Fermented Pu-erh Tea Increases In Vitro Anticancer Activities in HT-29 Cells and Has Antiangiogenetic Effects on HUVECs

Abstract

Pu-erh tea is produced in China and known to possess medicinal properties. The anticancer and antiangiogenesis effects of fermented Pu-erh tea on HT-29 colon cancer cells and human umbilical vein endothelial cells, respectively, were examined. Two kinds of unfermented and fermented Pu-erh tea (Seven-son tea cake Pu-erh tea and Xiaguan bowl tea [X]) and green tea were used. An MTT assay showed fermented Pu-erh tea X (85% inhibition) possessed more potent anticancer activities than unfermented Pu-erh tea X (67% inhibition) and green tea (53% inhibition) (P < 0.05). Moreover, fermented Pu-erh tea X increased the number of apoptotic bodies determined through DAPI staining and flow cytometric analysis. Fermented Pu-erh tea X induced apoptosis indicated by increased expression of Bax, caspase-9, and caspase-3 messenger RNA and decreased expression of Bcl-2. Fermented Pu-erh tea X also had an anti-inflammation effect, shown in decreased expression of nuclear factor-κB-p65, inducible nitric oxide synthase, COX-2 messenger RNA and increased expression of IκB-α. Further, fermented Pu-erh teas showed stronger antiangiogenesis effects than the 2 other types of tea. After fermentation, the concentrations of gallic acid, resorcylic acid, quercetin, and kaempferol in Pu-erh tea were increased. These results collectively indicated that fermented and unfermented Pu-erh teas possess stronger anticancer and antiangiogenesis effects than green tea. Furthermore, fermented Pu-erh tea showed stronger functional activities than unfermented Pu-erh tea.

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Journal of Environmental Pathology, Toxicology and Oncology, 32(4):275-288 (2013)
275
0731-9989/13/$35.00 © 2013 Begell House, Inc. www.begellhouse.com
Fermented Pu-erh Tea Increases In Vitro Anticancer
Activities in HT-29 Cells and Has Antiangiogenetic
Effects on HUVECs
Xin Zhao,1,2 Jia-Le Song,1 Jong-Deog Kim,3 Jong-Suk Lee,4 Kun Young Park1
*1Department of Food Science and Nutrition, Pusan National University, Busan, Korea; 2Department of Biological and
Chemical Engineering, Chongqing University of Education, Chongqing, PR China; 3Department of Biotechnology, Re-
search Center on Anti-Obesity and Health Care, Chonnam National University, Yosu, Chonnam, Korea; 4Gyeonggi
Biocenter, Gyeonggi Institute of Science and Technology Promotion (GSTEP), Suwon, Gyeonggi-do, Korea
* Address all correspondence to: Prof. Kun Young Park, PhD, Department of Food Science and Nutrition, Pusan National University, 30 Jang
Jun-dong, Keum Jung-ku, Busan 609-735, Korea; Tel.: Tel: (82) 51-510-2839; Fax: (82) 51-514-3138; kunypark@pusan.ac.kr.
ABSTRACT: Pu-erh tea is produced in China and known to possess medicinal properties. The anticancer and an-
tiangiogenesis effects of fermented Pu-erh tea on HT-29 colon cancer cells and human umbilical vein endothelial
cells, respectively, were examined. Two kinds of unfermented and fermented Pu-erh tea (Seven-son tea cake Pu-erh
tea and Xiaguan bowl tea [X]) and green tea were used. An MTT assay showed fermented Pu-erh tea X (85% in-
hibition) possessed more potent anticancer activities than unfermented Pu-erh tea X (67% inhibition) and green tea
(53% inhibition) (P < 0.05). Moreover, fermented Pu-erh tea X increased the number of apoptotic bodies determined
through DAPI staining and ow cytometric analysis. Fermented Pu-erh tea X induced apoptosis indicated by in-
creased expression of Bax, caspase-9, and caspase-3 messenger RNA and decreased expression of Bcl-2. Fermented
Pu-erh tea X also had an anti-inammation effect, shown in decreased expression of nuclear factor-κB-p65, induc-
ible nitric oxide synthase, COX-2 messenger RNA and increased expression of IκB-α. Further, fermented Pu-erh
teas showed stronger antiangiogenesis effects than the 2 other types of tea. After fermentation, the concentrations
of gallic acid, resorcylic acid, quercetin, and kaempferol in Pu-erh tea were increased. These results collectively
indicated that fermented and unfermented Pu-erh teas possess stronger anticancer and antiangiogenesis effects than
green tea. Furthermore, fermented Pu-erh tea showed stronger functional activities than unfermented Pu-erh tea.
KEY WORDS: Camellia sinensis, fermented Pu-erh tea, green tea, anticancer, antiangiogenesis
I. INTRODUCTION
Tea is a beverage consumed worldwide. Green tea
(Camellia sinensis) has been used in traditional Chi-
nese medicine and has gained considerable attention
because of its antioxidant, anti-inammatory, anti-
hypertensive, antidiabetic, and antimutagenic prop-
erties.1 Chinese Pu-erh tea, named after Pu-erh City
in the Yunnan province of China, is one of the most
famous Chinese teas.2 This tea is made from the
large leaves of C. sinensis O. kuntze var. assamica
Kitamura. Pu-erh tea leaves are approximately 10–
26 cm in length, the longest of its kind. These leaves
are processed using a special fermentation proce-
dure based on the chemical components of raw or
nonfermented Pu-erh tea. With the action of extra-
cellular enzymes that occurs because of the damp-
ness and heat in the tea heap, the tea polyphenols
undergo a series of complex and dramatic chemical
changes that result in the special avor and taste of
Pu-erh tea.3–5 Many types of food undergo fermenta-
tion processes that increase their medicinal effects.
For example, fermented soybeans (doenjang) and
fermented cabbage (kimchi) have been shown to
exert stronger anticancer effects than raw or unfer-
mented products.6,7 Microorganisms predominantly
found in Yunnan Pu-erh tea undergoing fermenta-
tion are Aspergillus niger, Aspergillus gloucu, and
species of Penicillium, Rhizopus, Saccharomyces,
and Bacterium. A. niger is most predominant, fol-
lowed by Saccharomyces spp. and then by only a
few Bacillus spp.8,9 Because the quality of Pu-erh

