Proceedings of the Third International Scientic Symposium

on Tea and Human Health: Role of Flavonoids in the Diet

Black Tea Consumption Reduces Total and LDL Cholesterol in Mildly
Hypercholesterolemic Adults
1
Michael J. Davies,* Joseph T. Judd,*
2
David J. Baer,* Beverly A. Clevidence,*
David R. Paul,* Alison J. Edwards,* Sheila A. Wiseman,

Richard A. Muesing**
and Shirley C. Chen

*Beltsville Human Nutrition Research Center, ARS-U.S. Department of Agriculture, Beltsville, MD;

Unilever
Research Laboratory, Vlaardingen, The Netherlands; **The George Washington University Lipid Research
Clinic, Washington, DC;

Unilever Bestfoods NA, Englewood Cliffs, NJ
ABSTRACT Despite epidemiological evidence that tea consumption is associated with the reduced risk of
coronary heart disease, experimental studies designed to show that tea affects oxidative stress or blood choles-
terol concentration have been unsuccessful. We assessed the effects of black tea consumption on lipid and
lipoprotein concentrations in mildly hypercholesterolemic adults. Tea and other beverages were included in a
carefully controlled weight-maintaining diet. Five servings/d of tea were compared with a placebo beverage in a
blinded randomized crossover study (7 men and 8 women, consuming a controlled diet for 3 wk/treatment). The
caffeine-free placebo was prepared to match the tea in color and taste. In a third period, caffeine was added to the
placebo in an amount equal to that in the tea. Five servings/d of tea reduced total cholesterol 6.5%, LDL cholesterol
11.1%, apolipoprotein B 5% and lipoprotein(a) 16.4% compared with the placebo with added caffeine. Compared
with the placebo without added caffeine, total cholesterol was reduced 3.8% and LDL cholesterol was reduced
7.5% whereas apolipoprotein B, Lp(a), HDL cholesterol, apolipoprotein A-I and triglycerides were unchanged.
Plasma oxidized LDL, F2-isoprostanes, urinary 8-hydroxy-2-deoxyguanosine, ex vivo ferric ion reducing capacity
and thiobarbituric acid reactive substances in LDL were not affected by tea consumption compared with either
placebo. Thus, inclusion of tea in a diet moderately low in fat reduces total and LDL cholesterol by signicant
amounts and may, therefore, reduce the risk of coronary heart disease. Tea consumption did not affect antioxidant
status in this study. J. Nutr. 133: 3298S3302S, 2003.
KEY WORDS: black tea cholesterol lipoproteins antioxidant status caffeine
Flavonoids, polyphenolic compounds found naturally in
various plant materials, possess antioxidant properties in vitro
and ex vivo and cholesterol-lowering effects in humans and
animals (14). Black tea is a major source of avonoids in
Western diets (3). Several recent epidemiological studies have
examined the relationship between black tea or avonoid
consumption and the risk of cardiovascular disease (CVD)
3
(1) including coronary heart disease (CHD) or ischemic stroke
(5), but the results from these studies are not consistent. Most
studies report an apparent protective effect for CHD or stroke
with high intakes of black tea or avonoids (610). Con-
versely, no protective effects from tea intake were noted in a
large cohort study (11), and in another study an increased risk
of death from CHD was found with increased tea consumption
(12).
Most free-living studies have observed that ingestion of
black tea did not improve plasma or lipoprotein antioxidant
status (1317). However, some researchers have found that
acute consumption of black tea increases antioxidant activity
(1820). Moreover, chronic consumption of tea reduced the
susceptibility of LDL to oxidation ex vivo in a cross-sectional
study (21). Two randomized trials did not detect an effect of
tea drinking on total LDL or HDL cholesterol (15,22). These
studies did not control for diet and thus may not have been
able to detect small alterations in antioxidant status and blood
lipids. Therefore, the purpose of the present study was to
examine the effects of black tea ingestion on blood lipid
proles and markers of oxidative stress and antioxidant status
1
Presented as part of The Third International Scientic Symposium on Tea
and Human Health: Role of Flavonoids in the Diet, given at the United States
Department of Agriculture, September 23, 2002. This conference was sponsored
by the American Cancer Society, American College of Nutrition, American Health
Foundation, American Society for Nutritional Sciences, Food and Agriculture
Organization, and the Linus Pauling Institute at Oregon State University and was
supported by a grant from the Tea Council of the U.S.A. Guest editor for this
symposium was Jeffrey Blumberg, PhD, Jean Mayer USDA Human Nutrition
Research Center on Aging, Tufts University, Boston, MA 02111.
