The Journal of Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Green Tea, Black Tea, and Oolong Tea

Polyphenols Reduce Visceral Fat and
Inflammation in Mice Fed High-Fat, High-
Sucrose Obesogenic Diets1–3

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David Heber,* Yanjun Zhang, Jieping Yang, Janice E. Ma, Susanne M. Henning, and Zhaoping Li

Center for Human Nutrition, David Geffen School of Medicine, University of California, Los Angeles, CA

Abstract
Green tea (GT) and caffeine in combination were shown to increase energy expenditure and fat oxidation, but less is
known about the effects of black tea (BT) and oolong tea (OT). This study investigated whether decaffeinated polyphenol
extracts from GT, BT, and OT decrease body fat and inflammation in male C57BL/6J mice fed high-fat/high-sucrose [HF/
HS (32% energy from fat, 25% energy from sucrose)] diets. Mice were fed either an HF/HS diet with 0.25% of polyphenol
from GT, OT, or BT or a low-fat/high-sucrose [LF/HS (10.6% energy from fat, 25% energy from sucrose)] diet for 20 wk.
Monomeric tea polyphenols were found in the liver and adipose tissue of mice fed the HF/HS diet with GT polyphenols
(GTPs) and OT polyphenols (OTPs) but not BT polyphenols (BTPs). Treatment with GTPs, OTPs, BTPs, and an LF/HS diet
led to significantly lower body weight, total visceral fat volume by MRI, and liver lipid weight compared with mice in the HF/
HS control group. Only GTPs reduced food intake significantly by ;10%. GTP, BTP, and LF/HS-diet treatments
significantly reduced serum monocyte chemotactic protein-1 (MCP-1) compared with HF/HS controls. In mesenteric fat,
monocyte chemotactic protein-1 (Mcp1) gene expression was significantly decreased by treatment with GTPs, BTPs,
OTPs, and an LF/HS diet and in liver tissue by GTP and BTP treatments. Mcp1 gene expression in epididymal fat was
significantly decreased by the BTP and LF/HS diet interventions. In epididymal fat, consistent with an anti-inflammatory
effect, adiponectin gene expression was significantly increased by GTPs and OTPs. Angiogenesis during adipose tissue
expansion is anti-inflammatory by maintaining adipocyte perfusion. We observed significantly increased gene
expression of vascular endothelial growth factor A by GTPs and vascular endothelial growth factor receptor 2 by
BTPs and the LF/HS diet and a decrease in pigment epithelium-derived factor gene expression by OTPs and BTPs. In
summary, all 3 tea polyphenol extracts induced weight loss and anti-inflammatory and angiogenic effects, although the
tissue content of polyphenols differed significantly. J. Nutr. 144: 1385–1393, 2014.

Introduction high-fat/high-sucrose (HF/HS)4 diets (5). However, the effects of

Energy-dense, nutrient-poor diets containing high amounts of the tea polyphenols found in green tea (GT), black tea (BT), and
fat and refined carbohydrates combined with sedentary lifestyles oolong tea (OT) on fat deposition and inflammation, as well as
are believed to be the major drivers of the global obesity potential mechanisms of action, were not adequately studied. All
epidemic (1–3). The consumption of tea containing polyphenols teas are derived from the leaves of Camellia sinensis, but
and caffeine was shown in numerous clinical trials to affect body different processing methods produce different types of tea.
weight and fat metabolism in humans (4) and in rodents fed Fresh tea leaves are rich in polyphenols known as flavan-3-ols,

4
Abbreviations used: Adipoq, adiponectin; BT, black tea; BTP, black tea polyphenol;
1
Supported by departmental funds from the Center for Human Nutrition, C/ebpa, CCAAT/enhancer binding protein a; Cpt1, carnitine palmitoyltransferase I;
Department of Medicine, David Geffen School of Medicine, University of ECG, (2)-epicatechin-3-gallate; EGC, (2)-epigallocatechin; EGCG, (2)-epigallocatechin
California, Los Angeles. gallate; FAS, FA synthase; Gpat, glycerol-3-phosphate acyltransferase; GT, green
2
Author disclosures: D. Heber, Y. Zhang, J. Yang, J. E. Ma, S. M. Henning, and tea; GTP, green tea polyphenol; HF/HS, high-fat/high-sucrose; Hmgcoa, 3-hydroxy-
Z. Li, no conflicts of interest. 3-methylglutaryl-CoA; Klf7, Kruppel-like factor 7; LF/HS, low-fat/high-sucrose; MCP-1,
3
Supplemental Tables 1 and 2, and Supplemental Figures 1–3 are available from monocyte chemotactic protein-1; OT, oolong tea; OTP, oolong tea polyphenol; Pedf,
the ‘‘Online Supporting Material’’ link in the online posting of the article and from pigment epithelium-derived factor; SCD-1, stearoyl-CoA desaturase; SREBP1c, sterol
the same link in the online table of contents at http://jn.nutrition.org. regulatory element binding protein-1c; Vegfa, vascular endothelial growth factor A;
* To whom correspondence should be addressed. E-mail: [email protected]. Vegfr2, vascular endothelial growth factor receptor 2; 4##-MeEGCG, 4##-O-methyl (–)-
edu. epigallocatechin gallate.

