Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and transcription factors involved in lipid catabolism

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Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and
transcription factors involved in lipid catabolism
Donghe Fu
a,1
, Elizabeth P. Ryan
b,
⁎
,1
, Jianan Huang
a
, Zhonghua Liu
a,
⁎⁎, Tiffany L. Weir
c
,
Randall L. Snook
d
, Timothy P. Ryan
e
a
Key Lab of Education Ministry for Tea Science, National Research Center of Engineering Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University,
Changsha 410128, China
b
Department of Clinical Sciences, Animal Cancer Center, Colorado State University, Fort Collins CO 80523, USA
c
Center for Rhizosphere Biology, Colorado State University, Fort Collins CO 80523, USA
d
Advanced Integrative Medicine, Lone Tree, CO 80124, USA
e
Governor's Office, Occupational Health Epidemiology, Cheyenne WY 82002, USA
abstractarticle info
Article history:
Received 21 February 2011
Accepted 8 July 2011
Available online xxxx
Keywords:
Post fermentation tea
Blood lipids
Transcriptional regulation
Emerging evidence supports health-promoting properties of post-fermented Chinese Brick Tea. Fu Zhuan Tea,
fermented with the fungus, Erotium cristatum, contains a unique phytochemical profile attributed to its
unique method of processing. Fu Zhuan Tea has been shown to activate pancreatic enzymes and regulate
blood lipids in laboratory models. Regulation of blood lipid levels by Fu Zhuan Tea consumption was
examined in an observational pilot study of volunteers with elevated LDL cholesterol that were not taking any
prescription lipid lowering medications. Significant changes in blood lipids were detected after 120 days of
daily consumption. Fu Zhuan Tea fractionation led to the investigation of six compounds for regulation of
transcription factors involved in lipid metabolism, including Farnesoid X receptor (FXR), Liver X-activated
Receptor (LXR) and two isoforms of the Peroxisome Proliferator-Activated Receptors (PPARγand PPARδ).
Reporter gene assays with liver cells revealed dose dependent differences in regulation of transcription factor
activation. These data reveal potential therapeutic efficacy and candidate molecular targets for Fu Zhuan Tea,
and provide rationale for chemical characterization of bioactive fractions and investigation of therapeutic
efficacy in cardiovascular disease and type 2 diabetes.
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
A tremendous body of literature suggests that consuming tea
(Camellia sinensis L.) provides important disease fighting activities, yet
results from clinical and epidemiological studies to support a relation-
ship between tea consumption and chronic disease risk are not clear
(Frank et al., 2009; Steptoe et al., 2007; Stote & Baer, 2008). Socio-
economic demographics and lifestyle differences among human
populations are important factors contributing to inconsistent results,
as well as variation in tea constituents, which are mainly a result of
processing rather than botanical varieties. While the purported benefits
and phytochemicals in green and black teas have been extensively
reviewed (Khan & Mukhtar, 2007; Saito, Gosmann, Pungartnik, &
Brendel, 2009; Sharma & Rao, 2009; Wang & Ho, 2009; Zaveri, 2006),
post-fermented teas have received considerably less research attention
even though they are widely consumed in China. Similar to green tea,
the oxidation of post-fermented teas is halted by steaming, which
inactivates enzymes responsible for breaking down chlorophyll and
producing tannins. This is followed by one or more microbial
fermentation steps that give the leaves a darker color and changes
their chemical composition (Xie et al., 2009). Recent metabolic profiling
studies also reveal that processing of post-fermented teas changed the
chemical contents when aged for 1–10 years (Jeng, Chen, Fang, Hou, &
Chen, 2007). Fungal fermentation of Fu Zhuan tea resultedin changes in
total tea polyphenols, catechins, amino acids, polysaccharides, and
organic acids during the fermentation process and these changes
corresponded with stimulation of pancreatic enzymes (Wu et al., 2010).
