Original Paper
Ann Nutr Metab 2008;53:33–42
DOI: 10.1159/000153006
Efficacy and Safety of Chinese Black
Tea (Pu-Ehr) Extract in Healthy and
Hypercholesterolemic Subjects
Hiroyuki Fujita Tomohide Yamagami
Research and Development Department, Nippon Supplement, Inc., Osaka, Japan
Key Words
Black tea extract  Cholesterol  Hypercholesterolemia 
Pu-Ehr  Metabolic syndrome
Abstract
Background/Aims: Water-extracted Chinese black tea (Pu-
Ehr) exerts a precipitating effect on mixed bile salt micelles
in foods. The amount of black tea extract (BTE) effective for
hypercholesterolemia (HC) was examined in humans. Mate-
rials and Methods: Animals (test 1) and humans (tests 2–5)
were given BTE in the following studies: (test 1) an acute 2-
week toxicity test conducted in mice with 2,000 mg/kg BTE
or saline; (test 2) a dose-finding 8-week toxicity study with
excessive BTE ingestion (0.75 or 1 g/day) in 10 healthy and 10
borderline HC subjects; (test 3) a 1-day acute toxicity test us-
ing a (10 g) single-bolus study (n = 10); (test 4) a 5-week long-
term safety test (5 g/day, n = 11), and (test 5) a 4-month non-
comparative study in 21 HC patients ingesting 1 g/day.
Results: The safety study showed no changes in hemato-
logical or relevant biochemical parameters in both mice and
humans in the acute and long-term toxicity tests. In test 5,
significant reductions in total and low-density lipoprotein
cholesterol levels accompanied by significant decreases in
body weight were observed without affecting other bio-
chemical parameters. Conclusions: BTE significantly re-
© 2008 S. Karger AG, Basel
0250–6807/08/0531–0033$24.50/0
Fax +41 61 306 12 34
E-Mail karger@karger.ch Accessible online at:
www.karger.com www.karger.com/anm
Received: August 23, 2007
Accepted: May 6, 2008
Published online: September 4, 2008
duced blood cholesterol levels in humans and may prove
safe and useful in preventing and improving metabolic syn-
drome-induced arteriosclerosis and/or obesity.
Copyright © 2008 S. Karger AG, Basel
Introduction
Several studies have reported a positive correlation be-
tween serum cholesterol levels and risk of coronary heart
disease [1, 2]. A recent meta-analysis using both univari-
ate and multivariate analyses has demonstrated a signifi-
cantly increased risk of cardiovascular disease or athero-
sclerosis in humans with hypercholesterolemia, indepen-
dent of other blood lipid levels [3]. Therefore, reducing
serum cholesterol levels in patients with hypercholester-
olemia, which has mainly been accomplished by inhibit-
ing cholesterol synthesis [4, 5] or by blocking the absorp-
tion of dietary cholesterol [6, 7], is desirable.
In recent years, there has been an increased interest
in the importance of functional and/or biologically ac-
tive substances in foods, with health-relevant properties
being identified in various foods [8, 9]. The Ministry of
Health, Labor and Welfare (MHLW) of Japan has intro-
duced a ‘Food for Specified Health Uses’ (FOSHU) sta-
tus to prevent exaggerated and misleading claims re-
Fujita Hiroyuki, PhD
Research and Development Department
Nippon Supplement, Inc.
19-19, Chayamachi, Kita-Ku, Osaka 530-0031 (Japan)
Tel. +81 6 6376 2220, Fax +81 6 6376 2234, E-Mail fujita@nippon-sapuri.co.jp
garding the efficacy of various food substances for dis-
eased and health-deficient subjects. In particular, foods
and beverages containing plant sterols [10, 11], fibers
[12, 13], soy [14], teas [15–17] and roughage [18] have
been identified to lower blood cholesterols. Among the
beverages, green tea has also been widely reported to
display cholesterol-lowering and anti-oxidative effects,
as well as exhibiting the ability to reduce abdominal fat.
All these effects have been attributable to catechins,
such as epigallocatechin-3-gallate (EGCG) [19]. In addi-
tion, green tea catechins have been found to precipitate
mixed bile salt micelles and lower blood cholesterol lev-
els in rats [20]. A fermented black tea (Pu-Ehr) extract
(BTE) has been demonstrated to indicate a similar pre-
cipitating effect on mixed bile salt micelles [21]. Chinese
black tea (Pu-Ehr), a traditional beverage in China and
its vicinities, is obtained by first parching green tea
leaves and then fermenting them with microorganisms
such as Aspergillus sp. Pu-Ehr tea is therefore different
from the other more popular teas such as green tea, oth-
er black teas and oolong tea. In a previous study, we
demonstrated that a single oral BTE administration
(135 mg/kg/day) inhibits cholesterol absorption in mice
and significantly reduces serum cholesterol levels and
abdominal fat after 3 weeks’ administration in rats [22].
In drug efficacy, an effective dose is thought to be pro-
portional to the body surface and not to the body weight.
Therefore, a daily BTE intake of ca. 333 mg/meal (or 1
g/day) would be effective in humans. Following the
guidelines of FOSHU safety standards, we studied the
acute and long-term safety aspects of BTE using a single
bolus dose (10-fold the daily intake) and repeated dosing
at 5 times the daily intake (i.e. 5 g/day) over a 5-week
period, respectively.
The safety of BTE after a single-bolus intake and re-
peated long-term ingestion (5 weeks) was tested in healthy
and borderline hypercholesterolemic (BHC) subjects.
Furthermore, we monitored the effects of BTE on the se-