ã 2014 American Society for Nutrition.

Manuscript received January 17, 2014. Initial review completed February 1, 2014. Revision accepted June 27, 2014. 1385
First published online July 16, 2014; doi:10.3945/jn.114.191007.
including (2)-epicatechin, (2)-epigallocatechin gallate (EGCG), was repeated twice. The ethanol was evaporated in a rotary evaporator
(2)-epigallocatechin (EGC), and (2)-epicatechin-3-gallate (ECG) under reduced pressure at 40°C. The dried extract was suspended in 500
(6). The 3 major types of tea are GT, OT, and BT, with different mL of pure water and extracted with dichloromethane to remove
degrees of fermentation during processing (6). During fermenta- caffeine. The decaffeinated water solution of tea extract was subjected to
an XAD-16 resin column separation, rinsed with 5 bed volumes of water,
tion to manufacture BT, monomeric polyphenols are converted to
and eluted with pure ethanol. The extract was dried using the rotary
polymeric polyphenols called theaflavins and thearubigins, which evaporator.
are not absorbed from the gastrointestinal tract like smaller
catechins, such as EGCG (7). BT contains lower amounts of Gallic acid equivalent. The assays were performed as reported
monomeric polyphenols (3–10% of solids) and higher concen- previously with some modification using Folin-Ciocalteau reagent (17).
trations of polymers (23–25% of solids) compared with GT (6). In The absorbance was read at 755 nm in a ThermoMax microplate reader
addition, a higher concentration of gallic acid is found in BT (8). (Molecular Devices) at room temperature. The standard curves were
The chemical composition of thearubigins is under investigation used to convert the average absorbance of each sample into milligrams
per gram gallic acid equivalent.