Fu Zhuan Tea (aka PHatea®), is a unique post-fermented tea
product from the Hunan Province in China that is distinguished from
other post-fermented teas by deliberate fermentation with the
fungus, Eurotium cristatum (Huang, 2007). Fu Zhuan Tea was recently
introduced for consumption in the United States because of the
purported human health benefits and findings from animal studies
showing Fu Zhuan Tea mediated regulation of blood lipids (Xiao,
2007a). Epidemiological translations from Chinese literature support
Food Research International xxx (2011) xxx–xxx
⁎Correspondence to: E. P. Ryan, Department of Clinical Sciences, Colorado State
University, Fort Collins CO 80523, USA.
⁎⁎ Correspondence to: Z. Liu. Key Lab of Education Ministry for Tea Science, National
Research Center of Engineering Technology for Utilization of Botanical Functional
Ingredients, Hunan Agricultural University, Hunan, Changsha410128, China.
E-mail addresses: e.p.ryan@colostate.edu (E.P. Ryan), larkin-liu@163.com (Z. Liu).
1
Indicates co-first authors.
FRIN-03808; No of Pages 7
0963-9969/$ –see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.07.008
Contents lists available at ScienceDirect
Food Research International
journal homepage: www.elsevier.com/locate/foodres
Please cite this article as: Fu, D., et al., Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and transcription factors involved
in lipid catabolism, Food Research International (2011), doi:10.1016/j.foodres.2011.07.008

low prevalence of hyperlipidemia among individuals that co-consume
high levels of dietary saturated animal fats with Fu Zhuan Tea.
However, the bioactive components and mechanisms responsible for
regulating blood lipids are not yet well understood.
Results from a human study conducted in China of daily Fu Zhuan
Tea consumption for reduced blood LDL cholesterol prompted an
observational study in a small group of US volunteers with elevated
cholesterol that were not taking lipid lowering medications. Findings
from this pilot cohort reported herein support the feasibility of
studying Fu Zhuan Tea consumption for changes in blood lipid
parameters. These findings prompted further fractionation and
isolation of functional compounds from Fu Zhuan Tea to test the
hypothesis that microbial fermented tea derived compounds exhibit
bioactivity on transcriptional regulators of lipid catabolism. Regula-
tion of Farnesoid X receptor (FXR, activating and restraining), Liver X-
activated Receptor (LXR) and Peroxisome Proliferator-Activated
Receptors (PPARγand PPARδ) were identified as candidate molecular
targets that can be screened using reporter gene assays, and may
advance identification of bioactive compounds in microbial fermented
tea. The findings reported herein reveal bioactivity of Fu Zhuan Tea
consumption for regulation of blood lipids in humans and led to
identification of transcriptional targets for future investigations.
Metabolite profiling studies and high throughput screening of Fu
Zhuan Tea fractions compared to other teas is a nascent field and may
reveal novel components as recently identified for antimicrobial
functions (Ling et al., 2010).
2. Materials and methods
2.1. Volunteers
Ten individuals with baseline LDL between 100 and 200 mg/dl and
hemoglobin A1c (HbA1c) ≥6% volunteered to participate in a prospec-
tive observational study that took place in the Advanced Integrative
Medicine clinic supervised by Dr. Randy Snook. The main obje ctive was to
assess changes in blood lipid panel parameters and HbA1c after 120 days
of daily Fu Zhuan Tea consumption. A control cohort-receiving medical
oversight was randomly selected from the same clinic and followed for
the same time period. Volunteer demographics and blood parameters
were provided to researchers in a de-identified manner and in
accordance with human subjects review board policies. Both tea
consuming individuals and controls were not prescribed medications
for management of blood cholesterol or Type II diabetes. The intervention
involved daily consumption of 1 l of tea, prepared by adding 1 l of boiling
water to a pre-weighed 5 g Fu Zhuan Tea brick coin stored at room
temperature. Participant compliance was monitored by weekly phone
calls and monthly check up visits. Fasting blood was collected at baseline
and on the last day of tea consumption. Volunteers were provided a copy
of their blood lipid panel results at baseline and at the end of the
observational period. Body weight and BMI were recorded at baseline
and 120 days post-consumption. Volunteers consuming the tea during or
after the 120-day period reported no adverse effects.