Journal of Environmental Pathology, Toxicology and Oncology
Zhao et al.
276
tea is closely related to the postfermentation pro-
cess, the polyphenol and the γ-aminobutyric acid
content of the tea leaves are assessed, along with
the free radical scavenging activity, at regular in-
tervals during the fermentation. A particular micro-
organism (Aspergillus) has been identied as the
most benecial for enhancing the quality of the tea
during the fermentation process.10
Drinking Pu-erh tea for a long period of time
can help maintain both mental and physical health.
This tea is believed to help reduce high blood pres-
sure and cholesterol levels and to play an impor-
tant role in preventing heart disease and cancer.11
Gallic acid is an important component of Pu-erh
tea and may have antifungal and antiviral proper-
ties. Gallic acid acts as an antioxidant and helps to
protect cells from oxidative damage.12 This com-
pound was found to have cytotoxic activity against
cancer cells without harming healthy cells and is
used as an astringent to treat internal hemorrhage.13
Some ointments used to treat psoriasis and external
hemorrhoids contain gallic acid.14
Pu-erh tea can also protect against colon can-
cer.15 Various phytochemicals, especially glyco-
side polyphenols, can be converted into aglycone
and other fermented products by benecial micro-
organisms. Thus, fermented products can help im-
prove health, including lowering the risk of cancer.
The known bioactive components of Pu-erh tea are
epicatechin and epigallocatechin.16 In this study,
we investigated the anticancer effects of ferment-
ed Pu-erh tea on human colon cancer cells and its
antiangiogenic effects on human umbilical vein
endothelial cells (HUVECs). These effects were
compared with those of unfermented Pu-erh tea
and green tea. We also tried to identify active com-
pounds in fermented Pu-erh tea by liquid chroma-
tography–mass spectrometry (LC-MS) analysis.
II. MATERIALS AND METHODS
A. Sources of Pu-erh Teas
Seven-son tea cake Pu-erh tea (Pu-erh tea S) (Yu-
nan Province Menghai County Yunhai Tea Fac-
tory, Yunan, China) and Xiaguan bowl tea (Pu-erh
tea X) (Yunnan Xiaguan Tuocha Co., Ltd., Yunan,
China) were purchased in Yunnan, China. Two
types of unfermented (U) Pu-erh tea (U-Pu-erh tea
S and U-Pu-erh tea X) and 2 types of fermented (F)
Pu-erh tea (naturally fermented for 1 year; F-Pu-
erh tea S and F-Pu-erh tea X) also were purchased
in Yunnan, China. Green tea (Longjing tea; Hang-
zhou Longjing Tea Industry Co., Ltd., Zhejiang,
China) was used as a control sample.
B. Preparation of Tea Extract
The Pu-erh tea and green tea were stored at -80°C
and freeze-dried to produce a powder. A 20-fold
volume of methanol was added to the powdered
samples and extracted twice by stirring overnight.
The methanol extracts were evaporated using a
rotary evaporator (N-1100; Eywla, Tokyo, Japan),
concentrated, and then dissolved in dimethylsulf-
oxide (DMSO) (Amresco, Solon, OH) to adjust to
the stock concentration (20% w/v).
C. Cancer Cell Preparation
HT-29 human colon adenocarcinoma cells were
obtained from the Korean Cell Line Bank (Seoul,
South Korea). The cancer cells were cultured in
RPMI-1640 medium (Welgene Inc., Daegu, South
Korea) and supplemented with 10% fetal bovine
serum and 1% penicillin-streptomycin (Gibco Co.,
Birmingham, MI) at 37°C in a humidied atmo-
sphere containing 5% carbon dioxide (CO2) (mod-
el 311; Forma, Waltham, MA). The medium was
changed 2 or 3 times each week.
D. MTT Assay
The anticancer effects of Pu-erh tea was assessed
by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltet-
razolium bromide (MTT) assay. The HT-29 human
colon carcinoma cells were seeded into a 96-well
plate, so that each well contained 180 µL of a cell
suspension composed of 2 × 104 cells per well. Ac-
cording to the proportion of cells in the suspen-
sion, 20 µL of specimen was added to a vessel and
cultured for 48 hours at 37°C in a humidied at-