rum cholesterol levels and body weight in hypercholes-
terolemic (HC) patients after long-term (4 months) inges-
tion.
Materials and Methods
BTE Preparation
The broad-leaf-categorized Pu-Ehr tea cultivated in the Yun-
nan Highlands of China was used as the raw material. Harvest-
ed Pu-Ehr tea leaves were heated, dried, and dampened before
fermentation with Aspergillus niger for 6–12 months. The leaves
were then fermented, and the maximal temperature was estab-
34 Ann Nutr Metab 2008;53:33–42
lished during the middle phase of fermentation, i.e. maturing
the leaves to yield the desired Pu-Ehr tea. Pu-Ehr tea leaves
(100 g) were milled, suspended in 900 ml water, and boiled for
60 min at 100 ° C before centrifugation at 2,050 g for 30 min at
room temperature. The supernatant thus obtained was filtered
(filter paper No. 5C; Toyo Roshi Co., Kawasaki, Japan), which
was then concentrated before drying under a stream of air to
yield a powder.
Bacterial Reverse Mutation Test
BTE was tested on Salmonella typhimurium strains (TA98,
TA1537, TA100, TA1535) and Escherichia coli WP2 uvrA. Based
on the results of a concentration-range finding (data not shown),
BTE was prepared by dissolution in water (50 mg/ml) before being
repeatedly tested at five different doses (6.25, 12.5, 25, 50 and 100
l/plate) with or without metabolic activation. The positive con-
trol substances were AF2 (Wako Pure Chemical Co., Ltd.), 9-ami-
noacridine (ICH Biomedicals), 2-aminoanthracene (Wako), S9
mix (Oriental Yeast Co., Ltd.) and sodium azide (Wako); water
was used as the vehicle (controls). Tests were performed by the
preincubation method, and three plates per dose were used.
After the 48-hour incubation period, revertant colony counts
were measured manually. A colony analyzer (CA-11D, System Sci-
ence Inc.) was used for counting 100 or more colonies (TA100
strain and positive control group). Precipitation of test articles on
the plate was checked grossly, and the state of growth inhibition
(background lawn) was examined under stereoscopy. The rever-
tant colony counts, the mean 8 SD for test article groups (each
dose), negative and positive control groups were tabulated for
each cell strain. The concentration-response curves were plotted
for the respective test article groups. The test was repeated to de-
termine reproducibility of the results.
Statistical analysis of data was not performed. Instead, the test
results were judged as positive when (1) the revertant colony count
in the test article group were double or more than that in the neg-
ative control; (2) a dose-response relationship was found in at least
one strain with or without the metabolic activation system, and
(3) reproducibility was obtained.
Acute (2-Week) Toxicity Test in Mice (Test 1)
Male and female mice (age: 5 weeks) of the ICR strain (Japan
SLC Inc., Shizuoka) housed in plastic cages (5 mice/cage) and ac-
climatized under standard laboratory conditions (room tempera-
ture: 23 8 2 ° C; illumination: an alternating 12-hour light-dark
cycle) were provided with Labo MR Stock diet (Nihon Nosanko-
gyo Co., Ltd.) and drinking water ad libitum for 1 week before
experiments.
Mice were randomized into four groups according to gender
(male, female, male control, female control groups), and each
mouse was weighed. The mice were starved for 4 h before drug
administration. In the experimental groups, BTE samples sus-
pended in distilled water were administered orally to animals
with a stomach tube at a dose of 2,000 mg/kg (volume: 20 ml/kg).
As for the control groups, an equivolume of distilled water per
mouse was administered as described above.
Clinical and behavioral observations were monitored at the
same time (12:00 h) before and after BTE administration on days
7 and 14 (final day). The mice were weighed on a weekly basis, and
the mean body weight values of the experimental and control
groups were statistically analyzed by the paired and nonpaired
Fujita/Yamagami
Student’s t test (p ! 0.05). Mice were sacrificed for necropsy study
at the end of the test period (day 14 after BTE intake).
Safety/Toxicity Tests in Humans
All human studies were performed in accordance with the
Helsinki Declaration of 1975 (revised in 1983; http://www.wma.
net/e/policy/b3.htm).
Dose-Finding (8-Week) Toxicity Test with Doses of 0.75 g and
1 g per Day (Test 2)
The dose of BTE that produces significant effects in humans
has been investigated prior to conducting the safety study. Given
that BTE effectively lowered serum cholesterol levels at 135 mg/
kg/day in rats [22], the corresponding dose was administered to
healthy and BHC subjects, since the efficacy or toxicity of drugs
is proportional to the body surface; namely, subjects ingested 250
mg (0.75 g/day) or 333 mg (1 g/day) of BTE (derived from the
minimum effective dosage of 135 mg/kg/day in mice [22] based
on surface area) for 8 weeks. Ten healthy and ten BHC volunteers
ranging from 23 to 62 years of age (mean age: 40.5 8 2.3 years)
were enrolled in the study. Written informed consent was ob-
tained from all volunteers after having explained to them the na-
ture, purpose, and possible risks of the study. The study was ap-
proved by the Institutional Review Board in February 2005. All
enrolled subjects completed the study. None of the subjects had
any significant medical history. Subjects were divided without