2
To whom correspondence should be addressed.
E-mail: judd@bhnrc.arsusda.gov.
3
Abbreviations used: 8OhdG, 8-hydroxy-2-deoxyguanosine; CHD, coronary
heart disease; CVD, cardiovascular disease; FRAP, ferric reducing ability of
plasma; MDA, malondialdehyde; NCEP, National Cholesterol Education Program;
P, placebo having no added caffeine; PC, placebo with caffeine added; T, black
tea.
0022-3166/03 $3.00 2003 American Society for Nutritional Sciences.
3298S

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in a controlled dietary setting with mildly hypercholester-
olemic volunteers.
METHODS
Subjects. Men and women were selected based on the following
criteria: mildly elevated total cholesterol, 35 y-of-age or older, 90
140% of ideal body weight, no major health problems such as diabe-
tes, heart disease, stroke, or cancer and not taking prescription
medications that could interfere with lipid metabolism. Women had
to be postmenopausal (last menses at least 1 y earlier) and not
undergoing hormone replacement therapy. Volunteers had to be
willing to consume all foods and beverages supplied by the study. The
Committee on Human Research, Johns Hopkins University School of
Hygiene and Public Health approved the study. All volunteers pro-
vided written consent for participation in the study. They also re-
ceived monetary compensation commensurate with the effort re-
quired of them by the study.
Study design. Volunteers were recruited to participate in a ran-
domized double-blind crossover study of black tea (T) compared with
a placebo having no added caffeine (P). To assess caffeine effects, a
third period was added post hoc in which all subjects received the
placebo with caffeine added (PC) at a concentration equivalent to
that in T. Volunteers for the third study period were recruited from
participants who completed periods 1 and 2 of the study. Beverages
for T, P and PC treatments were prepared from dry powders similar to
instant tea.
Prior to the rst treatment period volunteers were placed in a 2-wk
run-in period. Additionally, treatments were separated by a 4-wk
washout period. During these times, alcohol and tea consumption
were not allowed. During all phases of the study (run-in, wash-out
and treatment periods) volunteers ingested either one cup of caffein-
ated coffee or two caffeinated diet sodas daily thus establishing a
consistent baseline level of caffeine intake and preventing possible
caffeine withdrawal symptoms. Subjects eliminated other caffeine-
containing foods and medications throughout the study. As an indi-
cator of compliance, intake of caffeine-containing beverages and
medications was assessed from daily forms completed by the volun-
teers. Urinary caffeine excretion was determined as an indirect
marker of intake.
Controlled diet. During the three treatment periods, volunteers
consumed the same background-controlled diet. All foods and bev-
erages were prepared and supplied by the Human Studies Facility at
the Beltsville Human Nutrition Research Center (Beltsville, MD).
Food items were weighed, served in proportion to caloric require-
ments and color-coded according to the treatment beverage. Dieti-
tians monitored food and treatment beverage selections at each meal.
Composites of foods in the 7-d menu cycle were prepared and ana-
lyzed for macronutrients and fatty acids (Covance Laboratories, Mad-
ison, WI). Seven-day menus were prepared in 200-kcal increments
and designed to follow a National Cholesterol Education Program
(NCEP) Step I-type diet (23). The diets provided 58% of calories
from carbohydrates, 26% from fat and 16% from protein. The fat had
a ratio of polyunsaturated to monounsaturated to saturated fatty acids
of 1:1:0.8. The diet provided 71 mg of cholesterol, 13.6 g of dietary
ber and 8.5 mg of iron per 1000 kcal. At the average energy intake
for the study of 2760 kcal, this translates to a daily intake of 196 mg
of cholesterol, 33.6 g of dietary ber and 23.5 g of iron. The amount
of dietary caffeine in the background diet, i.e., not associated with the
treatment beverages, coffee or sodas, was 26.3 mg/1000 kcal (72.6
mg/d). During P, caffeine excretion was approximately equal in the
prestudy baseline level. During T and PC, caffeine excretion in-
creased by 2.6 and 3.0 times the level of excretion during P. Except
for calcium and iron when prescribed by the volunteers personal
physician, vitamin and mineral supplementation was not permitted.
