Tang L, Wang F, Song X, et al. (2012) Mitigation of Oxidative Damage by Green Tea Polyphenols and Tai Chi Exercise in Postmenopausal
Women with Osteopenia. PLoS ONE 7(10): e48090. doi:10.1371/journal.pone.0048090
Editor: D William Cameron, University of Ottawa, Canada
Received May 15, 2012; Accepted September 19, 2012; Published October 31, 2012
Copyright: ? 2012 Qian et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Center for Complementary and Alternative Medicine (R21AT003735)(nccam.nih.gov/) and National Cancer
Institute, National Institutes of Health (RO1CA90997)(www.cancer.gov). The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Osteoporosis is a degenerative bone disease characterized by
low bone mass and structural deterioration of bone tissue. Bone
fragility and increased susceptibilities to bone fracture especially in
the hip, spine and wrist are common outcomes . Worldwide,
osteoporosis is a huge health and social concern as approximately
200 million women suffer from this chronic disease . In the
United States, it is estimated that about 44 million people at age of
50 and older suffer from osteoporosis or low bone mass .
Postmenopausal women have four times the risk to develop
osteoporosis than men due to decreased estrogen levels after
menopause and represent the highest risk population .
Etiological risk factors associated with osteoporosis include poor
nutrition, imbalanced cytokines and hormones, and the aging
process [5,6]. It has been noticed that reactive oxygen species
(ROS) plays a key role in the aging process, and contributes greatly
to osteoporosis [7–9]. Roles of ROS in the pathology of
osteoporosis have been reviewed in detail by Manolagas ,
including influences of the generation and survival of osteoclasts,
osteoblasts, and osteocytes, disturbance of FoxOs in early
mesenchymal progenitors and Wnt signaling pathway, which led
to decreased osteoblastogenesis. Excess ROS can damage DNA to
form 8-hydroxy-29-deoxyguanosine (8-OHdG), an oxidative bio-
PLOS ONE | www.plosone.org1October 2012 | Volume 7 | Issue 10 | e48090
marker that has been widely used in human studies to indicate the
oxidative stress status [11,12].
Green tea polyphenols (GTP, extract of green tea) have shown
its osteo-protective effects via decreasing oxidative stress, increas-
ing activity of antioxidant enzymes, and decreasing expression of
proinflammatory mediators in rodent models , and the
beneficial effects of GTP on bone health has been reviewed
recently . Biomarkers of green tea consumption using green
tea components have been validated in human studies , and
the GTP supplementation has demonstrated chemopreventive
effects on cancer , cardiovascular diseases , and neurode-
generative diseases . However, limited information is available
about the beneficial effect of consumption of tea or its bioactive
components (i.e., GTP) on bone health in postmenopausal women.
On the other hand, Tai Chi (TC) exercise, a form of low to
moderate intensity mind-body exercise with aerobic and muscular
fitness activity, has been demonstrated to potentially benefit bone
health in several human studies [19–21]. The potential mecha-
nisms include slowing the decrease of bone mineral density (BMD)
, reducing the oxidative damage, and enhancing the enzyme
activity of superoxide dismutase (SOD) . Nevertheless, the
information on effects of GTP and TC, especially their combined
effects, on oxidative stress status of postmenopausal women is
In this study, a 6-month placebo-controlled randomized trial
was conducted to examine the pharmacokinetics of GTP and
effects of GTP and TC on mitigation of the oxidative damage
biomarker in 171 study participants, who were recruited from a
large community pool including 1,065 postmenopausal women. As
for the whole project of this clinical trial, the detailed study
protocol , the data on the safety issues and life quality of study
participants , and clinical outcomes on bone health  have
been published previously.
The protocol for this trial and supporting CONSORT checklist
are available as supporting information; see Checklist S1 and
1. Study Participants and Design
The detailed flow of the trial is described in Figure 1. The study
participants were screened and enrolled in 2007 from Lubbock,
Texas and the surrounding area. The trial was conducted in 2007–
2008. The study followed the randomized and placebo-controlled
clinical trial guidelines from National Institute of Health (NIH).