Volume 32, Number 4, 2013
Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 277
mosphere containing 5% CO2. We added 200 µL
of MTT solution (5 mg/mL), and the cells were
cultured for another 4 hours under the same condi-
tions. After removing the supernatant, 150 µL of
DMSO was added to each well and mixed for 30
minutes. Finally, the absorbance of each well was
measured by an enzyme-linked immunosorbant as-
say reader (model 680; Bio-Rad, Hercules, CA) at
540 nm.17
E. Nuclear Staining with 4,6-Diamidino-
2-Phenylindole (DAPI)
Untreated control cells and cells treated with tea
extracts (200 µg/mL) were harvested, washed with
phosphate-buffered saline (PBS), and xed with
3.7% paraformaldehyde (Sigma, St. Louis, MO)
in PBS for 10 minutes at room temperature. Fixed
cells were washed with PBS and stained with a
DAPI solution (1 mg/mL) (Sigma) for 10 minutes
at room temperature.18 The cells were washed 2
more times with PBS and examined with a uores-
cence microscope (model BX50; Olympus, Tokyo,
Japan).
F. Flow Cytometry Analysis
After treatment with tea samples, the cells were
collected by trypsinization; they were washed with
cold PBS and resuspended in PBS. The DNA con-
tents of the cells were measured using a DNA stain-
ing kit (CycleTESTTM PLUS kit; Becton Dick-
inson, Heidelberg, Germany). Nuclear fractions
stained with propidium iodide were obtained by
following the kit protocol. Fluorescence intensity
was determined using a FACScan ow cytometer
(EPICS XL-MCL; Beckman Coulter KK, Tokyo,
Japan) and was analyzed using CellQuest software
(Becton Dickinson).19
G. Reverse Transcriptase
Polymerase Chain Reaction of Various
Messenger RNAs
Total RNA was isolated from HT-29 cells using
Trizol reagent (Invitrogen, Carlsbad, CA) accord-
ing to the manufacturer’s recommendations. The
RNA was digested with RNase-free DNase (Roche,
Basel, Switzerland) for 15 minutes at 37°C and pu-
ried using an RNeasy kit (Qiagen, Hilden, Ger-
many) according to the manufacturer’s protocol.
Complementary DNA was synthesized from 2 µg
of total RNA by incubation at 37°C for l hour with
avian myeloblastosis virus reverse transcriptase
(GE Healthcare, Little Chalfont, UK) with random
hexanucleotides according to the manufacturer’s
instructions. Primers used to specically amplify
the genes of interest are shown in Table 1. Ampli-
cation was performed in a thermal cycler (Ep-
pendorf, Hamburg, Germany) with cycles of de-
naturation. The polymerase chain reaction (PCR)
products were separated in 1.0% agarose gels and
visualized with ethidium bromide staining.20
H. In Vitro Antiangiogenesis Test
The HUVECs used in this study were obtained
from Young Science Inc. (Seoul, South Korea),
with cells from passages 3 to 5. HUVECs were
cultured in EBM-2 growth medium (Cambrex,
Hopkinton, MA), containing hydrocortisone, epi-
dermal growth factor, basic broblast growth fac-
tor, insulin-like growth factor-1, vascular endothe-
lial growth factor, ascorbic acid, heparin, and 2%
fetal bovine serum at 37°C in a humidied atmo-
sphere containing 5% CO2. The cells were seeded
in culture asks coated with 2% gelatin (Sigma)
and allowed to grow to conuence before the ex-
perimental treatment. The formation of tubular
structures by HUVECs in Matrigel was used to as-
sess the antiangiogenesis effect of the tea samples.
Culture plates (24-well) were coated with 150 µL
of Matrigel, which was allowed to solidify at 37°C
for 1 hour. HUVECs were seeded on Matrigel-
coated wells (25,000 cells/well) and treated with
tea samples at a concentration of 50 µg/mL for 24
hours at 37°C in an atmosphere humidied with
5% CO2. Digital images were captured in random-
ly selected areas of the wells (5 each) using a digi-
tal camera (CoolPix 4500; Nikon, Tokyo, Japan).
The formation of tube networks was quantied
by summing up the arbitrary length of tubes from