Each weekday, volunteers were weighed and energy intake was
adjusted as needed to keep body weight constant. Dinner and break-
fast were consumed at the Center during the week; carryout lunches
and snacks were provided. Weekend foods and treatment beverages
were packaged with instructions for home consumption. Blood pres-
sure was monitored weekly to assess the effects of T, P and PC.
Blinding. During the rst two periods of the study, investigators,
volunteers and kitchen staff were blinded to the T or P treatments.
Because of the post hoc addition of a third treatment period, only the
kitchen staff and volunteers were blinded to the PC treatment in the
third study period. Treatments were blinded to investigators and
kitchen staff by placing powdered drink mixtures in numbered and
color-coded packets and to volunteers by color coding and the addi-
tion of articial fruit avors (apple or lemon), articial sweetener and
coloring to mask the appearance and distinctive taste of black tea.
Treatment packets were coded, analyzed and supplied by Unilever
Bestfoods NA [Englewood Cliffs, NJ (Table 1)]. Black tea was pro-
vided as lyophilized tea solids in packets that contained the amount
of tea solids that approximated that which would come from one
regular tea bag, i.e., one serving of tea. Black tea and P were prepared
daily by the kitchen staff and served with breakfast and dinner.
Volunteers consumed two servings of T or P (apple-avored) with
breakfast and the equivalent of three servings of T or P (lemon-
avored) with dinner for a total of ve servings per day. All beverages
were prepared with 180 mL of room temperature spring water per
serving. Volunteers were allowed to chill, heat or add additional
articial sweetener to their treatment beverage as desired. Volunteers
consumed 71% of the treatment beverages (25 out of 35 drinks per
week) under supervision of a dietitian in the Beltsville human study
facility during breakfast and dinner on weekdays. The remaining 10
drinks per week were consumed at home during the weekends.
Sample Collection. Procedures for blood sampling and processing
were those described in the protocol for the Lipid Research Clinics
Program (24). Blood samples were drawn during the last week of the
study on two different days and after an overnight fast (minimum
12 h). Plasma (from EDTA tubes) or serum was harvested from whole
blood collected by venipuncture and divided into cryogenic vials for
storage at 80C. LDL, for use in oxidation assays, was isolated from
4 mL of fresh plasma. Briey, the triglyceride-rich (d 1.0063 g/mL)
and LDL (d 1.065 g/mL) fractions of plasma were isolated and
removed sequentially by standard ultracentrifugation techniques
(25). Both centrifugation runs were performed at 10C and 187,000
g for 14 h using a 50.4 Ti rotor (Beckman Instruments, Palo Alto,
CA). Once isolated, LDL was brought to 4 mL, portioned as one mL
aliquots, purged with nitrogen and stored at 80C. For the TBARS
assay, glutathione was added to the LDL fraction to minimize oxida-
TABLE 1
Composition of one serving of black tea, placebo without
caffeine, and placebo with caffeine
1
Component
Black
tea
Placebo
Without
caffeine
With
caffeine
Tea solids, mg 700 0 0
Epigallocatechin, mg (% of
polyphenols) 6.6 (3.8) 0 0
Epigallocatechin gallate, mg
(% of polyphenols) 7.8 (4.5) 0 0
Epicatechin, mg (% of
polyphenols) 4.6 (2.7) 0 0
Epicatechin gallate, mg (%
of polyphenols) 5.3 (3.1) 0 0
Theaavin, mg (% of
polyphenols) 2.0 (1.2) 0 0
Theaavin 3-gallate, mg (%
of polyphenols) 2.0 (1.2) 0 0
Theaavin 3-gallate, mg (%
of polyphenols) 0.5 (0.3) 0 0
Theaavin3,3-digallate, mg
(% of polyphenols) 1.6 (0.9) 0 0
Polyphenols, mg 172 0 0
Caffeine, mg 40.6 0 44
Carbohydrates, g 2.75 2.75 2.75
Sugar, g 0.25 0.25 0.25
Other, g 2.50 2.50 2.50
1
Volunteers consumed ve servings of treatment beverage per day.