Briefly, a total of 171 community-dwelling postmenopausal
women were recruited to participate in this 6-month trial. The
recruited participants were randomized in a stratified method
based on age ($65 or ,65 years old), history of green tea
consumption, and history of mind-body exercise and assigned to
one of the four treatment groups: Placebo group (age: 57.6 (7.5),
mean (SD)): medicinal starch 500 mg daily; GTP group (age: 56.5
(5.5), mean (SD)): GTP 500 mg daily; Placebo+TC group (age:
58.3 (7.7), mean (SD)): medicinal starch 500 mg daily and 24-
move simplified Yang-style TC training (60 minutes per session, 3
sessions per week), and GTP+TC group (age: 57.6 (6.7), mean
(SD)): GTP 500 mg daily and 24-move simplified Yang-style TC
training (60 minutes per session, 3 sessions per week). A daily dose
of GTP or placebo material was administered as two capsules
(250 mg each). Daily consumption of such a GTP dose (500 mg)
was equivalent to 4 cups of green tea (about 500 mL). During
intervention, all participants were provided with 500 mg elemental
calcium and 200 IU vitamin D (as cholecalciferol) daily. The
complete study protocol has been reported in detail previously
. Inclusion criteria in this study included: postmenopausal
women (at least 2 years after menopause) with osteopenia (mean
lumbar spine and/or hip BMD T-score between 1 and 2.5
standard deviation (SD) below the normal sex-matched areal
BMD of the reference database; normal function of thyroid, liver,
and kidney; and serum 25-hydroxy vitamin D (25-OH-D,
$20 ng/mL)). Women were excluded if they had a disease
condition or were taking medication known to affect bone
metabolism; history of cancer except for treated superficial basal
or squamous cell carcinoma of the skin; uncontrolled intercurrent
illness or physical condition that would be a contraindication to
exercise; depression; cognitive impairment; and if unwilling to
accept randomization. Written informed consent was obtained
from all the participants before enrollment and the study was
approved by the Texas Tech University Health Sciences Center
Institutional Review Board.
2. Randomization and Blinding
A central allocation schedule for randomization was prepared
through a computer-generated random sequence of the four
treatment allocations. Treatment assignments were then made
from separate randomization sequences created for each stratum.
Placebo or GTP capsules dispersion was conducted by the site
research pharmacist according to the patient’s randomization
assignment. Research staff and medical staff were unaware of the
treatment assignment. Sample analysis, data collection and
statistical analysis were blinded to the technical personnel.
3. Sample Size
Data from previous studies were used for calculation of sample
size. The effect size of 0.75 was assumed based on the baseline
mean and standard deviation (SD) values for the primary outcome
of urinary 8-OHdG (7.562.5 pmol/mL) . Equal sample size
for each group was assumed. The main comparisons are expected
to be made as follows: placebo vs. GTP; placebo vs. placebo+TC;
and placebo vs. GTP+TC. The sample size was calculated based
on detecting a 25% change in urinary 8-OHdG, as compared to
placebo group, with a power of 0.8 at a=0.05. An assumed
correlation coefficient of 0.80 between baseline and follow-up
measurements within a treatment group was used. The sample size
for each group was estimated to be 32 for urinary 8-OHdG based
on a previously reported method . Therefore, a total sample
size of 128 participants is needed which requires 151 participants
at an attrition rate of 15%.
4. Chemicals and Instruments
GTP capsules and placebo agents were supplied by Zhejiang
Yixin Pharmaceutical Co., Ltd. China (GTP IND no. 77,470 by
FDA of USA). The main GTP components were 46.5% of
epigallocatechin-3-gallate (EGCG), 21.25% of epigallocatechin
(ECG), 10% of epicatechin (EC), 7.5% of epicatechin-3-gallate
(EGC), 9.5% of gallocatechingallate (GCG), and 4.5% of catechin
with the purity higher than 98.5%. Authentic standards of EC,
ECG, EGC, and EGCG, beta-glucuronidase, sulfatase, and
tetrahydroxyfuran (THF) were purchased from Sigma Chemical
Co. (St. Louis, MO). Acetonitrile (ACN) was purchased from
Burdick & Jackson (Muskegon, MI). Ethyl acetate, methylene
chloride, and sodium dihydrophosphate (NaH2PO4) were prod-
ucts of Fisher Scientific (Pittsburgh, PA). An ESA HPLC-
CoulArray system (Chelmsford, MA) was used for detection of
GTP components and metabolites as well as urinary 8-OHdG.