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(9). OTs are partially oxidized, leading to an intermediate tea with
a lower concentration of polymeric polyphenols and higher
HPLC condition for analysis of catechins and caffeine. A Water
concentrations of EGCG than BT.
Alliance 2695 HPLC system coupled with a PDA detector and Empower
Preparations of GT are used as aids in weight loss and weight 2 software was used to analyze the catechins and caffeine. The
maintenance. Catechins and caffeine, both contained in GT, are separation of catechins and caffeine was conducted on an Agilent
each believed to have a role in increasing energy metabolism and Zorbax SB C18 column with a gradient of acetonitrile and 0.4%
fat oxidation, which may lead to loss of body fat and weight loss. phosphoric acid in water. The detection wavelength was 280 nm.
A number of randomized controlled trials evaluating the role of
GT in weight loss were published previously (5). Dulloo et al. Experimental mouse and body composition studies
(10), using a whole-body energy expenditure chamber, found All mouse procedures were approved by the University of California, Los
that a combination of GT extract and caffeine led to an increase Angeles Animal Research Committee in compliance with the Association
in energy expenditure in the range of 2–4% of total daily energy for Assessment and Accreditation of Laboratory Care International.
Male C57BL/6J mice (strain JAX 000664) were received from The
expenditure in normal volunteers. Conversely, in a study by
Jackson Laboratory at 6–7 wk of age (body weight: 16–18 g). After 1 wk
Hursel et al. (11), GT alone did not prevent weight regain after of acclimation, 28-d-old male C57BL/6J mice were assigned to 5 groups
weight loss in obese individuals. Maki et al. (12) found that GT with similar body weight distribution in each group (Supplemental
extract consumption in combination with exercise led to a Fig. 1) and were fed a low-fat/high-sucrose (LF/HS) diet (D12489B;
decrease in abdominal visceral fat and TGs. A recent meta- Research Diets), an HF/HS diet (D12266B; Research Diets), or an HF/
analysis by Hursel et al. (13) concluded that a combination of HS diet supplemented with GT polyphenols (GTPs), OT polyphenols
GT and caffeine led to increased energy expenditure and fat (OTPs), or BT polyphenols (BTPs) (Table 1) at 0.5 g/100 g diet providing
oxidation in humans. To our knowledge, there are numerous 0.25 g polyphenols/100 g diet. Tea extracts were mixed into the diet by
studies of pure ECGC but no previous studies of the effects of tea Research Diets. Body weights were recorded weekly and food consumption
polyphenols from the 3 major types of tea (GT, BT, and OT) on 3 times per week. Groups of 3–5 mice from each group were killed after 4,
8, 12, 16, and 20 wk of dietary treatments (Supplemental Fig. 1). Tissues
body fat in mice consuming an obesogenic HF/HS diet.
were collected, weighed, and stored at 280°C until analysis. Body fat
Another potential benefit of weight loss is the reduction of
obesity-associated inflammation, which was implicated as a
major factor in age-related chronic diseases. Abdominal visceral TABLE 1 Composition of LF/HS diet, HF/HS diet, and HF/HS
diets containing different tea polyphenols fed to male C57BL/6J
fat, but not lower body fat, is implicated in obesity-associated
mice for 20 wk1
inflammation (14). A decrease in microvessel density and blood
circulation in visceral adipose tissue leads to hypoxia and LF/HS HF/HS HF/HS-GTP HF/HS-OTP HF/HS-BTP
adipocyte necrosis, leading to the release of proinflammatory Ingredients diet diet diet diet diet
cytokines into the circulation (15,16). Weight loss was shown to
be associated with an increase in adiponectin (Adipoq) forma- g/kg g/kg g/kg g/kg g/kg
tion, which results in increased angiogenesis in visceral fat that Casein 161.2 182.2 181.3 181.3 181.3
counteracts the hypoxia due to decreased microvessel density, DL-Methionine 2.5 2.8 2.9 2.9 2.9
and can inhibit inflammation (14). Corn starch 423.1 206.2 205.1 205.1 205.1
The present study was designed to compare the activities of Maltodextrin 10 29.7 71.9 74.4 74.4 74.4
the caffeine-free extracts of polyphenols from GT, BT, and OT in Sucrose 246.1 278.1 276.7 276.7 276.7
inhibiting fat deposition and systemic inflammation during Cellulose 25.5 28.8 28.6 28.6 28.6
weight gain in wild-type C57BL/6J mice fed an HF/HS diet. Butter fat 12.5 42.4 42.2 42.2 42.2
Corn oil 33.4 113.2 112.6 112.6 112.6
Mineral mix S10001 34.0 38.4 38.2 38.2 38.2
Methods and Materials Calcium carbonate 4.7 5.3 5.2 5.2 5.2
Tea polyphenol extracts Sodium chloride 4.7 5.3 5.2 5.2 5.2
Chemical reagents and plant materials. All solvents were HPLC Potassium citrate 11.5 12.9 12.4 12.4 12.4
grade and purchased from Fisher Scientific. Gallic acid (>98%), tea Vitamin mix V10001 9.3 10.5 10.5 10.5 10.5
polyphenol, and caffeine standards were purchased from Sigma-Aldrich. Choline bitartrate 1.6 1.9 1.9 1.9 1.9
All GT, OT, and BT leaves were collected and purchased in a selected GTPs 5
location in Sichuan province, China. The samples were kept in sealed
OTPs 5
bags at room temperature before extraction.
BTPs 5
Tea polyphenol extract preparation. A total of 500 g of tea leaves 1
A total of 0.25 g of GTPs, OTPs, and BTPs was added to 1 kg of diet based on gallic
were extracted with 4 L of 75% ethanol in room temperature for 3 h. acid equivalents. BTP, black tea polyphenol; GTP, green tea polyphenol; HF/HS, high-
The leaves were separated and extracted with ethanol. The procedure fat/high-sucrose; LF/HS, low-fat/high-sucrose; OTP, oolong tea polyphenol.

1386 Heber et al.

composition was measured at week 20. Five mice from each group were 1-factor ANOVA, with the factor diet. The Tukey-Kramer multiple
anesthetized by isoflurane inhalation and imaged using an Aspect imaging comparison procedure was used for post hoc comparisons. P values
M2 MRI system. Total adipose tissue and abdominal adipose tissue were <0.05 were considered statistically significant.
quantified using the T1-weighted spin-echo data (VivoQuant; inviCRO).

Measurement of liver total lipid content, TGs, and FA Results

composition
Total lipid content. Total hepatic lipids were quantified by chloroform Body weight and composition in mice fed the HF/HS diet
methanol extraction following a modification of the method by Bligh supplemented with polyphenol-enriched tea extracts.
and Dyer (18). During the 20-wk dietary intervention, the HF/HS-treated
mice had significantly higher body weight and subcutaneous
FA analysis by GC. The FA content of 6 FAs was determined as methyl fat by weight (Fig. 1A, B) and total visceral fat by weight
esters by a GC–flame ionization detector method as published previously compared with the LF/HS group (Supplemental Fig. 2B). As

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(19). The analysis was performed on an Agilent 7890 A GC as described shown in Figure 1A, B and Supplemental Figure 2B, body weight
previously (19). The FFAs were identified and quantified by comparing gain and visceral fat and subcutaneous fat gain normalized to
the retention times and area of peaks with those of commercial FFA
body weight were decreased significantly in mice fed the HF/HS
methyl ester standards purchased from Sigma-Aldrich.
diets supplemented with all 3 types of tea polyphenols compared
Liver Oil Red O staining. Liver tissue was fixed in 10% neutral-buffered with HF/HS controls. We also assessed total visceral fat of mice by
formalin (VWR) and embedded in optimal cutting temperature com- MRI at week 20. The HF/HS-induced gain in total visceral fat was
pound. Sections were processed for Oil Red O staining as described significantly reduced by GTP, OTP, and BTP supplementation
previously (20). Liver specimens were evaluated by light microscopy, and
the Oil Red–positive area was analyzed using VivoQuant data processing.