any significant differences into two groups of 10 subjects (healthy:
5; BHC: 5 cases) each: while 10 subjects ingested 250 mg of BTE
before each meal (i.e. 3 meals/day or 0.75 g/day), the remaining 10
subjects ingested 333 mg of BTE before each meal (i.e. 3 meals/day
or 1 g/day) for 8 weeks. Hematological and relevant biochemical
parameters were examined before and 8 weeks after BTE intake;
the period after intake did not indicate any abnormal events. All
subjects were instructed to maintain their usual way of life
throughout the study period to avoid undue stress, and each kept
a diary to account for events/symptoms encountered. Participants
were also asked to comment on any unexpected deviations in di-
etary intake or bowel habits.
Acute Toxicity Test with a 10-g Single-Bolus Administration
(Test 3)
For the single-bolus (BTE: 10 g) ingestion study, 10 healthy
male (n = 6) and female (n = 4) volunteers (age range: 21–58 years;
mean age: 39.6 8 2.2 years) consented in writing to participate in
the study after the nature, purpose and possible risks were ex-
plained to them. The study was approved by the Institutional Re-
view Board in June 2005. Subjects were given 10 g of BTE in a
single-bolus administration, and their hematological and rele-
vant biochemical parameters were examined for 24 h after intake.
None of the subjects had any significant medical history. Only
healthy subjects were used because if any unwanted adverse ef-
fects should occur, any further test on unhealthy BHC or HC sub-
jects would have to be aborted.
Long-Term (5-Week) Safety Test with 5 g BTE/Day (Test 4)
In the long-term (5-week) safety study, 11 subjects (male: 6;
female: 5) comprising 5 healthy and 6 BHC volunteers (age range:
23–62 years; mean age: 41.3 8 2.6 years) consented in writing to
participate in the study after the nature, purpose, and possible
risks had beed explained to them. The study was approved by the
Hypocholesterolemic Effect of Black Tea
Extract in Humans
Institutional Review Board in June 2005. The gender allocation
for the healthy and BHC groups was impartially distributed to the
nearest uniformity. None of the subjects had any significant med-
ical history, and all subjects completed the study. Hematological
and relevant biochemical parameters, body weight and body mass
index (BMI) were monitored before and 5 weeks after BTE intake;
the post-intake observations did not reveal any abnormal events.
All subjects maintained their usual life activity throughout the
study period, faithfully recorded any unusual events/symptoms
encountered, and commented on any unexpected deviations in
dietary intake or bowel movements in a diary. Subjects were given
1.67 g of BTE before each meal (i.e. 3 meals daily or 5 g/day) for a
5-week period.
A Noncomparative (4-Month) Study of 1 g BTE/Day in
HC Patients (Test 5)
A noncomparative study was conducted between December
2005 and May 2006 at a hospital in Osaka, Japan. Twenty-one
fully developed HC patients (male: 5; female: 16) with (10/21 cas-
es) and without (11/21 cases) medications participated in this
study; the former and latter indicated respective fasting total cho-
lesterol (TCHO) and LDL cholesterol levels of 15.69 mmol/l
(mean: 7.25 8 0.15) and 13.88 mmol/l (4.88 8 0.11). All patients
gave their written informed consent for the study after the nature,
purpose, and possible risks had been explained to them. The
study was approved by the Institutional Review Board in Novem-
ber 2005. Patients treated with insulin, as well as those with a his-
tory of serious cardiac, renal, hepatic diseases, gastrectomy, en-
terectomy, other gastrointestinal surgeries or hypothyroidism
and those judged by the attending physician to be incompatible
were excluded from the study. Although 10 of 21 patients under
treatment with HMG-CoA reductase inhibitors (Atorvastatin)
were included, their dosages did not change during the study pe-
riod. All patients maintained normal physical activities and food
intake throughout the study. Blood was sampled for analysis of
hematological and biochemical parameters at 0 (immediately be-
fore BTE intake), 1, 2 and 4 months after BTE intake. All patients
kept a diary to account for events/symptoms noted, and they were
also instructed to comment on any unexpected deviations in di-
etary intake or bowel movements. The patients were given 3 meals
a day. Patients were given 333 mg of BTE before each meal (i.e. 3
meals a day or 1 g/day) for 4 months. All enrolled patients com-
pleted the study.
Blood Chemistry Analysis of Subjects
White and red blood cell counts and hemoglobin levels were
measured with an SE-9000 Multi Hematological Analysis System
(Sismex, Kobe, Japan). The following biochemical parameters
were measured at blood analysis laboratory (BML Co., Osaka, Ja-
pan) using a 7150 AutoAnalyze System (Hitachi Ltd., Tokyo, Ja-
pan): TG, total cholesterol, HDL cholesterol, free fatty acid, fast-
ing blood glucose (FBG), GOT, GPT, -GTP, creatinine and
CRP.
Statistical Analysis
Results are expressed as the mean 8 SE. Differences where
p ! 0.05 before and after BTE intake assessed with the paired
Student’s t test (Stat View, Abacus Concepts Inc., Berkeley, Calif.,
USA) were considered significant.
Ann Nutr Metab 2008;53:33–42 35
Table 1. Results of bacterial reverse mutation test of BTE