Journal of Environmental Pathology, Toxicology and Oncology
Zhao et al.
278
each image using the National Institutes of Heath
image program.21
I. LC-MS Analysis
The tea extracts were dissolved in DMSO to pro-
duce a nal concentration of 10 mg/mL and then
diluted with 50% methanol to make a nal con-
centration of 2 mg/mL. The diluted sample (5 µL)
was analyzed by liquid chromatography followed
by tandem mass spectrometry (LC-MS/MS). LC-
MS/MS was performed using a Finnigan LCQ Ad-
vantage MAX ion trap mass spectrometer (Ther-
mo Electron Co., Waltham, MA) equipped with
an electrospray ionization source. Separation by
high-performance liquid chromatography was per-
formed with a Finnigan Surveyor Modular HPLC
System (Thermo Electron Co.) using an Xterra MS
C18 column (5 µm, 2.1 × 150 mm; Waters, Mil-
ford, MA). Mobile phase A was water, and mobile
phase B was acetonitrile; both contained 0.1% for-
mic acid. Gradient elution at a ow rate of 0.2 mL/
min was carried out as follows: 0–5 minutes with
0–40% B (linear gradient), 5–20 minutes with 40–
80% B (linear gradient), 20–25 minutes with 80–
100% B (linear gradient), and 25–30 minutes with
100% B (isocratic). Full-scan mass spectra were
obtained in the positive and negative ion modes at
a range of m/z values of 100–1000. For identify-
ing compound structures, data obtained from the
MS/MS analysis were compared with those from
an MS/MS spectral library search.22
J. Statistical Analysis
An analysis of variance was performed using data
from the experiments for each specimen. The re-
sults are presented as the mean ± standard devia-
tion (SD). Differences between the mean of the in-
dividual groups were assessed by one-way analysis
TABLE 1: Primers of Reverse Transcriptase Polymerase Chain Reaction
Gene name Sequence
Bax Forward: 5′-AAG CTG AGC GAG TGT CTC CGG CG-3′
Reverse: 5′-CAG ATG CCG GTT CAG GTA CTC AGT C-3′
Bcl-2 Forward: 5′-CTC GTC GCT ACC GTC GTG ACT TGG-3′
Reverse: 5′-CAG ATG CCG GTT CAG GTA CTC AGT C-3′
Caspase-9 Forward: 5′-GGC CCT TCC TCG CTT CAT CTC-3′
Reverse: 5′-GGT CCT TGG GCC TTC CTG GTA T-3′
Caspase-3 Forward: 5′-CAA ACT TTT TCA GAG GGG ATC G-3′
Reverse: 5′-GCA TAC TGT TTC AGC ATG GCA-3′
NF-κB-p65 Forward: 5′-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3′
Reverse: 5′-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3′
IκB-α Forward: 5′-GCT GAA GAA GGA GCG GCT ACT-3′
Reverse: 5′-TCG TAC TCC TCG TCT TTC ATG GA-3′
Inducible nitric
oxide synthase
Forward: 5′-AGA GAG ATC GGG TTC ACA-3′
Reverse: 5′-CAC AGA ACT GAG GGT ACA-3′
COX-2 Forward: 5′-TTA AAA TGA GAT TGT CCG AA-3′
Reverse: 5′-AGA TCA CCT CTG CCT GAG TA-3′
GAPDH Forward: 5′-CGG AGT CAA CGG ATT TGG TC-3′
Reverse: 5′-AGC CTT CTC CAT GGT CGT GA-3′