Liver and adipose tissue tea polyphenol analysis

Extraction and digestion (b-glucuronidase and sulfatase; Sigma-Aldrich)
of liver and adipose tissue tea polyphenols was performed as described
previously (21). Dried extracts were reconstituted in 50% methanol:
water for detection by LC–MS. The LC–MS analysis was performed on a
Zorbax SB C18 column (Agilent). Analyses were performed using the
HPLC–electrospray ionization–MS system (Thermo Finnigan LCQ
advantage) at negative mode. MS2 spectra were automatically
performed with helium as the collision gas (EGC: m/z 305/219;
EGCG: m/z 457/331; methyl-EGCG: m/z 471/287; and ECG: m/z
441/289). Concentrations were calculated by comparison of sample
peak area with the commercial standard peak area (21).

Real-time qPCR
Liver and adipose tissues of mice after 16 wk of dietary treatment were
dissected and immediately preserved in RNALater Solution (Life
Technologies). Total RNA was isolated using an RNeasy mini kit and
an RNeasy lipid tissue mini kit (Qiagen). RNA treated with deoxyribo-
nuclease I was quantified, and equal amounts of RNA were reverse
transcripted into cDNA using a first-strand cDNA synthesis kit
(Clontech). qRT-PCR was performed using a SYBR green PCR master
mix (Clontech) and HT7900 Fast Real-Time PCR (Applied Biosystems).
The mRNA levels of all genes were normalized using GAPDH as internal
control. The primers were designed to evaluate the expressions of the
pigment epithelium-derived factor (Pedf), vascular endothelial growth
factor A (Vegfa), vascular endothelial growth factor receptor 2 (Vegfr2),
Gapdh, monocyte chemotactic protein-1 (Mcp1), and Adipoq genes
(Supplemental Table 1).

Analysis of serum MCP-1

The plasma concentration of mouse MCP-1 was measured with
Quantikine M mouse MCP-1 ELISA kits (R&D Systems). Intra-assay
and interassay precision indicated by percentage coefficient of variation FIGURE 1 Effects of polyphenol-enriched tea extracts on body
are 4.6–7.3 and 5.1–8.3, respectively. weight (A) and percentage subcutaneous fat normalized to body
weight (B) in male C57BL/6J mice fed an HF/HS, LF/HS, HF/HS-GTP,
Statistical analyses HF/HS-OTP, or HF/HS-BTP diet for 20 wk. Data are means 6 SEMs
All statistical analyses were conducted using IBM SPSS Statistics version (n = 3–5). Statistical significance as evaluated by 2-factor ANOVA (diet 3
21; mean values, SDs, and SEs were calculated using descriptive time), followed by the Tukey-Kramer multiple comparison procedure.
statistics. Energy intake, body weight, percentage visceral fat/body Both diet and time affected body weight and percentage subcuta-
weight, percentage subcutaneous fat/body weight, percentage liver neous fat normalized to body weight, but there was no interaction
weight/body weight, and percentage lipid weight/liver weight were between the effect of time and diet (Supplemental Table 2).
analyzed with 2-factor ANOVA, with the factors diet and time. The Labeled means of dietary interventions without a common letter
Tukey-Kramer multiple comparison procedure was used for post hoc differ by diet throughout the intervention time (4–20 wk), P , 0.05.
comparisons of diet means. Total visceral fat volume (MRI), hepatic FAs, BTP, black tea polyphenol; GTP, green tea polyphenol; HF/HS, high-
hepatic Oil Red–positive area, Mcp1, Adipoq, Pedf, Vegfa, and Vegfr2 fat/high-sucrose; LF/HS, low-fat/high-sucrose; OTP, oolong tea
gene expression, and MCP-1 protein expression were analyzed with polyphenol.

Tea polyphenol-induced reduction of weight gain 1387

(Fig. 2). Energy intake of the mice consuming the HF/HS-OTP
and HF/HS-BTP diets was not significantly different from the
HF/HS control mice, whereas HF/HS-GTP consumption was
associated with a significant decrease in energy intake compared
with HF/HS control mice (weeks 4–20) (Supplemental Fig. 2A).