With (+) or without (–) S9 mix BTE concentration Mean number of revertants (number of colonies/plate)
(ml/plate)
basepair substitution type frameshift type
TA100 TA1535 WP2 uvrA TA98 TA1537

S9 mix (–) 0 (negative control) 110 11 22 17 6

106 8 31 17 6
88 (101)* 11 (10) 14 (22) 17 (17) 6 (6)
6.25 103 11 18 19 7
97 (100) 10 (11) 29 (24) 13 (16) 4 (6)
12.5 117 11 27 27 9
121 (119) 16 (14) 14 (21) 16 (22) 10 (10)
25 113 13 25 14 8
111 (112) 10 (12) 27 (26) 13 (14) 10 (9)
50 104 13 22 12 10
107 (106) 8 (11) 26 (24) 21 (17) 8 (9)
100 108 9 13 15 8
110 (109) 12 (11) 14 (14) 18 (17) 7 (8)
S9 mix (+) 0 (negative control) 105 12 31 26 22
115 10 21 26 14
112 (111) 12 (11) 25 (26) 22 (25) 10 (15)
6.25 108 13 26 33 8
114 (111) 13 (13) 21 (24) 35 (34) 15 (12)
12.5 114 11 11 30 17
100 (107) 12 (12) 22 (17) 27 (29) 8 (13)
25 122 11 21 22 10
104 (113) 7 (9) 27 (24) 37 (30) 9 (10)
50 131 6 19 26 12
121 (126) 16 (11) 19 (19) 31 (29) 14 (13)
100 135 12 28 31 13
144 (140) 7 (10) 23 (26) 42 (37) 20 (17)
Positive control S9 mix (–) chemical AF-2 NaN3 AF-2 AF-2 9-AA
concentration (mg/plate) 0.01 0.5 0.01 0.1 80
colony/plates 285 524 112 265 207
290 520 107 227 256
292 (289) 539 (528) 113 (111) 246 (246) 273 (245)
Positive control S9 mix (+) chemical 2-AA 2-AA 2-AA 2-AA 2-AA
concentration (mg/plate) 1.00 2.0 10.00 0.5 2
colony/plates 2,412 420 197 642 567
1,839 398 177 177 572
1,530 (1,930) 390 (403) 211 (195) 211 (195) 591 (577)

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; 9-AA = 9-aminoacridine; 2-AA = 2-aminoanthracene.

* Average of numbers.

Results strain used (table 1). Microbial contamination was not

Bacterial Reverse Mutation Test
The mutagenicity test was conducted at five concen-
trations (6.25–100 l/plate), with a common ratio of 2.
With or without S9 mix, none of the revertants showed
an increase double the count of negative controls in any
detected in the test solution, even at the highest concen-
tration, or with S9 mix, in any of the tests. The mutagen-
icity of positive control substances was confirmed, and
revertants in the positive and negative controls were
within the range (mean 8 3 ! SD) of historical control
values (table 1). Moreover, neither precipitation of the test

36 Ann Nutr Metab 2008;53:33–42 Fujita/Yamagami

 6.5 4.0
6.4 3.9
6.3 3.8

Total-cholesterol (mmol/l)