Modulation of Oxidative Damage by GTP and Tai Chi
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5. Blood and Urine Sample Collection and Processing
All participants reported to Clinical Research Center at Texas
Tech University Medical Center for blood/urine collection. The
blood samples were collected between 7:00 and 10:00 a.m. after
an overnight fast with abstinence of food, drink, nicotine, and
caffeine. Blood was drawn from a superficial arm vein with a
syringe, transferred to a vacutainer, allowed to clot at room
temperature, and then centrifuged at 1500 g for 10 min within 2
hours of collection to separate plasma or serum. Urine samples
were also collected in acid-washed polyethylene containers. After
the total volume of the urine sample was measured to an accuracy
of 0.1 ml and recorded, urine was aliquoted. All plasma and urine
samples were stored at 280uC before analyses.
6. Extraction of Serum and Urinary GTP
The protocol for serum GTP extraction was previously
published . Briefly, 200 mL serum samples were incubated
with beta-glucuronidase (500 units) and sulfatase (20 units) at 37uC
for 45 min to release the conjugated GTP components before
repeated extraction with 400 mL methylene chloride to remove
proteins and lipids. The aqueous phases were pooled for twice
extraction with 700 mL ethyl acetate, and the organic phase was
vacuum-dried with a Labconco Centrivap concentrator (Kansas
City, MO) and reconstituted for HPLC-ECD analysis.
Urinary GTP extraction followed a previously established
protocol . Briefly, thawed urine samples were centrifuged
and 1 mL supernatant was taken for 60 min incubation with beta-
glucuronidase (500 units) and sulfatase (2 units) at 37uC to release
conjugated GTP, then directly extracted twice with ethyl acetate.
Organic phases were pooled, dried in vacuo, and reconstituted in
15% acetonitrile before analysis.
7. HPLC-ECD Analysis of Serum and Urinary GTP
The method for analyzing serum and urinary GTP conjugates
was modified from previously established protocols . GTP
analysis was conducted in the ESA HPLC-CoulArray system
(Chelmsford, MA). The mobile phases consisted of buffer A
(30 mM NaH2PO4:ACN:THF
pH 3.35) and buffer B (15 mM NaH2PO4:ACN:THF at 30%:
63%: 7%, v/v, pH 3.45). The Zorbax Eclipse XDB-C18 (5
micron, 4.66250 mm) was used and maintained at 35uC during
at 98%:1.8%: 0.2%,v/v,
Figure 1. Flow chart of the trial.
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separation. Flow rate was set at 1 mL/min and a gradient was
generated to separate the GTP components within 60 min. The 8
channels of the CoulArray detector were sequentially set at 290,
210, 70, 150, 230, 310, 380 and 450 mV potentials for the
detection of GTP components. The main peaks appeared at
210 mV (EGC), 70 mV (EC, EGCG), and 150 mV (ECG).
Calibration curves for individual standard GTP component were
generated separately, and EGC, EC, EGCG, and ECG were
eluted at around 14, 21, 24, and 29 min, respectively. Quality
assurance and quality control procedures included analysis of one
authentic standard for every set of five samples and simultaneous
analysis of a quality control sample daily. The limits of detection
were 1.0 ng/mL urine for EC and EGC and 0.5 ng/mL serum for
EGCG and ECG, respectively. Urinary GTP components were
adjusted by creatinine level to eliminate the variation in urine
volume. Urinary creatinine concentration was determined color-
imetrically with a Diagnostics Creatinine Kit (Sigma Co).