2.2. Post fermented tea supply
Fu Zhuan Tea used in all studies was supplied by Hunan Yiyang Tea
Factory (Hunan, Yiyang, China), and using their standard quality control
procedures. Each 5 g coin was individually wrapped and a 120-day
supply was provided to study volunteers. The aqueous preparations
consumed by participants were the same as those extracted for chemical
identification of compounds and for cell culture treatments.
2.3. Reagents and chemical extraction
Organic solvents including ethyl acetate, methanol, ethanol, 1-
butanol and chloroform were purchased from Changsha Chemical
Company (Changsha, China). Standards were purchased from Sigma
Chemicals, USA.
Fu Zhuan Tea was extracted sequentially using chloroform, 1-
butanol and ethyl acetate (Fig. 1). The ethyl acetate extract was
dissolved in 60% ethanol and separated using a poly-amidoamine
column (Nankai University Chemical Plant, Tianjin, China) with a flow
rate of 2 BV per hour. Ethanol extract was vacuum-dried and further
separated using Sephadex LH-20 column (Pharmacia, Stockholm,
Sweden) at a flow rate of 0.2 BV to yield six fractions. Compounds in
each of the fractions were detected using a Shimadzu SCL-10ATVP
system equipped with a model LC-10ATVP pump, SPD-M20A diode
array detector, a LC-solution data system and a Kromasil™C
18
column
(5 μm, 4.6 mm× 200 mm, Shimadzu, Japan). The column was operat-
ing at ambient temperature (≅40 °C). The mobile phase consisted of
water (Solvent A) and methanol (Solvent B), and an isocratic elution
of mobile phase A:mobile phase B (60:40, v/v) at a flow rate of
1.0 mL min
−1
.
2.4. Compound identification
The ultraviolet (UV) spectra of the separated individual peaks
were measured using a UV–vis spectrophotometer (model UV-2550,
Shimadzu, Japan). The infrared spectra were measured using Fourier-
transform infrared spectrometer (WQF-310, Beijing, China) with the
samples measured as potassium bromide disks. Purified compounds
were submitted for characterization by thin layer chromatography on
polyamide plates (TZSHSL, Taizhou, China) using a mobile phase of
95% ethanol. Chromatograms were evaluated under visible light to
detect the presence of each compound.
LC/MS spectra were measured with Agilent 1100 LC/MSD SL
(Agilent Inc., USA) equipped with an atmospheric pressure chemical
ionization (APCI) interface. LC/MS was performed on a Zarbax C
8
column, 200×4.6 mm i.d. (Agilent Inc., USA) at flow-rate of
1.0 mL min
−1
. The elution buffer was a mixture of methanol and
water (70:30, v/v) containing 4 g L
−1
ammonium formate. The
effluent from the LC column was delivered to the ion source
(150 °C) through heated nebulizer probe (400 °C) using nitrogen as
drying gas (5 L min
−1
, 350 °C) and nebulizer pressure was set to
60 psi. The mass spectrometer m/z ratio was 50 to 1000 in full scan
mode.
Nuclear magnetic resonance (NMR) spectrometry was carried out
using Varian Unity INOVA 300 NMR. Chemical shifts (δ) are reported
in mg kg
−1
relative to the residual solvent signals (δH 3.35 and δC
49.0 mg kg
−1
) and coupling constants (J) in Hz.
2.5. Cell culture
Human liver cancer (SMMC-7721) cells were maintained in RPMI
1640 supplemented with 15% heat inactivated fetal bovine serum
(FBS), gentamicin (40 mg mL
−1
), penicillin (100 units mL
−1
) and
streptomycin (10 mg mL
−1
).Cellsweregrownat37°Cina
humidified atmosphere of 5% CO
2
/95% air. All Fu Zhuan Tea isolated
compounds were resuspended in DMSO (final concentration 0.1%)
prior to addition to cell culture medium.