Liver weight and lipid content. When normalized to body

weight, no difference in liver weight was observed at the end of
the 20-wk dietary intervention with the HF/HS diet group
compared with the LF/HS diet group (Fig. 3A). However,
normalized liver weights of mice in the HF/HS-GTP and HF/HS-
BTP groups, but not the HF/HS-OTP group, were significantly

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lower compared with HF/HS-fed mice (Fig. 3A). Weights of the
spleen, heart, pancreas, and kidney were similar among all the
experimental groups (data not shown). None of the tea
polyphenol extracts caused liver toxicity. At 20 wk of interven-
tion, serum alanine transaminase activity was below 33 U/L,
which is in the normal range for mice (22). Hepatic total lipid
and TG content was increased significantly in the HF/HS diet
mice compared with the LF/HS diet mice. We observed that all 3
tea polyphenol extracts significantly decreased liver total lipid
and TG content compared with HF/HS control mice (Fig. 3B, C
and Supplemental Fig. 3).
The liver FA analysis at 20 wk demonstrated that the dietary
tea polyphenol treatment was associated with changes in hepatic
FA composition (Table 2). GTPs and BTPs significantly altered
the percentage palmitic acid (16:0) and DHA (22:6n–3) of total
FAs quantified compared with the HF/HS diet alone. All 3 tea
polyphenol extracts significantly regulated the percentage stearic
acid (18:0) and oleic acid (18:1n–9) compared with the HF/HS
diet alone. The percentage linoleic acid (18:2n–6) and arach-
idonic acid (20:4n–6) were significantly increased by GTPs only
compared with the HF/HS diet alone.

Effect of tea polyphenols on inflammatory responses in

white adipose tissue and liver of mice with HF/HS-induced
obesity. Mcp1 gene expression was evaluated in white adipose
tissue (epididymal and mesenteric fat) at the end of the 16-wk
dietary tea treatment. In mesenteric fat, all 3 tea extracts

FIGURE 3 Effects of polyphenol-enriched tea extracts on HF/HS-

induced fatty liver in male C57BL/6J mice fed an HF/HS, LF/HS, HF/
HS-GTP, HF/HS-OTP, or HF/HS-BTP diet for 20 wk. Liver weight was
normalized to body weight (A), and liver lipid weight was normalized to
liver weight (B) over 20 wk. Data are means 6 SEMs (n = 3–5).
Statistical significance as revealed by 2-factor ANOVA (diet 3 time),
followed by the Tukey-Kramer multiple comparison procedure. Both
diet- and time-affected liver weight were normalized to body weight
and liver lipid weight normalized to liver weight, but there was no
interaction between the effect of time and diet (Supplemental Table
2). Labeled means of dietary interventions without a common letter
differ by diet throughout the intervention time (4–20 wk), P , 0.05.
Quantification of TG by Oil Red staining in liver sections (C). Data are
means 6 SEMs (n = 5). Data were analyzed by 1-factor ANOVA,
followed by the Tukey-Kramer multiple comparison procedure.
FIGURE 2 Coronal T1-weighted spin-echo MRIs were obtained Labeled means without a common letter differ, P , 0.05. BTP, black
from male C57BL/6J mice fed an HF/HS, LF/HS, HF/HS-GTP, HF/HS- tea polyphenol; GTP, green tea polyphenol; HF/HS, high-fat/high-
OTP, or HF/HS-BTP diet for 20 wk. The images were processed with sucrose; LF/HS, low-fat/high-sucrose; OTP, oolong tea polyphenol.
volume segmentation for total visceral fat. Quantification of MR
images was performed using VivoQuant software. Data are means 6
SEMs (n = 5). Data were analyzed by 1-factor ANOVA, followed by the significantly decreased HF/HS diet–induced Mcp1 upregulation
Tukey-Kramer multiple comparison procedure. Labeled means with- (Fig. 4A), whereas BTP treatment significantly decreased HF/HS
out a common letter differ, P , 0.05. BTP, black tea polyphenol; GTP, diet–induced Mcp1 upregulation in epididymal fat (Fig. 4B). In
green tea polyphenol; HF/HS, high-fat/high-sucrose; LF/HS, low-fat/ liver, Mcp1 gene expression was inhibited significantly by the
high-sucrose; OTP, oolong tea polyphenol. addition of GTPs and BTPs but not OTPs (Fig. 4C). The serum
1388 Heber et al.
 TABLE 2 Effects of polyphenol-enriched tea extracts on hepatic FA composition expressed as a
percentage of the sum of all FAs analyzed in male C57BL/6J mice fed an HF/HS, LF/HS, HF/HS-GTP, HF/
HS-OTP, or HF/HS-BTP diet for 20 wk1