8. Measurement of Urinary 8-OHdG
The procedure for 8-OHdG analysis was modified from
established protocols . Urinary 8-OHdG was extracted from
1 mL urine with the OasisH HLB 3 cc (60 mg) cartridge. The
eluents were dried under ultra-pure N2stream and reconstituted in
buffer (10 mM ammonium acetate in 2% MeOH, pH 4.3) for
analysis with the ESA HPLC-CoulArray system. The HPLC
column for 8-OHdG analysis was Waters YMC basicTM column
(S3 mm, 4.66150 mm). The mobile phases consisted of buffer A
(10 mM ammonium acetate, pH 4.3) and buffer B (100%
methanol). Flow rate was kept at 0.8 mL/min and a linear
gradient (0–40% MeOH in 15 min) was applied for chromato-
graphic separation with the peak of 8-OHdG eluted at around
9.5 min. The CoulArray Detector was set at 270, 300, 330, and
360 mv, with the highest peak appeared at 330 mv channel.
Authentic standard 8-OHdG was used for qualification by
retention times and response patterns, and quantification by
calibration curves. Quality assurance and quality control proce-
dures included analysis of one authentic standard for every set of
five samples and simultaneous analysis of a spiked urine sample
daily. The limit of detection for 8-OHdG was 1 ng/mL urine. The
amount of 8-OHdG was adjusted by urinary creatinine concen-
trations, which was also determined with a Diagnostics Creatinine
Kit (Sigma Co).
9. Statistical Analysis
All of the data generated were stored in an Excel database and
analyzed with SAS software version 9.3 (SAS Institute Inc., Cary,
NC). Median, mean, standard deviations (SD) and range were
calculated for concentrations of 8-OHdG, EC and EGC in urine,
and EGCG and ECG in serum. The values were expressed as
median and mean 6 SD unless otherwise stated. To assess the
efficacy of GTP, TC, or GTP+TC intervention arms, the
statistical evaluation focused on the comparisons among different
treatment arms and different time points. For the parameters that
were normally distributed (serum EGCG and ECG), repeated
measures ANOVA and Bonferroni correction were used to
compare differences among means of different times separately
for serum EGCG and ECG. For the parameters that were not
normally distributed (urinary EC, EGC and 8-OHdG), the
Kruskal-Wallis test of one-way ANOVA followed by Wilcoxon
rank sum test with Bonferroni correction was used to compare the
differences among different treatment groups; and the Friedman’s
test followed by Wilcoxon signed rank test with Bonferroni
correction was used to compare the differences among different
time points (urinary EC, EGC and 8-OHdG) given the
dependence of measurements. For urinary EC and EGC, the test
of difference between GTP and GTP+TC groups at the same time
point was done by Wilcoxon rank sum test with Bonferroni
correction. To evaluate the effect of dose and time interactions on
treatment arms, a nonparametric model for analysis of repeated
measurements was applied as previously described . A P-value
of less than 0.05 (two-tailed) was considered statistically significant.
1. Overall Study Outcome
A total of 88% (150/171) participants completed for this 6-
month clinical trial with complete data. Seven (16%) participants
in the Placebo arm, 5 (12%) in the Placebo+TC arm, 8 (17%) in
the GTP arm, and 1 (3%) in the GTP+TC arm withdrew before
the end of the study due to accidental fall at home (1 participant),
relocation (2 participants), time conflicts (6 participants), lost to
follow-up (5 participants) and lost interest (7 participants). The
compliance rate was 89% for both GTP and placebo capsules and
the adherence rate for TC classes was 83%.
2. Serum GTP in Study Participants
To examine the adherence and compliance of study participants
in this intervention trial, serum and urinary GTP concentrations
were measured at different intervention intervals, 0, 1-, 3- and 6-
month. There were no detectable GTP components in the placebo
group and TC group over 4 collection times of serum samples.