Volume 32, Number 4, 2013
Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 279
of variance with Duncan’s multiple range tests.
Differences were considered signicant when P
< 0.05. SAS version 9.1 statistical software (SAS
Institute Inc., Cary, NC) was used to perform these
analyses.
III. RESULTS
A. In Vitro Anticancer Effect of Fermented
Pu-erh Tea in HT-29 Cells
An MTT assay was used to analyze the dose-de-
pendent effects on HT-29 cells. As the tea concen-
trations increased, HT-29 cell growth was increas-
ingly inhibited. At concentrations of 0–400 µg/mL,
the growth inhibitory effects of the F-Pu-erh tea
X sample increased. According to our preliminary
results, the growth inhibitory rate of Pu-erh tea was
100% for concentrations greater than 400 µg/mL.
Therefore, concentrations of 100 and 200 µg/mL
were chosen to evaluate the in vitro anticancer ef-
fect of Pu-erh tea.
The anticancer effects of Pu-erh tea and green
tea extracts on HT-29 cells were examined. The
growth inhibitory rates of HT-29 cells treated with
100 µg/mL of green tea, U-Pu-erh tea S, F-Pu-erh
tea S, U-Pu-erh tea X, and F-Pu-erh tea X were
12%, 13%, 37%, 25%, and 52%, respectively. At
200 µg/mL, the growth inhibitory rates of the cells
treated with these samples were 53%, 55%, 70%,
67%, and 85%, respectively (P < 0.05). The anti-
cancer effect of F-Pu-erh tea on the HT-29 cells
was clearly stronger than that of U-Pu-erh tea;
the effects of both types of F-Pu-erh tea also were
greater than that of green tea (Table 2).
B. Induction of Apoptosis by F-Pu-erh Tea
To determine whether the growth inhibitory ac-
tivity of Pu-erh tea in HT-29 human colon carci-
noma cells was related to the induction of apop-
tosis, chromatin condensation rst was analyzed
by uorescent microscopy using the DNA-binding
uorescent dye DAPI and ow cytometric analy-
sis. HT-29 cells normally contain nuclei with a ho-
mogeneous chromatin distribution. Treatment with
Pu-erh tea extracts (200 µg/mL) induced chromatin
condensation and nuclear fragmentation, suggest-
ing the presence of apoptotic cells (Fig. 1A). Chro-
matin condensation and the formation of apoptotic
bodies, characteristics of apoptosis, were observed
in the cells cultured with F-Pu-erh tea X and to a
lesser extent in those cultured with U-Pu-erh tea
X. These results suggest that F-Pu-erh tea X was
more effective than U-Pu-erh tea X in inducing the
condensation and formation of apoptotic bodies.
Green tea also was shown to induce the forma-
tion of apoptotic bodies, but the extent of forma-
tion was weaker than that of U-Pu-erh tea X. The
sub-G1 DNA content of the cancer cells was evalu-
ated by ow cytometric analysis, which revealed
enhanced apoptosis (increased apoptotic cells) of
HT-29 cancer cells when treated with F-Pu-erh tea
X compared with those treated with U-Pu-erh tea
X and green tea (Fig. 1B and C).
C. Expressions of the Apoptosis-Associat-
ed Bax, Bcl-2, and Caspase Genes
To determine which type of apoptotic pathways
was induced by treatment with tea, the expression
of the Bax, Bcl-2, and caspase genes in HT-29 cells
were examined by reverse transcriptase PCR (RT-
PCR). As shown in Fig. 2A, treatment with 200
µg/mL of F-Pu-erh tea signicantly changed the
expression of proapoptotic Bax and antiapoptotic
Bcl-2. These results suggest that the tea induced
apoptosis in HT-29 cells via Bax-dependent and
Bcl-2-dependent pathways. Moreover, the apop-
tosis induced by F-Pu-erh tea X was associated
with increased Bax and decreased Bcl-2 messenger
RNA (mRNA) expression when compared to the
other types of tea.
The levels of expression of caspase-9 and -3 in
mRNA were low in untreated control HT-29 cells
but were detected after cells were treated with 200
µg/mL of F-Pu-erh tea X. With the F-Pu-erh tea
treatment, the expression of caspase-9 and -3 in
mRNA were signicantly upregulated (Fig. 2B).
In particular, a 2- to 3-fold difference in the
expression of these genes was observed when cells
treated with F-Pu-erh tea X were compared with