2.6. Cell viability
Cytotoxicity of purified compounds from Fu Zhuan Tea was
assessed using the MTS reduction assay (CellTiter 96® Aqueous,
Promega, WI, USA). Liver cell line SMMC-7721 (2 × 10
5
/mL) was
incubated in 96-well plates and treated with a series of concentrations
of each compound for 24 h. Positive controls and blanks were run in
parallel under the same conditions. Cell viability was measured
according to the manufacturer's guidelines at an absorbance of
490 nm and cytotoxicity was calculated by (A
s
–A
b
)/(A
c
–A
b
), where
2D. Fu et al. / Food Research International xxx (2011) xxx–xxx
Please cite this article as: Fu, D., et al., Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and transcription factors involved
in lipid catabolism, Food Research International (2011), doi:10.1016/j.foodres.2011.07.008

A
s
,A
b
and A
c
are the Absorbance at 490 nm in the presence of sample,
blank, or control.
2.7. Reporter gene assay
Nuclear receptor activation by Fu Zhuan Tea compounds in human
liver cells was measured using reporter gene constructs. DNA
response elements for PPARγ, PPARδ, LXR and FXR were inserted
upstream of the luciferase reporter gene, such that the regulation of
nuclear receptor activation by tea extracts would correlate with
luciferase gene expression. Liver cells (2 ×10
5
) were plated in 96-well
plates with a series of concentrations of each compound for up to 24 h.
Positive controls and blank were run parallel under the same
conditions. Irradiant intensity of oxidated luciferin was used as a
measure of luciferase gene expression. In order to account for
transfection efficiency, the cell inoculation number, isolated tea
compounds, and a green fluorescent protein (GFP) plasmid were
added together, and the irradiance value was normalized to GFP.
The activation multiple was calculated from the following
equation:
= LUCs=GFPs
ðÞ=LUCb=GFPb
ðÞ
LUCs = the output of the luciferase regulated by sample, GFPs =
the output of the green fluorescence protein regulated by sample, s =
sample and b = blank. The agonists rosiglitazone, 2-bromohexade-
canoic acid, chenodeoxycholic acid, and 22-(R)-hydroxycholesterol
were used as positive controls of PPARγ, PPARδ, FXR and LXR
activation respectively.
2.8. Statistical analysis
Descriptive statistics are presented as means. Differences between
tea consuming individuals and non-intervention controls were
examined using student's ttests. Statistical significance was ascribed
to 2-tailed p valuesb0.05. Analysis was performed using SAS
windows, version 9.1. All experiments were performed in triplicate.
The significance of the inhibition rate against human liver cancer
(SMMC-7721) cells was tested using one-way ANOVA under the SPSS
software package (SPSS 13.0 for windows, SPSS Inc. 2004). A
probability level of 5% (p b0.05) or 1% (p b0.01) was considered
significant.
3. Results and discussion
3.1. Regulation of blood lipids by Fu Zhuan Tea intake
Given the safety profile for Fu Zhuan Tea consumption from prior
toxicity and clinical studies (Xiao, 2007a; Xiao, 2007b), a daily dose
of 5 g brick tea/l of water was used in a pilot observational study of
tea consumption in adults with elevated LDL cholesterol compared
to controls. There were 6 males and 4 females in the intervention
group and consisted of seven white Caucasians, with three non-
Caucasians (one Hispanic, one African American and one Indian) in
the intervention group. The control group contained 7 males and 3
females of the entire white Caucasian race. The mean age of
participants in the tea-consuming group was 50.7 ± 7.1 years old
and 54.3± 5.9 years old in the controls. The study participants in the
intervention group were an average of 3 years younger and had a
significantly higher baseline LDL and HbA1C than non intervention
controls. Table 1 shows the parameters and values that were
examined at baseline and 120 days post tea consumption and those
for controls. Significant differences were detected in HDL, LDL,
FuzhuanTea water extract
Extracted with CHCl3, EtOAc, BuOH in turn
CHCl3 extract EtOAc extract BuOH extract Water extract
poly-amidoamine column chromatograph (60% ethanol)
Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5 Fr. 6
GA MDBA DBA (-)-EGCG (-)-ECG
(+)-GC
Sephadex LH-20 column chromatograph (60% ethanol)
Recrystallization
compound 1 compound 3 compound 4 compound 5 compound 6compound 2
Fig. 1. Schematic of Fu Zhuan Tea extraction for isolation of bioactive compounds. The six compounds that were isolated from bioactive tea extracts were 1) GA = gallic acid, 2) (+)-
GC = (+)-gallocatecin, 3) MDBA = 3-methoxy-4,5-dihydroxy-benzoic acid, 4) DBA = 3,4-dihyddroxy-benzoic acid, 5) (−)EGCG = (−)-epigallocatecingallate, and 6) (−)ECG =
(−)-epicatechingallate.