There were no GTP components in the baseline of all 4 treatment
groups, which was consistent with the questionnaire data, i.e., no
green tea drinking habits in these study participants. Among GTP-
intervention groups (GTP and GTP+TC), only EGCG and ECG
were constantly detectable in serum samples of the study
participants after intervention. Serum EGCG concentrations in
both GTP and GTP+TC groups were elevated in study
participants at 1-month after intervention and kept at the similar
levels at 3- and 6-months (Figure 2A). No significant time effect on
serum EGCG concentrations was found in GTP+TC Group
P=0.413) while significant time effect were found in GTP group
(P=0.019) and between 3- and 6-months (P,0.050). Serum ECG
concentrations in both GTP and GTP+TC groups were also
elevated in study participants at 1-month after the intervention
and kept at similar concentrations at 3- and 6-months (Figure 2B.).
No statistically significant time effect on serum ECG concentra-
tions was found (P=0.321 in GTP group and P=0.438 in
GTP+TC group), though there were some fluctuations in data
between 3-month and 6-month time points.
3. Urinary GTP in Study Participants
There were no detectable green tea polyphenol components in
the placebo group and TC group participants over 4 time-points
collected urine samples. Among GTP intervention groups (GTP
and GTP+TC), only EC and EGC were constantly detectable in
urinary samples of the study participants. As shown in Figure 3A,
urinary EC concentrations in both GTP and GTP+TC groups
were elevated in study participants at 1-month after the
intervention and kept at higher concentrations at 3- and 6-
months. No significant time effect on urinary EC concentrations
was found (P=0.201 in GTP group and P=0.575 in GTP+TC
group). Urinary EGC concentrations in GTP-intervened group
were elevated in study participants at 1-month after the
intervention and kept at constantly higher concentrations at 3-
and 6-months (Figure 3B), whereas urinary EGC concentrations in
GTP+TC group were kept elevated linearly in study participants
in 1-, 3-, and 6-months. No significant time effect on urinary EGC
Modulation of Oxidative Damage by GTP and Tai Chi
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concentrations was found in GTP group (P=0.689), while
significant difference occurred in GTP+TC group (P=0.035)
with difference found between 1- and 3-months.
4. Urinary 8-OHdG Concentrations in Study Participants
Urinary concentrations of 8-OHdG in study participants from
each treatment group at the baseline, 1-, 3-, and 6-months of the
intervention trial were summarized in Table 1, Figures 4 and 5.
Data in Table 1 included the mean concentration of 8-OHdG
with standard deviation, median, and detectable range. Detailed
distribution of the whole dataset was shown in Figures 4 and 5. As
shown in Figure 4A, the mean baseline concentrations among 4
treatment groups, although varied, were comparable (P=0.299).
After 1-month intervention, though no statistical significance was
found among groups, the median concentration of 8-OHdG
decreased by 27.4%, 43.9%, and 40.1% in TC, GTP, and
GTP+TC treated groups, respectively (Figure 4B). After 3-months
intervention, the median concentration of 8-OHdG decreased by
44.1%, 62.4%, and 64.8% in TC, GTP, and GTP+TC treated
groups, respectively (Figure 4C), which showed statistical signifi-
cance among treatment groups (P,0.001). After 6-months
intervention, the median concentration of 8-OHdG decreased
by 40.1%, 60.1%, and 73.3% in TC, GTP, and GTP+TC treated
groups, respectively (Figure 4D), which also showed statistical
significance among treatment groups (P,0.001).
Over the 6-month period, urinary 8-OHdG concentrations in
the placebo group showed minimal changes as compared with the
baseline level and no significant time effect was found (P=0.133,
Figure 5A). As shown in Figure 5B, urinary 8-OHdG concentra-
tions in TC group significantly decreased over intervention time
(P,0.001). The time effect was even more striking in GTP group
(P,0.001, Figure 5C) and GTP+TC group (P,0.001, Figure 5D),
suggesting that treatment with TC, GTP, or GTP+TC signifi-
cantly decreased 8-OHdG concentrations in study participants.
5. Non-parametric Model Analysis of Treatment and Time
on Concentrations of Urinary 8-OHdG
The effects of treatment and time interactions on urinary 8-
OHdG concentrations were analyzed in a non-parametric model
for repeated measures, which were summarized in Table 2.
Statistical significances in the main effect of treatment and time
and their interaction were found by this model analysis (P#0.001).