Journal of Environmental Pathology, Toxicology and Oncology
Zhao et al.
280
TABLE 2: Inhibition of the Growth of HT-29 Human Colon Carcinoma Cells by
Methanol Extracts of Tea Samples, as Evaluated by an MTT Assay
Treatment
OD540 (µg/mL)
100 200
Control (untreated) 0.929 ± 0.004a,A 0.929 ± 0.004a,A
Green tea 0.820 ± 0.014 (12)b0.434 ± 0.012 (53)B
U-Pu-erh tea S 0.809 ± 0.005 (13)b0.423 ± 0.009 (55)B
F-Pu-erh tea S 0.589 ± 0.009 (37)c0.275 ± 0.008 (70)C
U-Pu-erh tea X 0.696 ± 0.018 (25)d0.309 ± 0.008 (67)D
F-Pu-erh tea X 0.443 ± 0.015 (52)e0.139 ± 0.007 (85)E
The values in parentheses are inhibition rates (%). Mean values with different letters (a–e,
A–E) in the same column are signicantly different (P < 0.05) according to Duncan’s multiple
range test.
F, fermented; MTT, 3-(4,5-dimetyl-thiazol)-2,5-diphenyl tetrazolium; OD540, optical density at 540
nm (concentration of sample); S, Seven-son tea cake Pu-erh tea; U, unfermented; X, Xiaguan
bowl tea.
those treated with green tea. The results suggested
that F-Pu-erh tea X had the strongest anticancer ef-
fect, that U-Pu-erh tea X exerted a stronger effect
than the green tea control, and that green tea had a
slight anticancer effect on HT-29 cells.
D. Expressions of Inammation-Related
NF-κB-p65, IκB-α, iNOS, and COX-2
Genes
The inammation induced by F-Pu-erh tea X was
associated with decreased NF-κB-p65 and in-
creased IκB-α expressions in mRNA when com-
pared to the other types of teas (Fig. 3A).
Inhibition of iNOS and COX-2 activity pre-
disposes a number of human cell lines to inam-
mation stimuli. Therefore, the anticancer actions
of F-Pu-erh tea associated with the inhibition of
the activation of iNOS and COX-2 gene expression
were examined. As shown in Fig. 3B, the expres-
sion of COX-2 and iNOS in mRNA was detected in
untreated control HT-29 cells but was not detected
after cells were treated with 200 µg/mL of F-Pu-
erh teas. With the tea treatment, the expression
of both COX-2 and iNOS in mRNA was reduced
gradually. These ndings indicated that F-Pu-erh
tea X may contribute to the prevention of cancer
by reducing susceptibility to inammation. The re-
sults also suggested that F-Pu-erh tea X has stron-
ger anti-inammatory properties than U-Pu-erh tea
X and green tea.
E. Antiangiogenesis Effect of Pu-erh Tea
Tube formation by endothelial cells in Matrigel
was analyzed quantitatively by examining 5 pho-
tomicrographs obtained from random elds in cell
cultures in each well. Incubation of HUVECs with
Pu-erh tea inhibited the formation of microtubu-
lar structures. Fig. 4 shows the antiangiogenetic
effect of the tea at a concentration of 50 µg/mL.
Incubation with the 2 kinds of U-Pu-erh tea (U-
Pu-erh tea S and U-Pu-erh tea X) and green tea
inhibited the formation of microtubular structures
to similar degrees (53.0%, 63.5%, and 67.2%, re-
spectively). The 2 types of F-Pu-erh tea (F-Pu-erh
tea S and F-Pu-erh tea X) signicantly inhibited
tube formation (86.3% and 89.3%, respectively;
P < 0.05).
In this study, Pu-erh tea was found to have
stronger antiangiogenetic effects than green tea;
thus, Pu-erh tea is able to inhibit the growth of

Volume 32, Number 4, 2013
Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 281
FIG 1: Induction of apoptosis in HT-29 human colon carcinoma cells by treatment with green tea and Pu-erh tea.
A: Cells were incubated with tea extracts (200 μg/mL) for 48 hours and then stained with DAPI. After 10 minutes of
incubation at room temperature, the cells were washed with phosphate-buffered saline and photographed with a
uorescence microscope using a blue lter (400× original magnication). The arrows in Fig. 1 mean the apoptotic
cells. B: To quantify the degree of apoptosis induced by tea samples (200 μg/mL), the cells were evaluated for the
sub-G1 DNA (apoptotic cells) content using a ow cytometer. C: Amount of the apoptotic cells in each group. The let-
ters (a–c) mean the signicant differences (P < 0.05) of apoptotic cells in each group according to Duncan’s multiple
range test. GT, green tea; FPX, fermented Pu-erh tea X; UPX, unfermented Pu-erh tea X.
solid tumors. This nding implies that drinking
Pu-erh tea, especially F-Pu-erh tea, may be quite
benecial for preventing cancer that can be consid-
ered an angiogenesis-dependent disease.
F. Analysis of Pu-erh Tea Composition
An initial LC-MS/MS analysis of the methanol
extracts of U-Pu-erh tea X and F-Pu-erh tea X
showed that there were 12 important peaks for
U-Pu-erh tea X. These peaks (Fig. 5) were for
gallic acid (1.70 minutes, m/z 169), epigallocat-
echin (9.10 minutes, m/z 305), catechin (9.30
minutes, m/z 289), caffeine (9.62 minutes, m/z
195), epicatechin (10.73 minutes, m/z 289), epi-
gallocatechin gallate (10.87 minutes, m/z 457),
epicatechin gallate (12.51 minutes, m/z 441),
quercetin-3-galactoside (12.74 minutes, m/z
463), kaempferol-3-rutinoside (13.24 minutes,
m/z 593), kaempferol-3-glucoside (13.51 min-