LDL-cholesterol (mmol/l)
6.2 3.7
6.1 3.6
6.0 3.5
5.9 3.4
Fig. 1. Effects of BTE on TCHO and LDL- 5.8 3.3
cholesterol levels in BHC subjects. Chang- 5.7 3.2
es in TCHO (a) and LDL-cholesterol levels 5.6 3.1
5.5 3.0
(b) in HC subjects given 250 mg (circle; 5.4 2.9
0.75 g/day) and 333 mg (square; 1 g/day) of 5.3 2.8
BTE before each meal (i.e. 3 times daily) 5.2 2.7 **
for an 8-week period. Values for each 5.1 ** 2.6
5.0 2.5
group (n = 10) are expressed as the mean
Before 8 weeks post-BTE Before 8 weeks post-BTE
8 SE, and differences (vs. pre-intake val-
a ingestion b ingestion
ues) where p ! 0.01 (**) were considered
significant.

Table 2. Body weight changes after oral

administration of BTE in male and Group Body weight, g
female mice pre-administration day 7 day 14

Experimental group (male; n = 5) 32.780.8 36.280.8 38.681.2

Control group (male; n = 5) 32.580.7 36.181.2 38.581.4
Experimental group (female; n = 5) 25.680.5 27.781.4 31.481.9
Control group (female; n = 5) 25.680.8 27.781.4 30.581.6

Test 1: Five mice were administered with BTE at a dose of 2,000 mg/kg.
Values are expressed as the mean 8 SD.

article nor growth inhibition was observed in any cell
strains. Furthermore, BTE did not increase colony counts
on the plate even at the highest dose. Data of the test were
judged as rational and therefore acceptable because: (1)
the negative control values were appropriate in compari-
son with the background values; (2) the positive control
value was more than twice the negative control value and
appropriate in comparison with the background values,
and (3) there were no abnormalities in the sterility test.
Test 1: Acute (2-Week) Toxicity Test in Mice
None of the mice of either sex indicated abnormal be-
havioral findings or died during the experimental peri-
od. Clinical observations indicated that the effects of BTE
consumption were gender independent. No significant
difference in body weight gain of either sex was noted in
the experimental and control groups (table 2). Necropsy
studies revealed no abnormal findings in any particular
organ of either sex in both groups.
Safety/Toxicity Tests in Humans
Test 2: Dose-Finding (8-Week) Toxicity Test with
Doses of 0.75 g and 1 g per Day
Although BTE reduced the serum TCHO (fig. 1a) and
LDL-cholesterol (fig. 1b) levels in BHC subjects in a dose-
dependent manner, a significant effect was established
only at a dose of 333 mg/meal (i.e. 3 meals daily or 1 g/day;
fig. 1). No significant effects on the hematological and
relevant biochemical data were observed in healthy sub-
jects. Both healthy and BHC subjects did not complain of
any adverse side effects (e.g. distension, abdominal pain,
diarrhea, retching, flatulence) or abnormal events/symp-
toms.
Test 3: Acute Toxicity Test with 10 g Single-Bolus
Administration
In the single-bolus ingestion study, none of the 10
healthy subjects who ingested the 10-g bolus of BTE com-
plained of any side effects (such as distension, abdominal
pain, diarrhea, retching or flatulence) or abnormal events/