The treatment of TC, GTP and GTP+TC all had significant
effects on urinary 8-OHdG concentrations, as compared with the
Figure 2. Mean (±SEM) serum GTP concentrations in study
participants in the clinical trial. A: serum EGCG concentrations; B:
serum ECG concentrations. Starting from first month, serum EGCG and
ECG kept constant with minor fluctuations in the GTP and GTP+TC
groups. The differences among different time points (1-, 3-, or 6-
months) within each group and between groups were tested with
repeated measures ANOVA followed by Bonferroni correction for
multiple comparisons for each outcome and the significant difference
was found in EGCG concentrations in the GTP group between 3- and 6-
months (P,0.050). No significant differences were found between
these two groups at each time point (P.0.050).
Figure 3. Mean (±SEM) urinary GTP concentrations in study
participants in intervention groups. A: urinary EC concentrations;
B: urinary EGC concentrations. Starting from first month, urinary EC and
EGC concentrations kept constant with minor fluctuations in GTP and
GTP+TC groups. No significant differences were found between these
two groups at same time points (P.0.050, tested with Wilcoxon rank
sum test). The differences among differnt time points within each
group were tested with Friedman’s test followed by Wilcoxon signed
rank test with Bonferroni correction for multiple comparisons for each
outcome and the significant difference only existed in the EGC
concentrations in GTP+TC group between 1- and 3-months (P,0.050).
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Table 1. Urinary 8-OHdG concentrations in study participants in the intervention trial.
Urinary 8-OHdG (ng/mg creatinine)
Groups No. of participants Baseline1-month 3-month6-month
Placebon=37 64.5642.7 65.8648.568.3648.173.5651.0 0.133
58.0 (3.5–197.4)55.4 (3.0–241.6) 66.5 (2.6–256.2)62.6 (4.9–258.2)
Placebo + TC n=37 64.1623.6 45.6625.43,4
65.1 (23.5–154.9)40.2 (9.6–132.6)37.2 (4.0–104.8) 37.5 (8.9–93.6)
65.4 (7.6–290.6) 31.1 (2.5–175.1)25.0 (1.1–154.4)25.0 (0.5–164.5)
GTP + TC n=3775.8661.0 45.7643.53,4
65.9 (4.2–249.8)33.2 (1.2–194.5) 23.4 (0.5–167.2) 16.7 (0.5–76.8)
Urinary 8-OHdG values are expressed as Mean6SD, median (range).
1P-values (Treatment) were from Kruskal-Wallis test of one-way ANOVA.
2P-values (Time) were from Friedman’s test.
3Significantly different from Placebo at the same time point, tested by Wilcoxon rank sum test with Bonferroni correction for multiple comparisons, P,0.050.
4Significantly different from baseline in the same group, tested by Wilcoxon signed rank test with Bonferroni correction for multiple comparisons, P,0.001.
Figure 4. Urinary 8-OHdG concentrations in study participants in different groups. A: baseline, no significant differences in urinary 8-
OHdG were found (P=0.299); B: 1 month, urinary 8-OHdG concentrations were reduced by all treatments, but no significant differences were
observed (P=0.110); C: 3 month, urinary 8-OHdG concentrations were significantly reduced in all treatment groups (P,0.001); D: 6 month, urinary 8-
OHdG concentrations were significantly reduced in all treatment groups (P,0.001).
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placebo group (P,0.050); the urinary concentrations of 8-OHdG
were also significantly modified by time for each of the treatment
group (P,0.050). For each group, the duration of treatment
proved to have cumulative effects on urinary 8-OHdG in the six
month period. These results demonstrated the efficacy of
treatment and time on lowering urinary 8-OHdG concentrations
in study participants in this trial.