Palmitic acid Stearic acid Oleic acid Linoleic acid Arachidonic acid DHA

Weight % Weight % Weight % Weight % Weight % Weight %

LF/HS diet 25.3 6 1.0a,b 6.0 6 1.8a,b 30.9 6 6.7a 8.9 6 1.6c 5.6 6 3.1a,b 1.4 6 0.7a,b
HF/HS diet 26.3 6 0.8a 4.1 6 1.1b 30.3 6 3.6a 16.9 6 1.9b 4.1 6 1.1b 1.0 6 0.2b
HF/HS-GTP diet 20.8 6 2.2c 7.7 6 2.6a 19.2 6 4.9b 21.1 6 4.4a 7.6 6 2.5a 2.0 6 0.8a
HF/HS-OTP diet 25.6 6 1.2a,b 6.9 6 1.4a 22.5 6 4.2b 16.6 6 2.0b 6.2 6 1.2a,b 1.4 6 0.3a,b
HF/HS-BTP diet 23.5 6 2.5b 7.4 6 2.9a 22.4 6 7.0b 17.7 6 1.9b 6.9 6 2.9a,b 1.9 6 0.9a
1
Values are means 6 SDs (n = 5). Data were analyzed by 1-factor ANOVA, followed by the Tukey-Kramer multiple comparison procedure.

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Hepatic FA profiles of mice fed the indicated diets for 20 wk were determined by GC analysis. Data were expressed as percentage of the
sum of all FAs. Means in a column without a common letter differ, P , 0.05. BTP, black tea polyphenol; GTP, green tea polyphenol; HF/HS,
high-fat/high-sucrose; LF/HS, low-fat/high-sucrose; OTP, oolong tea polyphenol.

concentration of MCP-1 in the HF/HS group was increased

compared with the LF/HS group; BTPs and GTPs significantly
inhibited HF/HS-induced serum MCP-1 upregulation (Fig. 5).

Gene expression of Adipoq, Pedf, Vegfa, and Vegfr2 in

white adipose tissue. During initial adipose expansion (HF
diet–induced obesity model), proangiogenic activities were
shown to be associated with a reduction of inflammation and
insulin resistance (15,16,23). Feeding the HF/HS-GTP and HF/
HS-OTP diets significantly increased Adipoq, whereas HF/HS-
OTP and HF/HS-BTP diets decreased Pedf gene expression in
epididymal fat compared with HF/HS diet mice (Fig. 6A, B). In
addition, gene expression of 2 proangiogenic markers, Vegfa and
Vegfr2, was determined. Compared with the HF/HS diet, the
HF/HS-GTP diet significantly increased Vegfa gene expression in
epididymal fat (Fig. 6C), and the HF/HS-BTP diet was associ-
ated with significantly increased Vegfr2 gene expression in
epididymal fat (Fig. 6D).

Concentration of tea polyphenols and metabolites in liver

and adipose tissue. Polyphenol extracts of GT, OT, and BT
contained 21%, 14%, and 3.6% of EGCG, respectively (Table 3).
EGCG was found in liver and white adipose tissue of mice fed the
GTP and OTP diets, with considerable individual variation (Fig.
7A, B). 4##-O-methyl EGCG (4##-MeEGCG) was only found in
liver of mice in the HF/HS-GTP and HF/HS-OTP groups (Fig.
7A). ECG was found in liver and white adipose tissue of mice in
all 3 groups (Fig. 7A, B). In the liver, EGC was detected in 1 of 3
mice fed the HF/HS-GTP and HF/HS-OTP diets (Fig. 7B).
Adipose EGC was detected in GTP-fed mice (Fig. 7B).

Discussion
In the present study, the addition of standardized GTP-, BTP-,
and OTP-enriched extracts to an HF/HS diet significantly
decreased body weight, abdominal visceral fat volume, and
biomarkers of inflammation compared with an HF/HS obeso-
genic diet alone. FIGURE 4 Effects of polyphenol-enriched tea extracts on Mcp1
In addition, gene expression of angiogenesis markers and gene expression in white adipose tissue and liver in male C57BL/6J
Adipoq were modulated by GTP-, BTP-, and OTP-enriched mice fed an HF/HS, LF/HS, HF/HS-GTP, HF/HS-OTP, or HF/HS-BTP
extracts. The final body weight in mice fed tea extracts with an diet for 16 wk. Mesenteric fat (A), epididymal fat (B), and liver (C). Data
are means 6 SEMs (n = 5). Data were analyzed by 1-factor ANOVA,
HF/HS diet was similar to mice fed an LF/HS diet, except that
followed by the Tukey-Kramer multiple comparison procedure.
liver fat in the GTP-fed group was greater than in the LF/HS diet. Labeled means without a common letter differ, P , 0.05. BTP, black
However, energy intake was decreased by ;10% with GTPs but tea polyphenol; E-Fat, epididymal fat; GTP, green tea polyphenol; HF/
unchanged with BTPs or OTPs added to the HF/HS diet. HS, high-fat/high-sucrose; LF/HS, low-fat/high-sucrose; M-Fat, mes-
Although all 3 tea polyphenol extracts were standardized to enteric fat; Mcp1, monocyte chemotactic protein-1; OTP, oolong tea
similar phenolic contents, the GTP extract had the highest polyphenol.