Journal of Environmental Pathology, Toxicology and Oncology
Zhao et al.
282
FIG. 2: Effects of tea (200 μg/mL) on the expression of Bax and Bcl-2 (A), and caspase-9 and caspase-3 (B) in HT-
29 human colon carcinoma cells. Cells were incubated with tea samples for 48 hours. Reverse transcriptase poly-
merase chain reaction (PCR) was performed on isolated total RNA to measure Bax and Bcl-2 expression. The PCR
products were separated on a 1% agarose gel and visualized by ethidium bromide staining. GAPDH was used as
a housekeeping control gene. Band intensity was measured with a densitometer and expressed as the fold change
over the control. Fold ratio was determined using the following formula: gene expression/GAPDH × control numeri-
cal value (control fold ratio of 1) Mean values with different letters (a–d) are signicantly different (P < 0.05) accord-
ing to Duncan’s multiple range test. GT, green tea; FPX, fermented Pu-erh tea X; UPX, unfermented Pu-erh tea X.
utes, m/z 447), quercetin (15.39 minutes, m/z
301), and lutein (25.09 minutes, m/z 568). The
spectra for F-Pu-erh tea X also contained the 12
peaks observed for the U-Pu-erh tea X extract.
In addition, 2 peaks occurred at 3.43 and 17.37
minutes on the LC chromatogram (Fig. 6). The
2 new compounds had molecular weights of 154
(m/z 153, [M-H]-) and 286 (m/z 285, [M-H]-) and
were identied as resorcylic acid (3.43 minutes)
and kaempferol (17.37 minutes), respectively
(Fig. 5). This analysis demonstrated that gallic
acid, resorcylic acid, quercetin, and kaempferol
concentrations were increased in F-Pu-erh tea X
but that epigallocatechin gallate levels were de-
creased. Furthermore, glycosides such as quer-
cetin-3-galactoside, kaempferol-3-rutinoside,
and kaempferol-3-glucoside were converted into
aglycones during fermentation (Fig. 5B). In this

Volume 32, Number 4, 2013
Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 283
FIG. 3: Effects of tea (200 µg/mL) on the expression of nuclear factor (NF)-κB-p65, IκB-α, inducible nitric oxide
synthase (iNOS), and COX-2 in HT-29 human colon carcinoma cells. Cells were incubated with tea samples for 48
hours. Reverse transcriptase polymerase chain reaction (PCR) was performed on isolated total RNA to measure
Bax and Bcl-2 expression. The PCR products were separated on a 1% agarose gel and visualized by ethidium bro-
mide staining. GAPDH was used as a housekeeping control gene. Band intensity was measured with a densitometer
and expressed as the fold change over the control. Fold ratio was determined using the following formula: gene
expression/GAPDH × control numerical value (control fold ratio of 1). Mean values with different letters (a–d) are
signicantly different (P < 0.05) according to Duncan’s multiple range test. GT, green tea; FPX, fermented Pu-erh
tea X; UPX, unfermented Pu-erh tea X.
study we performed an LC-MS analysis to con-
rm that the fermentation process increased gallic
acid, resorcylic acid, quercetin, and kaempferol
concentrations in Pu-erh tea.
IV. DISCUSSION
Apoptosis is a fundamental cell event, and un-
derstanding its mechanisms of action will have a
signicant effect on antitumor therapy. The Bcl-2
family, which includes promoters (Bax and Bid)
and inhibitors (Bcl-2 and Bcl-xL), is a key regu-
lator in mitochondria-mediated apoptosis.23 Bcl-2
is especially important to preserve the integrity of
the outer mitochondrial membrane, thereby pre-
venting the release of proapoptotic factors from
mitochondria and inhibiting apoptosis.24,25 In this
study, apoptosis induced by F-Pu-erh tea is relat-

Journal of Environmental Pathology, Toxicology and Oncology
Zhao et al.
284
FIG. 4: Antiangiogenesis effects of the different tea extracts (50 µg/mL). Mean values with different letters (a–f) are
signicantly different (P < 0.05) according to Duncan’s multiple range test. GT, green tea; FPS, fermented Pu-erh tea
S; FPX, fermented Pu-erh tea X; UPS: unfermented Pu-erh tea S; UPX, unfermented Pu-erh tea X.

Volume 32, Number 4, 2013
Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 285
FIG. 5: High-performance liquid chromatography chromatogram using a photodiode array detector (200–600 nm) for
unfermented Pu-erh tea X (A) and fermented Pu-erh tea X (B) extracts.
FIG. 6: Identication and structures of the new compounds discovered in fermented Pu-erh tea X extract. Mass
spectra (upper ones of each peak), mass spectrometry (MS)/MS spectra (lower ones of each peak), and molecular
structures (bottom) of resorcylic acid (A) and kaempferol (B).