Table 1
Parameters examined at baseline and after 120 days of daily Fu Zhuan Tea intake
compared to controls.
Study parameters Study arms Baseline 120 days p value
HDL (mg/dl) Control 56.8± 18.88 55.6±17.66 b0.0009
Intervention 43.0±8.84 49.6±9.11
LDL (mg/dl) Control 122.4 ± 20.82 123.8 ±22.01 b0.0001
Intervention 145.7±20.45 113.1±20.82
HbA1C (%) Control 5.47± 0.24 5.47 ±0.25 b0.0034
Intervention 7.25±0.96 6.65±0.84
Weight (lbs) Control 216.0 ± 38.76 216.8 ±39.90 b0.0041
Intervention 242.3±47.72 233.6±50.56
Cholesterol (mg/dl) Control 201.5± 37.70 207.4± 42.72 b0.0005
Intervention 230.4±30.15 202.5±22.13
Triglycerides (mg/dl) Control 122.7 ± 33.91 123.9 ±33.68 b0.028
Intervention 167.2±70.12 153.4±65.76
Body Mass Index (BMI) Control 31.43± 2.81 32.35± 4.47 b0.0637
Intervention 35.76 ±6.34 34.44 ±6.99
3D. Fu et al. / Food Research International xxx (2011) xxx–xxx
Please cite this article as: Fu, D., et al., Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and transcription factors involved
in lipid catabolism, Food Research International (2011), doi:10.1016/j.foodres.2011.07.008

cholesterol, triglycerides and HbA1C between the control group and
the intervention group (pb0.05). Improvement in the blood lipid
parameters and the decrease in HbA1C suggest that multiple
bioactive components and unique mechanisms exist for Fu Zhuan
tea bioactivity. There was a small, but non-significant decrease in
the body weight and BMI in 6 out of the 10 individuals consuming
the tea. Clinic staff asked participants about changes in appetite at
each monthly visit, and 6/10 tea consuming individuals verbally
reported a decrease in appetite. While future investigations are
required to validate this finding, we judge that changes in appetite
may be an important factor to consider for some individuals.
These findings demonstrate pilot feasibility testing in humans and
support current trends of Fu Zhuan Tea efficacy in hypercholesterimic
Wistar rats and Chinese populations (Xiao, 2007a; Xiao, 2007b).
Elevated blood cholesterols have also been associated with increased
blood HbA1c (Allen et al., 2011; Chao et al., 2010), which is an
advanced glycation end product and biomarker for altered glucose
homeostasis and diabetes risk. Decreased HbA1c levels are generally
associated with pharmacologic management of Type II diabetes (e.g.
metformin) (Fonseca et al., 2010), and these data from an observa-
tional cohort first demonstrate significant reductions after 120 days in
people consuming Fu Zhuan Tea over baseline and compared to
control group. These data provide critical information regarding an
achievable effect size following daily Fu Zhuan Tea consumption and
will be useful for sample size and power calculations needed to assess
tea efficacy in a larger study cohort. Future investigations should be
directed towards examining the safety and efficacy of consuming the
Fu Zhuan Tea/PHatea® daily in combination with other prescription
drug medications.