Tea polyphenol-induced reduction of weight gain 1389

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FIGURE 5 Effects of polyphenol-enriched tea extracts on serum
concentrations of MCP-1 in male C57BL/6J mice fed an HF/HS, LF/
HS, HF/HS-GTP, HF/HS-OTP, or HF/HS-BTP diet for 16 wk. Data are
means 6 SEMs (n = 5). Data were analyzed by 1-factor ANOVA,
followed by the Tukey-Kramer multiple comparison procedure.
Labeled means without a common letter differ, P , 0.05. BTP, black
tea polyphenol; GTP, green tea polyphenol; HF/HS, high-fat/high-
sucrose; LF/HS, low-fat/high-sucrose; MCP-1, monocyte chemotactic
protein-1; OTP, oolong tea polyphenol.

content of EGCG, which could explain the decreased energy

intake through its known effects of increasing hepatic FA
oxidation (4). Monomeric GTPs are absorbed in the small
intestine, whereas the larger polymeric polyphenols in BT and
OT are only marginally absorbed even after the administration
of large amounts equivalent to 6 L (30 cups) of BT (24). GT or
EGCG administration with HF diets can cause weight loss
independent of effects on energy intake (25,26) through in-
creased thermogenesis, increased lipid oxidation, and/or re-
duced lipogenesis (4,5). In addition i.p. injection of EGCG
(85 mg/kg body weight) in rats is associated with a decrease in
energy intake of ;15% along with decreases in serum leptin and
luteinizing hormone concentrations (27).
Fewer studies examined the effect of BT on weight loss in
humans and animals (28–30). The large-molecular-weight tea
polyphenols in BT suggest that the antiobesity effects of BTPs
may be through inhibition of pancreatic lipase, leading to
decreased lipid absorption (31–33). OTPs also cause weight loss
in mice fed an HF diet (34), most likely due to effects similar to
those of BT because OT also contains less catechin and EGCG
than GT.
Rapid weight gain in humans and C57BL/6J mice leads to
accumulation of intra-abdominal or visceral fat (35), which is
accompanied by apoptosis, immune system activation, and
recruitment of macrophages, leading to an increase in blood
concentrations of circulating chemokines, MCP-1, IL-6, TNF-a,
and other cytokines (36). MCP-1 is a small cytokine that recruits
monocytes, memory T cells, and dendritic cells to the site of FIGURE 6 Effects of polyphenol-enriched tea extracts on Adipoq and
injury. Plasma concentrations of MCP-1 are increased with angiogenesis-related gene expression in epididymal fat from male C57BL/
obesity (37). In the present study, all 3 tea polyphenol extracts at 6J mice fed an HF/HS, LF/HS, HF/HS-GTP, HF/HS-OTP, or HF/HS-BTP diet
the same total polyphenol content led to a decrease in MCP- for 16 wk: Adipoq (A), Pedf (B), Vegfa (C), and Vegfr2 (D) gene expression.
1 concentrations in serum. Similar results were observed in mice Data are means 6 SEMs (n = 5). Data were analyzed by 1-factor ANOVA,
fed 60% energy from fat supplemented with 1% and 2% of GT followed by the Tukey-Kramer multiple comparison procedure. Labeled
extract or 0.37% EGCG (35,36,38). Treatment with low means without a common letter differ, P , 0.05. Adipoq, adiponectin;
concentrations of gallic acid (0.1 and 1 mmol/L) inhibited BTP, black tea polyphenol; GTP, green tea polyphenol; HF/HS, high-fat/
high-sucrose; LF/HS, low-fat/high-sucrose; OTP, oolong tea polyphenol;
proinflammatory cytokine gene expression in vitro, which may
Pedf, pigment epithelium-derived factor; Vegfa, vascular endothelial
explain the anti-inflammatory effect of BTPs (39).
growth factor A; Vegfr2, vascular endothelial growth factor receptor 2.
Tissue FA composition reflects the FA composition of the diet
(40). Corn oil is rich in linoleic acid and oleic acid. A 2-fold
relative increase of linoleic acid was seen in mice consuming the acid and oleic acid were observed in the hepatic fat of mice
HF/HS diet compared with the LF/HS diet. Decreases in palmitic consuming the HF/HS diet with tea polyphenols compared with
1390 Heber et al.
TABLE 3 GAE, caffeine, and EGCG concentrations of GTPs,
OTPs, and BTPs1

GAE Caffeine EGCG

mg/g mg/g mg/g
GTPs 565 6 24 0.5 6 0.1 214 6 4.5
OTPs 588 6 29 0.6 6 0.1 142 6 0.6
BTPs 532 6 25 1.4 6 0.1 36 6 0.3
1
Values are means 6 SDs (n = 6). Total phenolic content was expressed as GAE. BTP,
black tea polyphenol; EGCG, (2)-epigallocatechin gallate; GAE, gallic acid equivalent;
GTP, green tea polyphenol; OTP, oolong tea polyphenol.