Hypocholesterolemic Effect of Black Tea Ann Nutr Metab 2008;53:33–42 37

Extract in Humans
Table 3. Safety of BTE examined by blood analysis after single- Table 4. Safety of BTE examined by blood analysis after long-term
bolus (10 g) ingestion in 10 healthy subjects ingestion in healthy and borderline hypercholesterolemic sub-
jects
Before Post-BTE
ingestion Before 5 weeks after
BTE ingestion
Hematology
White blood cell, cells/nl 5.780.5 5.980.4 Hematology
Red blood cell, cells/pl 5.080.1 5.180.1 White blood cell, cells/nl 5.480.5 5.980.6
Hemoglobin, g/l 1.5680.04 1.5980.04 Red blood cell, cells/pl 4.680.2 4.680.2
Hematocrit, % 48.281.2 47.881.0 Hemoglobin, g/l 1.4780.04 1.4680.06
MCV, fl 96.581.7 94.381.2 Hematocrit, % 44.281.2 43.681.8
MCH, pg 31.380.5 31.480.5 MCV, fl 95.981.3 95.381.1
MCHC, % 32.580.6 33.280.5 MCH, pg 31.880.4 31.980.5
Platelet, cells/pl 0.2080.02 0.2180.02 MCHC, % 33.280.3 33.580.3
Biochemistry Platelet, cells/pl 0.2380.03 0.2380.02
GOT, U/l 27.580.6 29.883.4 Biochemistry
GPT, U/l 24.081.2 24.381.3 GOT, U/l 25.384.6 23.584.8
ALP, U/l 205.2822.4 204.0823.6 GPT, U/l 22.685.0 20.987.0
-GTP, U/l 33.285.0 34.085.6 ALP, U/l 195.0815.4 191.4817.3
Total protein, g/l 73.581.6 74.081.1 -GTP, U/l 29.984.7 25.883.6
Amylase, U/l 124.8817.6 121.3816.8 Total protein, g/l 73.581.2 72.881.0
Blood glucose, mmol/l 4.8480.25 4.1480.79 Amylase, U/l 130.6818.2 129.6817.3
Total cholesterol, mmol/l 4.9480.26 4.8680.28 Blood glucose, mmol/l 4.9580.26 4.7580.12
LDL cholesterol, mmol/l 3.1280.12 3.0480.16 Total cholesterol, mmol/l 5.8980.27 5.3580.25*
HDL cholesterol, mmol/l 1.5880.11 1.6080.09 LDL cholesterol, mmol/l 3.8980.05 3.3280.01
Triacylglycerol, mmol/l 1.1780.18 1.1280.13 HDL cholesterol, mmol/l 1.7680.18 1.7980.22
Urea, g/l 0.05480.005 0.06080.005 Triacylglycerol, mmol/l 1.1880.15 1.1980.11
Blood urea nitrogen, mmol/l 5.980.5 5.880.3 Urea, g/l 0.05580.005 0.05480.004
Creatinine, mmol/l 74.084.2 81.083.2 Blood urea nitrogen, mmol/l 5.980.6 5.680.7
CRP, mg/l 0.8080.24 0.7780.25 Creatinine, mmol/l 71.685.9 69.184.4
CRP, mg/l 0.5980.06 0.5080.04
Test 3: Healthy human subjects (male: 6; female: 4) ingested a Body weight, kg 59.584.9 57.684.2
10-gram bolus of BTE each. Hematological and biochemical data Body mass index 21.581.3 20.981.1
were measured 24 h after BTE intake. Values are expressed as the
mean 8 SE. Test 4: A total of 11 human subjects comprising 5 healthy
(male: 3; female: 2) and 6 BHC (male: 3; female: 3) subjects in-
gested BTE (1.67 g/meal) 3 times daily before meals for 5 weeks.
Values are expressed as the mean 8 SE. * p < 0.05.

symptoms 24 h after intake. Moreover, neither the hema-

tological nor the relevant biochemical data indicated sig-
nificant changes (table 3).
Test 4: Long-Term (5-Week) Safety Test with 5 g
BTE/Day
In the 5-week ingestion study, the results indicated
that neither hematological nor relevant biochemical data
revealed any significant changes before and after BTE in-
take. BTE significantly (p ! 0.05) reduced TCHO levels
after BTE intake (5.35 8 0.25 mmol/l) compared with
pre-intake (5.89 8 0.27 mmol/l; table 4). However, nei-
ther the TCHO levels, body weights nor BMI values (20.9
8 1.1 vs. pre-intake value of 21.5 8 1.1) of the subjects
indicated significant changes after 5-week BTE intake
compared with pre-intake values (table 4). Apart from
the absence of other side effects, subjects did not com-
plain of any self-monitored symptoms (i.e. digestive tract-
related disorders such as abdominal distension, abdo-
minal pain, diarrhea, retching or flatulence; data not
shown).
Test 5: A Noncomparative (4-Month) Study of 1 g
BTE/Day in HC Patients
All 21 participating subjects (detailed physical partic-
ulars listed in table 5) completed the study. TCHO and
LDL-cholesterol levels of all fully developed HC patients
decreased at 1 month and were significantly (p ! 0.01)

38 Ann Nutr Metab 2008;53:33–42 Fujita/Yamagami

 Table 5. Safety of BTE by blood analysis after long-term ingestion in 21 HC patients