3.2. Fu Zhuan Tea fractionation and chemical analysis
Toxicity studies conducted in mice and rats support the safety
index for consuming Fu Zhuan tea extracts (Xiao, 2007b), however
characterization of tea constituents are still in their infancy (Zhu,
2006; Huang, 2007). Fig. 1 shows the series of tea fractionation steps
prior to the isolation of purified compounds. Six compounds isolated
from bioactive fractions of Fu Zhuan Tea were identified as 1) gallic
acid (GA), 2) (+)-gallocatechingallate (GC), 3) 3-methoxy-4,5-
dihydroxy-benzoic acid (MDBA) 4) 3,4-dihydroxy-benzoic acid
(DBA), 5) (−)-epigallocatechingallate (EGCG), and 6) (−)-epica-
techingallate (ECG) (Fig. 2). NMR chemical shifts were compared
between isolated compounds and commercially available standards
for confirmation of identity (data not shown). All six compounds
have been previously isolated in varying quantities from prepara-
tions of C. sinensis. Tea catechins have been widely studied for their
antioxidant capabilities (Intra & Kuo, 2007; Maatta-Riihinen,
Kahkonen, Torronen, & Heinonen, 2005; Torres, Lozano, & Maher,
2005) and MDBA, also called 4-O-methylgallic acid, has been used as
a biomarker for tea consumption (Hodgson et al., 2000)aswellas
having anti-inflammatory properties (Na et al., 2006). In animal
models, extracts enriched in tea catechins have been shown to
inhibit cholesterol absorption and lower plasma cholesterol
(T Murase, A Nagasawa, J Suzuki, T Hase & I Tokimitsu, 2002;
Yang, Wang, & Chen, 2001; Crespy & Williamson, 2004), but few
studies have examined these chemicals individually. The most
abundant tea catechin, EGCG, has been shown to increase LDL
receptor binding activity in cultured human liver cells by increasing
the conversion of sterol regulatory element binding protein-1
(SREBP-1) to its active form, resulting in lower cellular cholesterol
concentrations (Bursill & Roach, 2006). These compounds have also
been shown to suppress differentiation of 3 T3-L1 pre-adipocytes
(Kim et al., 2010). Thus, further assessment of bioactivity related to
lipid metabolism was applied herein related to transcription factor
activation in liver cells.
3.3. Cytotoxicity of isolated compounds
Isolated tea components did not significantly reduce viability of
liver cells at any of the doses examined (Fig. 3). These data suggest a
high safety profile for Fu Zhuan Tea and are consistent with prior
doses applied to Ames test and in toxicity studies following dietary
intake(Xiao, 2007b). The doses that did not show adverse effects on
liver cell toxicity were used for evaluation of transcriptional
activation. A nonsignificant reduction in viability (less the 80%) was
detected for GC, MDBA and DBA at the higher doses. These findings
supported the use of 30 μg/mL to assess bioactivity, whereas up to
50 μg/mL was applied for GA, ECGC and EGC.
3.4. Dose dependent regulation of FXR and PPARδtranscriptional
activation
To our knowledge there are no studies reporting the effects of
these isolated tea compounds on transcriptional regulators of lipid
metabolism in liver cells, and that studies to examine these
mechanisms with tea extracts are limited. Testing of Fu Zhuan tea
compounds GA, EGCG, ECG showed that GA demonstrated the lowest
activation potential for FXR and PPARδat all the concentrations tested
when compared to ECG and EGCG (10, 30, 50 μg/ml—Table 2). The
highest doses of ECG and EGCG (50 μg/mL) had an activation potential
of 3.22 and 6.00 respectively for FXR (Table 2). While all three
compounds were active on PPARδ, EGCG showed greatest magnitude
of activation at 2.67 ± 0.21, and that of ECG was 2.04 ± 0.25. This
report first demonstrated that specific tea constituents ECG and EGCG
can regulate the activation of liver FXR and PPARδ. These findings
support a recent report for Fu Zhuan tea extracts to activate FXR
(KunBo, 2009), however a specific compound contained in the
bioactive fractions was not previously described. Given that the FXR
is a key regulator of bile acids, lipids and glucose (Chen et al., 2011;
Trauner et al., 2010; Cariou et al., 2006) and that PPARδhas been
considered a therapeutic target for diabetes and obesity through
COOH
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
COOH
OCH
3
OH
OH
COOH
OH
OH
O
O
OH
OH
OH
OH
OH COH
O
OH
OH
O
O
OH
OH
OH
OH COH
O
OH
OH
2 (+)-GC1 GA
4 DBA 3 MDBA
5 (-)-EGCG 6 (-)-ECG
Fig. 2. Chemical structures of isolated compounds identified by Fu Zhuan Tea
fractionation. 1) GA = gallic acid, 2) (+)-GC = (+)-gallocatecin, 3) MDBA = 3-
methoxy-4,5-dihydroxy-benzoic acid, 4) DBA = 3,4-dihyddroxy-benzoic acid, 5) (−)
EGCG = (−)-epigallocatecingallate, and 6) (−)ECG = (−)-epicatechingallate.