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controls. These observations are consistent with the effect of
tea polyphenols inhibiting de novo fat synthesis from sucrose
through acyl-CoA synthase reaction and the conversion of
stearic acid to oleic acid by the stearoyl-CoA desaturase
(SCD-1) (41). In addition, GT, BT, and pu-erh (completely
fermented) tea were shown to inhibit FA synthase (FAS)
through AMP-activated protein kinase phosphorylation in rats
fed a high-fructose diet (42). GT extract also decreased the
expression of hepatic lipogenic genes, including sterol element
binding protein-1c (SREBP1c) and its downstream regulatory
target genes FAS and SCD-1, in fructose-fed ovariectomized
rats (43).
In adipose tissue, we focused on the gene expression of the
following: 1) the adipokines Adipoq, leptin, and Pedf; 2) the FIGURE 7 Effects of polyphenol-enriched tea extracts on tissue
concentrations of tea polyphenols and their metabolites in liver (A) and
angiogenesis-related genes Vegfa and Vegfr2; 3) the adipogenic
E-fat (B) in male mice fed an HF/HS, LF/HS, HF/HS-GTP, HF/HS-OTP,
factors Kruppel-like factor 7 (Klf7), CCAAT/enhancer binding or HF/HS-BTP diet for 16–20 wk. Data are means 6 SEMs (n = 3).
protein a (C/ebpa), Pparg, and Srebp1; and 4) the energy Data were analyzed by 1-factor ANOVA, followed by the Tukey-
expenditure genes carnitine palmitoyltransferase I (Cpt1) and Kramer multiple comparison procedure. Labeled means without a
glycerol-3-phosphate acyltransferase (Gpat). The adipocyte se- common letter differ, P , 0.05. No comparison was performed for E-
creted hormone ADIPOQ has multiple functions in lipid and fat 4##-MeEGCG because it was undetectable in all E-fat samples.
glucose metabolism, inflammation, and vascular remodeling (14), BTP, black tea polyphenol; ECG, (2)-epicatechin-3-gallate; EGC,
including increased angiogenesis (14). PEDF, an adipose secreted (2)-epigallocatechin; EGCG, (2)-epigallocatechin gallate; E-Fat, epidid-
factor, inhibits angiogenesis and is elevated in response to obesity ymal fat; GTP, green tea polyphenol; HF/HS, high-fat/high-sucrose; LF/
(44–47). In this study, a 2-fold increase of Adipoq gene expression HS, low-fat/high-sucrose; OTP, oolong tea polyphenol; 4##-MeEGCG,
4##-O-methyl (–)-epigallocatechin gallate.
by GTPs and 63% decrease of Pedf gene expression by BTPs were
associated with increased angiogenic factor gene expression by
Vegfa in the GTP group and significantly enhanced Vegfr2
expression in the BTP group. Our observations are consistent with Our observations of the effects of caffeine-free extracts of
the notion that tea polyphenols increase blood vessel formation in GT, BT, and OT demonstrate that the polyphenol fractions of
adipose tissue, which contributes to their anti-inflammatory teas are biologically active in inhibiting weight gain on an HF/
effects. This finding is in contrast to the traditional concept that HS diet. However, the induction of weight loss by the 3 types of
inhibition of angiogenesis results in weight loss (48) but is tea extracts was induced through different mechanisms. The
consistent with recent observations of proangiogenic activity combination of GTPs and BTPs, by working through different
during adipose tissue expansion mediating protective effects on mechanisms as suggested by the results of the present study, may
metabolism and inflammation (23). Expression of genes asso- provide an interesting combination approach for future human
ciated with adipogenesis, energy expenditure, and lipid meta- studies of the effects of tea polyphenols in obesity treatment and
bolism, including Klf7, C/ebpa, Pparg, Cpt1, Gpat, Srebp, and weight maintenance.
3-hydroxy-3-methylglutaryl-CoA (Hmgcoa), were not changed
by tea polyphenols (data not shown). Acknowledgments
The bioavailability and biotransformation of tea polyphenols The authors thank Mark Hsu for proofreading the manuscript.
are the limiting factor in mediating the biologic activity of tea D.H. and Y.Z. developed the overall research plan and had
polyphenols in target tissues (49). 4##-MeEGCG, the major study oversight; J.Y. and J.E.M. conducted the research; and
biotransformation product of EGCG, is formed by catechol-O- S.M.H., Z.L., and J.Y. assisted in the study design and
methyltransferase in the liver and in the current study was not interpretation of the data, and wrote the manuscript. All
found in adipose tissue (50). In the current study, EGCG, 4##- authors read and approved the final manuscript.
MeEGCG, EGC, and ECG were found in higher concentrations
in the livers of mice consuming GTPs compared with OTPs and
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Tea polyphenol-induced reduction of weight gain 1393