Before 1 month 2 months 4 months

Hematology
White blood cell, cells/nl 5.580.2 5.480.2 5.580.2 5.480.2
Red blood cell, cells/pl 4.680.7 4.580.6 4.780.9 4.580.7
Hemoglobin, g/l 1.480.2 1.380.3 1.480.3 1.480.2
Hematocrit, % 42.680.6 42.080.6 42.380.6 41.580.6
MCV, fl 93.680.8 93.680.9 93.380.8 93.480.8
MCH, pg 30.880.3 30.880.2 30.880.3 30.680.4
MCHC, % 32.880.2 32.780.4 32.880.2 32.880.2
Platelet, cells/pl 0.2480.02 0.2180.03 0.2380.02 0.2380.01
Biochemistry
GOT, U/l 25.581.3 25.281.6 25.281.2 26.381.7
GPT, U/l 25.982.5 25.682.4 25.982.3 26.582.9
ALP, U/l 233.5812.2 231.3813.0 232.5813.0 232.9811.2
-GTP, U/l 39.085.1 37.085.8 36.585.1 33.083.5
Total protein, g/l 75.080.7 74.280.8 74.080.6 73.980.7
Amylase, U/l 123.488.7 123.988.7 121.488.5 113.488.4
Blood glucose, mmol/l 5.6580.18 5.4580.12 5.6580.14 5.7580.21
Total cholesterol, mmol/l 7.2580.15 7.0480.11 6.4380.11** 6.3380.10**
LDL cholesterol, mmol/l 4.8880.11 4.6980.09 4.1480.11** 4.0380.10**
HDL cholesterol, mmol/l 1.5480.06 1.5580.06 1.5980.07 1.6180.06*
Triacylglycerol, mmol/l 1.6080.13 1.5480.11 1.3480.12 1.3280.11*
Urea, g/l 0.05580.003 0.05580.002 0.05680.005 0.05480.004
Blood urea nitrogen, mmol/l 2.380.8 2.380.8 2.280.7 2.180.6
Creatinine, mmol/l 65.083.4 64.583.6 64.483.0 64.583.6
CRP, mg/l 0.0980.01 0.1080.01 0.0980.02 0.0880.02
Body weight, kg 60.881.8 60.281.8 59.881.7* 58.381.5**
Body mass index 25.280.5 24.980.5 24.880.4* 24.280.4**

Test 5: A total of 21 HC patients (male: 5; female: 16) with (10/21 cases) and without medications partici-
pated in the study. HC patients ingested BTE (0.33 g/meal) 3 times daily before meals (or 1 g/day) for 4 months.
The mean age of patients was 62.5 8 1.5 years.
Values where p < 0.01 (**) or <0.05 (*) are expressed as the mean 8 SE.

reduced 2 and 4 months after BTE intake (fig. 2). On the
contrary, HDL-cholesterol levels significantly increased
(p ! 0.05) 4 months after BTE intake (fig. 3a). The HC ef-
fects of BTE (table 5) indicated that BTE effectively at-
tenuated the TCHO levels (!0.52 mmol/l) in 15 patients
(71.4%) and LDL-cholesterol levels (!0.39 mmol/l) in 16
patients (76.2%) 4 months after BTE intake. In a similar
tendency and with the same post-intake time intervals,
triglyceride contents in 10 HC cases of type IV mixed hy-
perlipidemia (hypertriglycerdemia: 11.5 g/l) were re-
duced significantly (p ! 0.05; fig. 3b).
Although slightly higher than the cutoff BMI level of
25 (according to the Japan Society for the Study of Obe-
sity [24]), the pre-intake body weight (60.8 8 1.8 kg) and
BMI (25.2 8 0.5) were significantly improved 4 months
after BTE ingestion (body weight: 58.3 8 1.5 kg; p ! 0.01,
BMI: 24.2 8 0.4; p ! 0.01; table 5). However, BTE neither
elicited side effects on the digestive tract system nor yield-
ed any abnormal hematological or relevant biochemical
data after intake.
Discussion
Although the efficacy and safety of BTE have been
well described in animals, very little is known about it in
humans. We confirmed the safety of BTE by the Ames
and acute toxicity tests and further investigated its safety
in human subjects in this study. We found that BTE low-
ered TCHO and LDL-cholesterol levels in BHC subjects
at a dose of 333 mg/meal or 1 g/day (fig. 1). The safety of
BTE was attempted with an intake 10 times the useful

Hypocholesterolemic Effect of Black Tea Ann Nutr Metab 2008;53:33–42 39

Extract in Humans
 7.5 5.3
7.4 5.2
7.3 5.1

Total-cholesterol (mmol/l)
7.2 5.0

LDL-cholesterol (mmol/l)
7.1 4.9
Fig. 2. Effects of BTE on TCHO and LDL- 7.0 4.8
cholesterol levels in HC patients (n = 21) 6.9 4.7
who consumed 333 mg of BTE before each 6.8 4.6
meal (i.e. 3 meals daily or 1 g/day) for a 4- 6.7 4.5
month period. The left (a) and right (b)
6.6
** 4.4

panels indicate TCHO and LDL-cholester-

6.5
6.4 ** 4.3
4.2
**
ol levels, respectively. All parameters were 6.3 4.1 **
monitored at 0 (immediately before in- 6.2 4.0
6.1 3.9
take), 1, 2 and 4 months after BTE intake. 6.0 3.8
The values are expressed as the mean 8 0 1 2 3 4 0 1 2 3 4
standard error, and differences (vs. pre-in- a Ingestion period (months) b Ingestion period (months)
take values) where p ! 0.01 (**) were con-
sidered significant.