4D. Fu et al. / Food Research International xxx (2011) xxx–xxx
Please cite this article as: Fu, D., et al., Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and transcription factors involved
in lipid catabolism, Food Research International (2011), doi:10.1016/j.foodres.2011.07.008

enhancement of fatty acid oxidation (Cao et al., 2010; Ye et al., 2011;
Yu et al., 2010), these findings suggest that future studies should be
designed to examine the in vivo liver activation of FXR and PPARδin
mice consuming Fu Zhuan Tea.
3.5. Screening GC, MDBA and DBA bioactivity for liver transcription
factor activation
A30μg/ml dose was determined from the liver cell viability assay
for comparative bioactivity of the isolated tea components GC, MDBA
and DBA (Fig. 2). Reporter gene assays for LXR were added to this high
throughput screen of tea compounds based on the role of this nuclear
receptor in cholesterol transport and metabolism, inflammation and
glucose metabolism (Michael, Schkeryantz, & Burris, 2005). Natural
compounds, including those from tea have also been screened for
PPARγagonism (Furuyashiki et al., 2004; Salam et al., 2008), and thus
this model was also included herein to compare levels of bioactivity.
The results in Table 3 showed that (+)-GC demonstrated the greatest
magnitude of transcription factor regulation for PPARδ, PPARγ, FXR
(activating) and LXR when compared to MDBA and DBA. The potential
for these tea compounds to activate multiple nuclear receptor
regulators of lipid catabolism in liver cells suggests promising novel
mechanisms of action to explore for Fu Zhuan Tea investigations in
vivo.
4. Conclusions
Some of the major tea producing countries in the world are
China, Sri Lanka, Kenya, India, Indonesia, Japan, and Vietnam. Fu
Zhuan Tea is one kind of post-fermented dark green tea with specific
microbes of E. cristatum that contributes towards the special
characteristics. Fu Zhuan Tea is produced in China and is a requisite
drink for the herdsman of China. The herdsman cannot digest their
traditional high fat food intake without drinking Fu Zhuan Tea.
Fig. 3. Cytotoxicity testing of isolated tea compounds on SMMC-7721 cells. Dose-dependent effects of six tea compounds [GA = gallic acid, GC = gallocatecin, MDBA = 3-methoxy-
4,5-dihydroxy-benzoic acid, DBA = 3,4-dihydroxy-benzoic acid, EGCG = (−)-epigallocatecingallate, and ECG = (−)-epicatechingallate] were examined on liver cells by MTS assay.
No statistically significant cytotoxic effects of tea components were demonstrated at 10–30 μg/ml for all compounds tested. GC, MDBA, and DBA exhibited a nonsignificant 20% cell
growth inhibition at the 40 and 50 μg/ml dose. Data is presented as mean ±SE.
Table 2
FXR and PPARδactivation by GA, EGCG and ECG.
Monomers FXR activation
a
PPARδ
a
10.00 μg/mL 30.00 μg/mL 50.00 μg/mL 10.00 μg/mL 30.00 μg/mL 50.00 μg/mL
GA
b
1.20±0.13⁎⁎ 1.57 ± 0.12⁎⁎ 1.77 ± 0.14⁎⁎ 1.30 ± 0.02⁎⁎ 1.50 ± 0.11⁎⁎ 1.67 ± 0.12⁎⁎
EGCG
c
1.78±0.32⁎⁎ 2.81 ± 0.35⁎⁎ 6.00 ± 0.45⁎⁎ 1.21 ± 0.02⁎⁎ 1.94 ± 0.11⁎⁎ 2.67 ± 0.21⁎⁎
ECG
d
1.21±0.0⁎⁎ 2.10 ± 0.1⁎⁎ 3.22 ± 0.0⁎⁎ 1.02 ± 0.00⁎⁎ 1.54 ± 0.10⁎⁎ 2.04 ± 0.25⁎⁎
2-Bro
e
ND ND ND 2.99±0.29 ND ND
CDCA
f
7.24±1.71 ND ND ND ND ND
a
Data shown as mean ± SE.