In this study, we assessed the adherence and compliance of the
6-month clinical trial by measuring GTP components in blood and
urine of the participants. Our previous studies have validated that
measurement of blood and urinary GTP components are good
biomarkers for determining green tea consumption and GTP
intervention in human population studies [15,27]. Among the four
major GTP components, all EGCG, EGC, EC, and ECG, were
detectable in blood and urine samples , but only EGCG and
ECG in serum and EC and EGC in urine showed dose-response
correlations . Results of serum concentrations of EGCG and
ECG and urinary excretion of EC and EGC in this study showed
significant elevation after GTP intervention and no detectable
concentrations in the baseline and the placebo participants over
the study period, which further confirmed the excellent adherence
and compliance of the overall trial. In addition, these results were
Figure 5. Temporal patterns of urinary 8-OHdG concentrations in study participants. A: Placebo, no significant differences was observed
in different treatment periods (P=0.133); B: TC, compared to baseline, urinary 8-OHdG concentrations were significantly reduced at 1-, 3-, and 6-
months treatment (P,0.001); C: GTP, compared to baseline, urinary 8-OHdG concentrations were all significantly reduced at 1-, 3-, and 6-months
treatment (P,0.001); and D: GTP+TC, compared to baseline, urinary 8-OHdG concentrations were all significantly reduced at 1-, 3- and 6-months
Table 2. Non-paramatric model analysis of treatment and
time on concentrations of urinary 8-OHdG.
Effect Test statistic (P-value)
Time 97.92 (P,0.001)
Treatment*Time 17.15 (P,0.001)
TC v.s. Placebo10.62 (P=0.001)
GTP v.s. Placebo3.76 (P=0.037)
(GTP+TC) v.s. Placebo4.80 (P=0.029)
TC*Time (time effect in TC group) 13.38 (P,0.001)
GTP*Time (time effect in GTP group)67.20 (P,0.001)
(time effect in (GTP+TC) group)
The test statistics and P-values shown here were generated from a non-
parametric model designed for repeated measurements using SAS macro
F1_LD_F1, the more detailed information on model assumption and hypothesis
is referred to reference #30, chapter 8.
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consistent with our previous findings, i.e., blood and urinary GTP
components are practical and reliable biomarkers for green tea
In this study, we further assessed the efficacy of GTP
supplement and TC exercise for their individual and combined
effects on the oxidative DNA damage biomarker, 8-OHdG, in
postmenopausal women with osteopenia in the 6-month clinical
trial. Results from this study showed that individual GTP
supplement, TC exercise, or the combination significantly
decreased urinary excretion of 8-OHdG after 1-, 3-, and 6-
months intervention in postmenopausal women, as compared with
the placebo group, which demonstrated the efficacy of this trial in
reducing the oxidative stress biomarker levels.
Several lines of evidence have indicated the role of ROS in
induction of osteoporosis [10,14]: ROS are capable of influencing
the generation and survival of osteoclasts, osteoblasts, and
osteocytes; ROS-activated FoxOs in early mesenchymal progen-
itors also leads to decreased osteoblastogenesis through disruption
of Wnt signaling pathway ; ROS also increases serum
osteopontin and transforming growth factor-b levels in iron
overloaded rats, suggesting osteoclast-mediated bone resorption
through the receptor activator of nuclear factor-kB/RANK ligand
(RANK/RANKL) mediated signaling pathway . In postmen-
opausal women, the reduced estrogen level might be a key factor
in boosting the ROS level because estrogen has been reported to
be able to diminish production of ROS and stimulate the activity
of glutathione reductase . The ability of green tea and GTP as
potent ROS scavengers has been well recognized as the basis for
its biological activity [16,32]. Previous studies by others and ours
showed strong evidence of protective effects of GTP on the
capacity of bone formation due to its anti-oxidative stress
potentials [33,34]. An animal study shows that urinary excretion
of 8-OHdG is a reliable indicator of oxidative stress in
postmenopausal rats . More importantly, urinary 8-OHdG
concentration has also been widely used as a DNA oxidative
damage biomarker in many human studies [11,35]. In this study
we observed a significant decrease in urinary 8-OHdG concen-
trations in postmenopausal women appearing at 1- to 6-months
after the GTP intervention as compared to the placebo control
group, which further suggests evidence of oxidative stress in the
study participants. The result of this study is consistent with the
previous finding in HBsAg carrier groups, which also showed
statistically significant reduced urinary 8-OHdG after 3-months
GTP intervention at 500 mg or 1,000 mg/day .