BLACK TEA AND CHOLESTEROL 3299S

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tion and stored at 80C under nitrogen. After the nal blood
collection, plasma, serum or LDL samples were analyzed with all
samples for a subject included in the same analytical run. Complete
urine collections were obtained on three consecutive days at the end
of each treatment period. An aliquot of urine was frozen each day.
Subsequently, samples were thawed on ice and an amount of urine
from each days collection proportional to that days total volume was
pooled for analysis.
Lipid and apolipoprotein proles. Prior to freezing, plasma ali-
quot was precipitated for HDL determination using the sequential
precipitation procedure of Gidez et al. (26). Lipid analyses were
performed at the Lipid Research Clinic Laboratory, The George
Washington University Medical Center, which maintains standard-
ization with the Centers for Disease Control and Prevention, U.S.
Department of Health and Human Services, for the analysis of total
cholesterol, triglycerides and HDL cholesterol. Plasma total choles-
terol, HDL cholesterol and triglycerides were determined enzymati-
cally using commercial kits (Sigma Chemical Company, St. Louis,
MO) on an Abbott VP analyzer (Abbott Laboratories, Chicago, IL).
LDL cholesterol was calculated by the Friedewald procedure (27).
Plasma apolipoprotein A-I and B concentrations were determined by
rate nephelometry (Beckman ICS Immunochemical Analyzer; Beck-
man Instruments, Fullerton, CA).
Lp(a) was analyzed as described previously (28,29) by using a
commercially available enzyme-linked immunosorbent assay (Strate-
gic Diagnostics, Newark, DE).
Oxidative stress. Oxidized LDL concentration was measured in
plasma with a commercial ELISA (Mercodia AB, Uppsala, Sweden).
Total F
2
-isoprostanes were measured in plasma with a commercial
ELISA (Cayman Chemical Co., Ann Arbor, MI). Urinary 8-hy-
droxy-2-deoxyguanosine (8OhdG) was determined using a commer-
cially available competitive ELISA assay (OxisResearch, Portland,
OR).
Antioxidant status. Plasma antioxidant capacity was measured
using the ferric-reducing ability of plasma (FRAP) assay as described
by Benzie and Strain (30). Lipid peroxidation in the LDL fraction was
determined spectrophometrically by measuring the amount of mal-
ondialdehyde (MDA) equivalents using thiobarbituric acid and ex-
pressed as TBARS according to the method of Fogelman et al. (31).
LDL protein content was determined by the Lowry method (32) and
used to normalize MDA equivalents for comparisons.
Statistics. Statistical analyses were performed using SAS-PC ver-
sion 8.2 (SAS Institute, Cary, NC). All variables from the start
(baseline) and end of each treatment period were compared with a
mixed model ANOVA that included xed terms for treatment (i.e.,
T, P or PC) and period with a repeated term for volunteer. BMI and
baseline value of the variable were included in the model as covari-
ates to adjust for differences among the subjects for these parameters.
Data are presented as least-square means SEE unless otherwise
stated in the text. Values were considered statistical signicant at P
0.05.
RESULTS
Physical characteristics and compliance. Sixteen volun-
teers (8 men and 8 women) completed the rst two random-
ized dietary periods and 13 out of 16 elected to participate in
the third treatment period. After lipid analyses, one male
volunteer was found to have had a hypertriglyceridemic re-
sponse to the reduced fat controlled diet and his data were
excluded from further analyses. Baseline physical characteris-
tics for the 15 volunteers included in the nal data are shown
in Table 2. Body weight was determined each weekday morn-
ing, and once the energy level needed to maintain weight was
established, stability of body weight without further caloric
change was considered to be an indicator of compliance.
Compliance was also assessed through a daily questionnaire
that included questions on general health and on consumption
of foods or drinks not provided by the study. Body weights did
not change signicantly from the prestudy level throughout
the study (data not shown). Weekly blood pressure measure-
ments were not different among treatments (data not shown).