b
Gallic acid.
c
(−)-epigallocatecingallate.
d
(−)-epicatechingallate.
e
2-bromohexadecanoic acid (positive control for PPARδactivation).
f
Chenodeoxycholic acid (positive control for FXR activation).
ND: not detected.
⁎⁎ pb0.01 when compared with that of the positive control.
5D. Fu et al. / Food Research International xxx (2011) xxx–xxx
Please cite this article as: Fu, D., et al., Fermented Camellia sinensis, Fu Zhuan Tea, regulates hyperlipidemia and transcription factors involved
in lipid catabolism, Food Research International (2011), doi:10.1016/j.foodres.2011.07.008

Furthermore, the herdsmen who eat high fat foods and drink Fu
Zhuan Tea do not suffer from hyperlipidemia. The compelling
observational findings in US volunteers of improved blood lipid
parameters reported herein are consistent with these traditional
uses. The decreased HbA1c levels following Fu Zhuan Tea consump-
tion for 120 days is a novel finding and provides strong rationale for
continued chemical characterization and assessment of bioactive
components that may influence production of advanced glycation
end products. The effects of Fu Zhuan Tea on insulin sensitivity and
glucose regulation are also highly relevant to novel therapeutic
applications for type II diabetes.
Fu Zhuan Tea is different from other post-fermented tea as a
result of the E. cristatum exposure during flowering and influence on
processing. While the differences in both chemical content and
bioactivity may be due to Fu Zhuan Tea processing (i.e., fermenta-
tion) and not to botanical varieties, further biomedical research is
warranted to substantiate these observed medicinal properties.
Based on reports from the bioactivity of Fu Zhuan Tea extracts, six
compounds were isolated and showed differential levels of
transcription factor regulation in liver cells. These data suggest a
role for Fu Zhuan Tea compounds to regulate lipid catabolism. Tea
and drug comparison study designs will be required to identify
unique health promoting and chronic disease fighting properties
across the different types of Chinese brick, post fermented teas.
Future Fu Zhuan Tea/ PHatea® investigations may assess bioavail-
ability of selected tea compounds, establish bioactivity of liver
transcriptional activation in vivo, and identify pharmacodynamics
mechanisms of synergy with current drug treatments, such as
statins and metformin. PHatea® has only recently become available
for consumption in the US and clinical research studies for the
chronic disease fighting properties in humans are still under
development. Epidemiological studies in various populations will
also be necessary to inform the utility of PHatea® for control and
management of metabolic syndromes, hyperlipidemia, and alter-
ations in glucose homeostasis/metabolism that may be seen in
obesity, pre-diabetes and non-insulin dependent type II diabetics.
Acknowledgments
The work was supported by the project from Hunan Provincial
Department of Education, China (10A048), Key Project of Hunan
Provincial Department of Science & Technology, China (08FJ1005) and
Fund of China National Tea Industry Technology Innovation System.
The authors thank the Denver Diabetes Foundation for providing Fu
Zhuan Tea from China Hunan Yiyang Tea Factory for human
consumption in the observational pilot study.
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a
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GC
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MDBA
c
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DBA
d
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e
ND ND ND ND 6.96±0.09⁎⁎
Ros
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ND ND ND 4.36±0.11 ND
2-Bro
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––2.25 ± 0.25 ––
CDCA
h
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f
Rosiglitazone, positive control of PPARγactivation.
g
2-Bromohexadecanoic acid, positive control of PPARδactivation.
h
Chenodeoxycholic acid, positive control of FXR activation.
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