TC exercise has been characterized as a moderate intensity
mind-body exercise, coupling muscular activity with an internally
directed focus, producing a temporary self-contemplative mental
state . In this study, the TC exercise significantly decreased
urinary 8-OHdG concentrations after 3-months intervention as
compared with the control group. The effect of exercise on
oxidative stress has been reviewed . It was reported that high-
intensity exercise increased oxidative stress biomarkers, including
8-OHdG and malondialdehyde (MDA)-modified low-density
lipoprotein in humans, whereas moderate exercise tended to
decrease both indices of oxidative stress . A recent study has
also demonstrated that TC exercise stimulated endogenous
antioxidant enzymes (superoxide dismutase, SOD) and reduced
oxidative damage markers (MDA) in middle-aged adults .
These reports support our observation that TC, a moderate
exercise, reduced oxidative stress in postmenopausal women.
This is the first study to investigate the combined effects of GTP
and TC on oxidative DNA damage in postmenopausal women
with osteopenia. An additive effect of GTP supplementation and
TC exercise on suppression of oxidative stress was found, as
evidenced by a 40% reduction of urinary 8-OHdG in the TC
group and 60% reduction in the GTP group alone and a 73%
reduction was observed in the GTP+TC group. Results of this
study further suggest that a combinative intervention strategy,
including dietary supplementation and moderate exercise, may be
more effective in promoting bone health in postmenopausal
women with osteopenia.
The strength of our study was in its design as a randomized and
placebo controlled clinical trial that could limit the biases usually
introduced through the design and data acquisition. Limitations to
this study included a relatively small sample size that might limit
the statistical power, such as parameters after 1-month of
intervention. A wide inter-individual variation found in concen-
tration of biomarkers measured may be due to the individual
variations in GTP metabolism enzymes, endogenous susceptibility
factors, and/or DNA repair capacity; the large variation in urinary
8-OHdG levels (43.9642.1 ng/mg creatinine) has also been
reported previously in females , which supports this finding
in our study. The stratified randomization and non-parametric
analysis used could limit potential biases (randomization) and
increase the power of detecting the differences (non-parametric
model analysis). The intent to treat (ITT) analysis could benefit the
comparison; however, considering the comparable compliance
and adherence rates for the treatment groups, the effect of
dropouts might be minimal. On the other hand, the exclusion of
dropouts into statistical analysis may not severely affect the
statistical power as an originally estimated 140 participants (34
participants for each group) would be sufficient for a power of
about 0.90 for the outcome of urinary 8-OHdG level.
In conclusion, this study provides evidence that 500 mg GTP
supplement, Tai Chi exercise, either alone or together, can
effectively reduce an oxidative stress biomarker in postmenopausal
women with osteopenia. Based on the putative role of oxidative
stress in osteoporosis and the observed beneficial effects on bone
health as published previously, these intervention strategies hold
the potential as an alternative tool to benefit the bone health in
postmenopausal women which deserves further long-term human
Safety and clinical outcomes S1.
Authors acknowledge all participants in this study, Dr. Jay Magaziner
(University of Maryland, MD) for his advice, and the assistance of Mary J.
Flores, Raul Y. Dagda, and Marisela Dagda in the clinical data collection.
We also thank Dr. Li Xu for her input in the statistical analysis.
Conceived and designed the experiments: JSW CLS MCC BCP.
Performed the experiments: GQ KX LT FW. Analyzed the data: XS
LT GQ. Contributed reagents/materials/analysis tools: JSW. Wrote the
paper: GQ. Read and approved the final manuscript: GQ KX LT FW XS
MCC BCP CLS JSW.
Modulation of Oxidative Damage by GTP and Tai Chi
PLOS ONE | www.plosone.org8 October 2012 | Volume 7 | Issue 10 | e48090
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Modulation of Oxidative Damage by GTP and Tai Chi
PLOS ONE | www.plosone.org9 October 2012 | Volume 7 | Issue 10 | e48090