Blood lipids and lipoproteins. Total and LDL cholesterol
concentrations were reduced by 3.8% (P 0.0589, trend) and
7.5% (P 0.0140), respectively, after consumption of 5 serv-
ings/d of T compared with P (Table 3). Compared with PC,
consumption of T reduced total and LDL cholesterol by 6.5%
(P 0.0007) and 11.1% (P 0.0002), respectively. Although
the total and LDL cholesterol concentrations were numeri-
cally higher after PC than P, the differences were not signif-
icant at the 0.05 probability level. Apolipoprotein B was not
signicantly different between T and P. However, compared
with PC, T lowered apo B by 5.0% (P 0.0322) and P by
4.7% (P 0.0371). There was no difference in Lp(a) concen-
tration between T and P. However, Lp(a) was signicantly
lower after T (16.4%, P 0.0085) and P (13.0%, P 0.0321)
than after PC. There were no effects of T, P or PC consump-
tion on HDL cholesterol, apolipoprotein A-I or triglyceride
concentrations.
Antioxidants and oxidative stress. No treatment effects
were found for plasma concentration of oxidized LDL, plasma
F2-isoprostane concentration or urinary excretion of 8OhdG
(Table 4). Antioxidant status or capacity indicated by FRAP
or TBARS in LDL were not different following consumption
of T, P or PC.
DISCUSSION
The present study was the rst controlled dietary investi-
gation to demonstrate that the consumption of black tea can
appreciably reduce total and LDL cholesterol. In our study, tea
was included as part of carefully controlled NCEP Step I-type
diet fed to mildly hypercholesterolemic volunteers. Whether
or not similar changes occur under different dietary conditions
such as with diets higher in fat or cholesterol or varying in
other nutrients, remains to be determined. However, because
our diets were composed of a variety of mixed foods and could
be considered typical of diets consumed by many people, it is
not unreasonable to speculate that the benecial effects of tea
can translate to other dietary conditions. Furthermore, we do
not know if tea can prevent the development of hypercholes-
terolemia because our study was conducted with subjects with
pre-existing moderately elevated cholesterol concentrations.
In the 1990 report of strategies for blood cholesterol reduction
from the NCEP (23), it was estimated that for every 1%
reduction in total cholesterol concentration, the risk of CVD
decreased by an average of 2%. In the present study, consump-
tion of black tea resulted in reductions in total cholesterol by
3.8 and 6.5% compared with P and PC, respectively. This may
translate to a decrease risk of CVD of from 8 to 13% when 5
TABLE 2
Characteristics of 15 volunteers at entry into the study
Variable Baseline values
1
n (m/f) 15 (7/8)
Age, yr 53.9 2.4
Weight, kg 86.8 3.6
BMI, kg/m
2
29.8 1.3
Total cholesterol, mg/dL 208.9 5.4
LDL cholesterol, mg/dL 135.0 4.3
HDL cholesterol, mg/dL 50.0 3.2
Triglyceride, mg/dL 119.7 11.2
1
Values are mean SE.
SUPPLEMENT 3300S

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servings of black tea per day are included as part of a prudent
diet moderately low in fat, saturated fatty acids and choles-
terol. These data are in agreement with the ndings of Stens-
vold et al. (7) from a large cohort study that noted reduced
total cholesterol concentration with increasing tea consump-
tion. Similar observations have been noted with green tea
ingestion (33). Our ndings are in conict with the results of
others that did not detect an effect of tea on total, LDL and
HDL cholesterol (13,15,21,22). These studies were all free-
living diet studies designed to assess the effect of consuming
tea on blood lipid proles in either a randomized crossover or
cross-sectional design. Moreover, three of the studies were
similar in length (4-wk) to the present study (3-wk), but they
were unable to detect a tea effect on blood lipid proles
(13,21,22). The controlled dietary regimens utilized in the
present study may have enabled us to detect the signicant
reduction in total and LDL cholesterol concentration with
black tea consumption.
A possible mechanism for the cholesterol-lowering effect of
tea may be that tea limits cholesterol absorption in the intes-
tine. Tea catechins, specically gallate esters, were shown to
be hypocholesterolemic in rats by reducing cholesterol absorp-
tion in vivo and by precipitating cholesterol from micelles in
vitro (34). The hypolipidemic effect of green tea extracts was
not associated with reduced cholesterol or fatty acid synthesis
in hamsters, thus leading the authors to suggest that the effect
of green tea extract on cholesterol reduction was on absorption
(35). It has not been determined if a similar mechanism occurs
in humans.