Journal of Environmental Pathology, Toxicology and Oncology
Zhao et al.
286
ed to the downregulation of Bcl-2 and increased
levels of Bax, thus promoting apoptosis in HT-29
cells. The caspase family of aspartate-specic
cystine protease also plays a critical role in regu-
lating apoptosis.24 Capase-3 is an especially criti-
cal executioner of apoptosis. Activated caspase-3
can cleave poly (ADP-ribose) polymerase, a cell
survival protein that is often deactivated by cleav-
age during apoptosis.25 F-Pu-erh tea increases the
mRNA expression of caspase-3 and -9 compared
with normal HT-29 cells. These data suggest that
apoptosis induced by F-Pu-erh tea is caused by
caspase-3-dependent cell death.
Nuclear factor (NF)-κB-p65 is a ubiquitous
transcription factor that presents in cytosol and
normally binds to its natural inhibitory protein, in-
hibitor of κB (IκB). Following its induction by a
variety of agents, NF-κB is released from IκB and
translocated to the nucleus and regulates the gene
expression required for cell proliferation, inam-
mation, and cell adhesion.26–28 In this study, F-Pu-
erh tea signicantly downregulated the expression
of NF-κB in mRNA and upregulated the expres-
sion of IκB in mRNA, showing anti-inammatory
activity in HT-29 human colon cancer cells (Fig.
3A). COX-2 and inducible nitric oxide synthase
are important enzymes that mediate inammatory
processes. Improper upregulation of COX-2 and/
or iNOS has been associated with the pathophysi-
ology of certain types of human cancers as well
as inammatory disorders. Since inammation is
closely linked to tumor promotion, inhibiting the
activities of COX-2 and iNOS may be a promising
approach for colon cancer chemoprevention.29,30
Treatment with tea samples, especially F-Pu-erh
tea, signicantly decreased the expression of COX-
2 and iNOS in mRNA (Fig. 3B). Thus F-Pu-erh tea
may contribute to the prevention of cancer by in-
creasing anti-inammatory effects as well.
A correlation has been noted between the level
of angiogenesis and the process of metastasis in
many varieties of malignant tumor31,32 Pathologi-
cal angiogenesis is a hallmark of cancer, as well as
various ischemic and inammatory diseases.33 An-
tiangiogenic therapy is one of the most promising
approaches for arresting tumor growth and can-
cer metastasis in cancer treatment.34 F-Pu-erh tea
signicantly inhibited angiogenesis in HUVECs
compared with U-Pu-erh tea and green tea. Al-
though the anticancer effect of 2 different F-Pu-erh
tea products were slightly different, F-Pu-erh tea
showed the highest anticancer activity, followed
by U-Pu-erh tea and green tea (Fig. 4).
Tea has been reported to exert anticancer ef-
fects, which are associated with its many func-
tional compounds, such as polypenols, catechines,
amino acids, and vitamins.35–38 The total catechin
content in F-Pu-erh tea was less than that of U-Pu-
erh tea, but the levels of gallic acid, resorcylic acid,
quercetin, and kaempferol were higher than those
in U-Pu-erh tea (Fig. 5). The amount of gallic acid
increased during fermentation because of its libera-
tion from catechin gallates. Gallic acid is cytotoxic
against cancer cells and induces apoptosis without
affecting normal cells.39 Resorcylic acid lactones
have been identied as a new class of kinase inhib-
itor and have been approved for the clinical treat-
ment of different types of cancers.40 Quercetin, a
natural plant product, may be an inhibitor of cancer
in tissues such as colon, lung, and intestine.41 Evi-
dence also suggests that kaempferol, which may be
converted to an aglycone during the fermentation
process, may have chemopreventative properties.42
After fermentation, kimchi (fermented cabbage)
and doenjang (fermented soybeans) had greater
functional composition than unfermented cabbage
and soybeans.6,7 New bioactive compounds and
aglycone avonoids could form during fermenta-
tion. These compounds can increase the anticancer
effects of the fermented foods.43
V. CONCLUSION
We used various in vitro experimental methods,
such as MTT assay, DAPI staining, and RT-PCR,
to evaluate the anticancer effect of teas, especially
F-Pu-erh tea. Our results showed that Pu-erh tea
had stronger anticancer activities after fermenta-
tion than did U-Pu-erh tea and green tea. Addi-
tional functional compounds were generated by
fermentation and may have contributed to these
ndings. The results of this study suggest that fu-

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Pu-erh Tea Increases Anticancer and Antiangiogenesis Effects 287
ture research should focus on understanding the
processes leading to the differences between F-Pu-
erh tea and U-Pu-erh tea. In a natural fermentation
process, the characteristics of Pu-erh tea and its
chemical compounds may vary, depending upon
environmental conditions such as humidity, tem-
perature, fermentation period, and microorgan-
isms. Therefore, standardization of such fermen-
tation conditions is highly recommended in the
future because the functionality and taste of F-Pu-
erh tea may be inuenced by the natural conditions.
An understanding of the functional changes in the
compound contents of Pu-erh tea during the fer-
mentation procedure and an adjustment in the fer-
mentation variables to produce optimal conditions
will be essential to improve the characteristics of
Pu-erh tea and maximize its anticancer effects.
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