2.0 2.0
1.9 1.9
HDL-cholesterol (mmol/l)

1.8 1.8

Triacylglycerol (mmol/l)
Fig. 3. Effects of BTE on HDL-cholesterol 1.7 * 1.7
and triacylglycerol levels in HC patients 1.6 1.6
(n = 21) who consumed 333 mg of BTE be- 1.5 1.5
fore each meal (i.e. 3 meals daily or 1 g/day)
1.4 1.4
*
for a 4-month period. The left (a) and right
(b) panels indicate HDL-cholesterol and 1.3 1.3
triacylglycerol levels, respectively. All pa- 1.2 1.2
rameters were monitored at 0 (immediate- 1.1 1.1
ly before intake), 1, 2 and 4 months after 1.0 1.0
BTE ingestion. The values are expressed as 0 1 2 3 4 0 1 2 3 4
the mean 8 SE, and differences where p ! a Ingestion period (months) b Ingestion period (months)
0.01 (*) were considered significant (vs.
pre-intake values).

dose of 1 g/day by single-bolus ingestion without abnor-
malities. Furthermore, based on the FOSHU guidelines
or the safety tests (http://www-bm.mhlw.go.jp/english/
topics/foodsafety/fhc/02.html), we orally administered 5
times the useful BTE dose or 5 g/day for 5 weeks without
encountering any abnormalities in hematological and
biochemical data after BTE intake. BTE significantly re-
duced but did not completely restore TCHO and LDL-
cholesterol levels to normal values in BHC after single-
bolus intake. These results suggest that BTE intake of 1 g/
day is safe.
Although current cholesterol-lowering drugs, such as
HMG-CoA reductase inhibitors (statins) are available,
they are not without side effects; namely, liver impair-
ments [25], myopathy [26] and rhabdomyolysis [27]. Un-
like fiber and plant sterols, which nondiscriminatively
inhibit absorption of nutrients and fat-soluble vitamins,
BTE selectively inhibits reabsorption of cholesterol with-
out affecting fat-soluble vitamins [22]. Therefore, BTE is
unlikely to deprive BTE consumers of useful nutrients
and fat-soluble vitamins. Our present study has con-
firmed that no abnormal behavioral or organic findings
(table 2) were noted in mice after long-term BTE con-
sumption, and the safety aspect of BTE is further advo-
cated by the fact that Pu-Ehr tea is a traditional Chinese
beverage that has long been enjoyed in Asia and in other
parts of the world.
In order to confirm the anti-HC properties of BTE, we
examined its effects in fully developed HC patients. BTE
significantly reduced TCHO and LDL-cholesterol levels
2 and 4 months after intake of 333 mg/meal (1 g/day).
While these favorable effects appeared as early as 2 weeks

40 Ann Nutr Metab 2008;53:33–42 Fujita/Yamagami

after intake in some patients (data not shown), significant
(p ! 0.01) reductions of TCHO and LDL-cholesterol lev-
els in HC patients (including those with hyperlipidemia)
were observed 2 and 4 months after ingestion (table 3).
These effects may be due to the precipitating effect on
mixed bile salt micelles and inhibitory effect on choles-
terol absorption of BTE [21, 22]. Based on a previous in
vitro study, we have found that BTE displays a precipitat-
ing effect on mixed bile salt micelles [22], suggesting that
lipid absorption from foods is attenuated with BTE in-
take.
Interestingly, the mean BMI of HC patients surpassed
the borderline obesity value (category I; defined accord-
ing to guidelines of the Japan Society for the Study of
Obesity). Apart from the above-mentioned effects, BTE
also significantly (p ! 0.01) decreased body weight and
BMI of subjects after 4 months of intake (table 5). A re-
cent study has reported that tea catechins exhibit anticor-
pulent effects, and epigallocatechin-3-gallate (EGCG)
appears to selectively activate -oxidation in the liver
[28]. However, as EGCG decomposes in BTE during fer-
mentation, its prevailing level is therefore only present in
negligible amounts [17].
Pu-Ehr tea is traditionally derived from fermented tea
leaves and has long been enjoyed by many people world-
wide. An effective daily intake of BTE (333 mg/meal or
1 g/day) is contained in 2 liters of black tea; namely, a safe
amount which slightly exceeds the usual quantity con-
sumed on a daily basis.
In conclusion, many studies have described the anti-
HC effects of teas, most of which inhibit the reabsorption
of bile acid in the ilium to eventually reduce blood cho-
lesterol levels. This study is the first to demonstrate that
BTE can reduce TCHO and LDL-cholesterol levels in
BHC and fully developed HC patients. Furthermore, BTE
significantly reduces triacylglycerol levels, body weight
and BMI without any adverse effects or toxicity after
long-term (4-month) intake. This study suggests that
BTE reduces LDL-cholesterol, TCHO and triacylglycerol
levels to eventually attenuate arteriosclerosis and/or obe-
sity attributable to metabolic syndrome in humans.
Acknowledgement
We are grateful to Dr. K. Ohshima (Director of Ohshima Clin-
ic) for performing the clinical tests.

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