Oxidative modication of LDL is thought to play an im-
portant role in the development of atherosclerosis (36). We
measured markers of antioxidant status and oxidative stress by
various methods: FRAP, LDL TBARS, oxidized LDL and
F2-isoprostanes and 8OhdG. Our ndings are not in agree-
ment with the ndings of those that have demonstrated that
acute and chronic consumption of tea improved antioxidant
status (1821). However, only one study included a caffeine
control group (19), and the other studies utilized water as the
control treatment (18,20,21). Furthermore, Hodgson et al.
(19) reported that lipid oxidation was similar between black
tea and caffeine treatments.
We conclude that the addition of ve servings of black tea
TABLE 3
Plasma lipid proles of volunteers consuming a controlled National Cholesterol Education Program Step I-type diet with ve
servings per day of either black tea, placebo without caffeine or placebo with caffeine equivalent to that in the tea
1
Variable Black tea
Placebo
Without caffeine With caffeine
n (m/f) 15 (7/8) 15 (7/8) 12 (6/6)
Total cholesterol, mg/dL 200.1 3.8
a
208.6 3.2
b
212.6 3.5
b
LDL cholesterol, mg/dL 122.0 3.1
a
131.9 3.1
bc
137.3 3.4
c
Apolipoprotein B, g/L 84.2 2.3
a
84.8 2.2
ab
88.6 2.5
c
Lipoprotein (a) 19.3 1.2
a
20.1 1.2
ab
23.1 1.3
c
HDL cholesterol, mg/dL 46.5 0.6
a
47.1 0.6
a
46.1 0.7
a
Apolipoprotein AI, g/L 152.2 2.8
a
151.1 2.8
a
153.2 3.0
a
Triglyceride, mg/dL 165.5 7.9
a
150.8 7.9
a
157.4 8.7
a
1
Data are presented as least-square means SEE estimated following a mixed model ANOVA for effects of diet, period and diet by period
interaction with statistical adjustment for covariance with BMI and baseline value of respective variable. Values with different superscripts are
signicantly different at P 0.05.
TABLE 4
Plasma and urine markers of oxidative stress and antioxidant status in volunteers consuming a National Cholesterol
Education Program step I-type diet with ve servings per day of either black tea, placebo without caffeine
or placebo with caffeine equivalent to that in the tea
1
Variable Black tea
Placebo
Without
caffeine
With
caffeine
n (m/f) 15 (7/8) 15 (7/8) 12 (6/6)
Oxidative stress
Plasma oxidized LDL, units/L 53 6 54 5 55 6
Plasma F2-isoprostanes, pg/mL 137 5 130 5 125 6
Urine 8-hydroxy-2-deoxyguanosine,
ug/mmol creatinine 0.6 0.01 0.4 0.01 0.5 0.02
Antioxidant status
Plasma FRAP,
2
mmol/L 1.5 0.1 1.4 0.1 1.5 0.1
LDL TBARS, nmol MDA/mg protein 2.5 0.3 2.6 0.4 2.5 0.3
1
There were no differences due to diet in a mixed model analysis of variance with adjustment for covariance with the baseline value of the
respective variable. Data are presented as least-square means SEE.
2
Abbreviations: FRAP, ferric-reducing ability of plasma; MDA, malondialdehyde.
BLACK TEA AND CHOLESTEROL 3301S

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per day to an NCEP Step I-type diet appreciably reduces total
and LDL cholesterol in mildly hypercholesterolemic volun-
teers. According to the new NCEP Adult Treatment Panel III
classication, consumption of black tea results in improving
LDL cholesterol classication from borderline high to near
optimal/above optimal (37). While questions remain regarding
some of the benecial effects tea may have as part of a prudent
diet, based on our study, the inclusion of tea in the diet has the
potential to signicantly reduce blood cholesterol and thereby
reduce the risk of CVD and should be encouraged.
ACKNOWLEDGMENTS
The authors thank Melanie Turgeon for technical assistance
throughout the investigation. We thank Evelyn Lashley and the staff
of the Beltsville Human Nutrition Research Center Human Studies
Facility for assistance in feeding the controlled diets. We thank
Unilever Bestfoods NA for partial nancial support and for prepara-
tion of the treatment beverages as well as for cooperation in the
performance of the study.
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